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: IDENTIFICATION OF KEYGENES, MIRNAS AND MIRNA-MRNA REGULATORY PATHWAYS FOR CHEMOTHERAPY RESISTANCE IN OVARIAN CANCER
Review round: 1
Reviewer: 2

Output:
Basic reporting: none
Experimental design: none
Validity of the findings: none
Additional comments: The presented work in this article is  incomplete and not clear.


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: IDENTIFICATION OF KEYGENES, MIRNAS AND MIRNA-MRNA REGULATORY PATHWAYS FOR CHEMOTHERAPY RESISTANCE IN OVARIAN CANCER
Review round: 1
Reviewer: 3

Output:
Basic reporting: - English language should be improved significantly. There still have some unclear and ambiguous parts.

- This manuscript lacks a literature review on bioinformatics-based ovarian cancer studies.

- Quality of figures should be improved.
Experimental design: - A critical concern is the use of a very small sample size of data (only 25 patients). This number was not enough to convince a significant result/finding.

- There must have more description of the methodology to support replicating the methods. Currently, it lacks detailed information.
Validity of the findings: - The authors did not have any validation data.

- What are the cut-off threshold and p-values of GO enrichment analysis?

- What are the limitations of the study and how to address them in future works?
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: IDENTIFICATION OF KEYGENES, MIRNAS AND MIRNA-MRNA REGULATORY PATHWAYS FOR CHEMOTHERAPY RESISTANCE IN OVARIAN CANCER
Review round: 2
Reviewer: 1

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


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

Input:
Title: HORSENETTLE (SOLANUM CAROLINENSE) FRUIT BACTERIAL COMMUNITIES ARE NOT VARIABLE ACROSS FINE SPATIAL SCALES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article titled “Horsenettle (Solanum carolinense) fruit bacterial communities are not variable across fine spatial scales (#62008)” by Heminger AR et al., studied the diversity of microbial communities in fine special scales present in fruits of Horsenettle and presented that there are no significant differences were observed within the range they have tested.
Experimental design: The study design justifies the rationale of this work and the experiments were well designed but for comparison a distant spatial scale could have been added. As a reviewer from a reader’s point of view the below comments were made to improve the overall reach of this work.
Validity of the findings: Overall, the manuscript draft is well written and presented with good statistical analysis. The findings were of interest to the peers working around microbial diversity in plants related studies. There were few minor areas that need authors attention and needs clarifications. I have the below comments and concerns regarding the article.
Additional comments: Comments:
1.	What is the effect of toxicity of Solinine glycoalkaloids present in the fruits of Solanum carolinense on the microbial communities? This is not discussed or considered in this study
2.	Although the rationale for choosing Solanum carolinense was provided in lines 118-120. It is not adequate or justified. Please elaborate.
3.	For comparison and to understand the microbial diversity, the authors could consider doing similar tests in distant spacial scales of Solanum carolinense or choose one more study site that is fine spatial scales. This would not only validate the observed results but also increases the significance of the study.
4.	Line 19: Delete “that”
5.	Line 143, 156, 157: Add a table for the primer sequences
6.	Line 240: “significant variation” variation of which factor?
7.	Line 270: correct to “fruit bacterial communities”
8.	Line 321: Add space between 17 m.
9.	Figure 1, 2 3: The authors have mentioned 9 sample locations. It is not clear from the map in Figure 1 as it shows only 8 locations.
10.	As authors have mentioned in the conclusions: The sample number (n#) is very low ranging from 2-5. It is very difficult to attain any statistical significance due to this low numbers. I would highly encourage to increase the sample number as well as plan a similar fine spatial study for significance.
11.	Figure 2: It is also “Enterobacterials” that make up high proportion of relative abundance. Why this is ignored in lines 227 and 228.
12.	The authors can comment on Figure 2. Site locations 2,3, 8 and 9 about the Micrococales abundance.
13.	Also, authors can write up briefly on other populations like Caulobacteriales, Betaptroteobacteriales and Flavibacterials abundance across different sites.
14.	Figure 3: For site location 5: why are the values for this site location are very low? Any reason for the same?
15.	Figure 4: A clustering is seen at -0.25 for site locations 2,8 and 3. This not discussed in the results. Rather no clustering was observed is stated (Line 245). Please explain.
16.	In the discussion and in the Introduction the authors have stressed on the influence of these microbial communities in “host fitness” but this was not discussed in their results. Please discuss.
17.	A limitations sections need to be added to this article. The points discussed in Discussion part like sample size etc need to be added.
18.	The sample location is fine and not well separated (no effect of environment, soil pH, wind direction, pollinators, etc), All the limitations and the experiments to address them were already stated by the authors in Discussion and conclusions, in that case, what is the exact rationale of this study to investigate microbial diversity within closely distributed plant communities?


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: HORSENETTLE (SOLANUM CAROLINENSE) FRUIT BACTERIAL COMMUNITIES ARE NOT VARIABLE ACROSS FINE SPATIAL SCALES
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1, There is no figure legend for the supplemental figure, the authors should consider including it.

2, The sites and sample information should also be included in the supplemental table 1.

3, The format of “p value” should be consistent in the manuscript, p should be lowercase and italicized.

4, The authors used boxplot in figure 4 to show the distribution of observed Richness, Shannon, and Faith’s phylogenetic diversity by sampling locations. Considering the sample size is small, the authors should consider using barplots or just points to show the results.

5, Compared to the result section,the discussion section in the current draft is too long and confusing. The authors should write it succinctly and just cover the key finding of this study, which is mainly that distance decay in the bacterial communities associated with fruit is not observed at small scales.

6, The raw sequencing data hasn't been deposited on a public archive.
Experimental design: No comment
Validity of the findings: 1, It seems that sample #73 (supplemental table 1 ) has very little input data, this might introduce extra bias for the analysis, maybe the authors should consider removing this sample if there are no other strong reasons to keep it.
Additional comments: The manuscript by Heminger et al., focuses on fruit house microbial communities in a perennial weed, Solanum carolinense. Previous studies from different labs were carried out mostly at large spatial scales. Compared to other tissues, the microbial microbiome in reproduction tissues are less studied. In the current study, the authors used 16S rRNA gene amplicon sequencing and studied the microbial communities in Solanum carolinense fruits at several different local sites within 0.5 km range. In contrast to the published observations in leaves, the bacterial community structure of these samples seems to be homogenous. There are no discernible differences observed regarding the richness or microbial community composition in this current study.

The results in this work challenged the "distance decay" in fruits at very fine scales. It's of interest to the field, if the author could address these points above, it will improve this manuscript.


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

Input:
Title: HORSENETTLE (SOLANUM CAROLINENSE) FRUIT BACTERIAL COMMUNITIES ARE NOT VARIABLE ACROSS FINE SPATIAL SCALES
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comments
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: RETURNING TO THE ROOTS: RESOLUTION, REPRODUCIBILITY, AND ROBUSTICITY IN THE PHYLOGENETIC INFERENCE OF DISSOROPHIDAE (AMPHIBIA: TEMNOSPONDYLI)
Review round: 1
Reviewer: 1

Output:
Basic reporting: Mostly good. The paper and literature coverage could be expanded to make the paper relevant outside the very small circle of dissorophoid experts. See the section General comments to the authors for details.
Experimental design: Again, mostly good. I have some suggestions to improve tree comparisons, especially when they were performed with different programs, which may report difference in lengths for the same tree because, among other things, of differences in treatment of polymorphism.  See the section General comments to the authors for details.
Validity of the findings: Results seem valid.
Additional comments: The draft by Gee is generally well-written and methodologically mostly sound (see some minor reservations below that probably require minor new analyses). I have not spotted technical, factual errors, but I am not a dissorophoid expert and I have not scrutinized the data matrices or the changes in character scores. I have annotated profusely the pdf file, which should be considered my main report (please make sure that Gee gets it), so here, I will only summarize the main key points.

The main thrust of the paper is that data matrices have often (read « nearly always ») been sloppily coded, with many mis-scores, including obvious shortcomings consisting in scores for characters for which the relevant part of the skeleton is not preserved in the relevant taxa. I agree entirely with him that this is totally unacceptable, and I find distressing that the author, reviewers, and editors of the original study did not spot them, and worse, that several colleagues have re-used the matrix (sometimes more than once) without spotting the problem (and again, neither reviewers nor editor spotted these mistakes). So, this main point is important to make. However, the author frames it in the very specialized perspective of dissorophoid phylogeny. There may be a dozen paleontologists worldwide who work regularly on this group, so this makes the paper relevant to a tiny community. Thus, I recommend that that author frames his findings in the bigger problem of putting more emphasis on data quality rather than data quantity. This concerns all systematists doing phylogenetics using phenotypic characters (mostly paleontologists now, but conceivably some working on the morphology, ecology and behavior of extant taxa too). Recent discussions of these issues can be found in Simões et al. (2017) and Laurin & Piñeiro (2018) and references cited therein. Framing the findings in such a broader perspective can be done in a sentence or two (or a paragraph), but it would greatly enhance the potential impact (and citation potential) of the paper, something that a young scientist looking for a tenure-track position ought to be preoccupied about.

The discussion of the nomenclatural (which he calls “taxonomic”) consequences of this work could be improved by reformulating the text to better reflect the fact that taxa are inherently undelimited in rank-based nomenclature and in fact, are not even required by the zoological code to be monophyletic. Thus, stating that (l 1206) “Cacops comprises three species.” is not entirely accurate. He means that we currently recognize three species. Surely, more were named at some point and put into subjective synonymy. As the term implies, this is a subjective decision that could be wrong. Also, there were probably other lineages in the clade that matches more or less the currently-known Cacops clade, so one day we may discover more of them. For a discussion of such points, which are also relevent elsewhere in the draft (l. 1220-1221; 1252; 1335; 1342), I suggest looking at Laurin (2008), though there is probably no need to cite it in the paper.

I believe that there is an unjustified inference on line 311, where the author indicates that “the higher character count of my matrix (44% more characters) made a branch-and-bound search intractable in PAUP*”. Search time increases linearly with character number, but exponentially with taxon number. Scores can affect search times too, especially with branch and bound, because fewer shortcuts can be taken if there is much homoplasy. So, the issue is certainly not character number, but there is nothing anomalous with the fact that he could not do branch-and-bound with that matrix.

On lines 619-621 and much more importantly 666-668, 1080-1090 and in Permutation 10, the author should verify if the trees of different lengths obtained by the different programs (TNT vs PAUP) reflect differences in calculation by the programs. I know that their treatment of polymorphism, among other things, varies, and ordering scheme can also make a difference. The proper way to do this is to import all trees into the same program and verify their length (I typically do this in Mesquite, which is very user-friendly). Because if one set of trees is shorter than a second set, the first one is preferable, whether their strict consensus is more or less resolved. Yet, this point is not addressed in the paper.

On lines 972-973, the author states that “A lack of resolution does not necessarily indicate that the matrix is compromised or poorly constructed”. I agree! This point could be strengthened by being explained in more detail. Irresolution is better than wrong resolution because the former gives a correct idea of uncertainty whereas the latter looks deceptively satisfactory. In fact, in our (mostly but not entirely simulation-based) work to assess the performance of phylogenetic methods, we penalized methods that yield wrong clades. About this, see Rineau et al. (2018: 19), where we present a mathematical formula to do this quantitatively using a metric derived from 3ia (other ways to do this exist and more could be developed). This point deserves a more extensive treatment because many systematist consider polytomies a failure and some are even tempted to “tinker” with their matrix to “solve the problem”, as the author points out.
This point is relevant on l. 1123 too.

On l. 1134-1135, the author states: “I have personally never run a PAUP* analysis in which a node recovered in the strict consensus was not recovered in the bootstrap tree.” That may well be true for the author's experience, but such differences do occur, including in Marjanovic & Laurin (2019), which he already cites (but not for this point). Compare their figures 10 and 11 (classical MP analysis of revised matrix from Ruta & Coates 2007, but with corrected scores) with the bootstrap analysis (B1, fig. 18). Note multiple differences, such as the placement of close relatives of Lissaphibia (there are probably some in temnospondyls took but these are complex with two sets of topologies depending on the position of anthracosaurs).

On l. 1162-1164, Gee comment again on Marjanovic & Laurin (2019) about their recovered paraphyly of Olsoniformes. Gee does not seem to realize that this paper presents many analyses, including some of the unmodified data by Ruta, some with scores corrected, some with an expanded taxonomic sampling, and the latter seem to refute (to an extant) Gee's suggestion that limited sampling explains this result. I suspect that the problem may be in scores in Ruta & Coates' (2007) matrix that Marjanovic (who did the bulk of the re-scoring) did not find when updating it. But this too is speculation. Perhaps Olsoniformes are paraphyletic? After all, we don't have the true tree of temnospondyls, as Gee himself emphasizes. See my more detailed comment on the annotated pdf file.

References:

Laurin M. 2008. The splendid isolation of biological nomenclature. Zoologica Scr 37:223–233. 10.1111/j.1463-6409.2007.00318.x

Laurin M, and Piñeiro G. 2018. Response: Commentary: A Reassessment of the Taxonomic Position of Mesosaurs, and a Surprising Phylogeny of Early Amniotes. Frontiers in Earth Science 6:1-9. 10.3389/feart.2018.00220

Rineau V, Zaragüeta I Bagils R, and Laurin M. 2018. Impact of errors on cladistic inference: simulation-based comparison between parsimony and three-taxon analysis. Contributions to Zoology 87:25-40.

Simões TR, Caldwell MW, Palci A, and Nydam RL. 2017. Giant taxon‐character matrices: quality of character constructions remains critical regardless of size. Cladistics 33:198-219. 10.1111/cla.12163


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: RETURNING TO THE ROOTS: RESOLUTION, REPRODUCIBILITY, AND ROBUSTICITY IN THE PHYLOGENETIC INFERENCE OF DISSOROPHIDAE (AMPHIBIA: TEMNOSPONDYLI)
Review round: 1
Reviewer: 2

Output:
Basic reporting: All good, except that the style is overwrought in places, making some passages difficult to read. See the attached file for details.
Experimental design: Good, but see the attached file for details.
Validity of the findings: Very good, see attached file.
Additional comments: My review is over 10% as long as the ms, and given that the ms has 142 pages, I provide my review as the attached file. I ask for "major revisions" only because I would like to see the next version; otherwise, I would not call the revisions I ask for "major", even though they are rather numerous.


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: RETURNING TO THE ROOTS: RESOLUTION, REPRODUCIBILITY, AND ROBUSTICITY IN THE PHYLOGENETIC INFERENCE OF DISSOROPHIDAE (AMPHIBIA: TEMNOSPONDYLI)
Review round: 1
Reviewer: 3

Output:
Basic reporting: The study by Gee represents an in-depth treatment of an important and complex clade of dissorophoid temnospondyls, the Family Dissorophidae. It builds extensively on the author's previous experience and excellent puplication record, revealing a fastidious eye for anatomical detail and a proficient use of analytical protocols. Much work remains to be done on this clade, but no stone is left untrurned in this work. Aside from providing a compendium of the history of research and revisions of dissorophids, this study also provides a much needed cladistic framework that could be redeployed in future analyses. The reference list is one of the best I have ever seen, up-to-date and comprehensive. It miswritten clearly and concisely, so the length is justified as this is a real tour de force through an elaborate topic. Figures are clear and essential, with more than adequate captions. The results and conclusions are amply justified.
Experimental design: The research is original and truly important and will be a useful primer for specialists in he field and anyone with interest in early tetrapods (in general) and temnospondyls (in particular) evolution. It demonstrates high standard of execution and fills many gaps in knowledge through attention to morphology and accurate revision, as far as I can tell, of selected characters and coding. Methodology is exhaustive and well written.
Validity of the findings: The findings are intriguing. Loss of resolution in some experiments is expected, but this is not a flaw. All results can be reproduced very easily, given the abundant supplementary material. There are no areas of wild speculation and all statements are justified.
Additional comments: I congratulate the authors on a masterclass delivery


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: RETURNING TO THE ROOTS: RESOLUTION, REPRODUCIBILITY, AND ROBUSTICITY IN THE PHYLOGENETIC INFERENCE OF DISSOROPHIDAE (AMPHIBIA: TEMNOSPONDYLI)
Review round: 1
Reviewer: 4

Output:
Basic reporting: I read this manuscript with great interest. Besides presenting a new phylogenetic analysis of Dissorophidae, the author critically assesses previous phylogenetic studies of the group.

I think this is an important manuscript as it presents a list of shortcomings of previous works that are also applicable beyond the study of Dissorophidae. Crucially, it highlights the importance of documenting and justifying the methodological decisions taken when scoring matrices and running the analyses, and of reporting the results in detail (e.g., reporting support metrics).

Even though there are other researchers in the field who might not agree with some of the statements made by the author (either based on personal opinions or due to the access to further data such as specimens not studied first-hand by Dr. Gee), the level of detail given throughout the manuscript paves the way for future discussions (e.g., in response papers).
Experimental design: - The study is mostly literature-based. This might be a shortcoming as first-hand observations of fossil material are more informative (when conducted by someone familiar with the taxonomic group). However, in this case as the scoring is based on the literature, it reinforces one of the key issues addressed in the manuscript: replicability. On the one hand, literature is more accessible than visiting collections in multiple countries; on the other hand, published and well supported descriptions are more transparent than non-published/non-photographed first-hand observations made by a researcher when scoring.

- I find the narrative structure of the manuscript particularly informative. It is useful to see how the overall goal of the project changed after the first results, and how the author attempted to reconstruct and reproduce previous studies, detailing the limitations he encountered. This is common practice in research, however it is rarely discussed in papers.

- The taxon and character sampling, and the phylogenetic analyses are explained in detail.
Validity of the findings: - The findings and conclusions are well supported by the numerous permutations and the extensive bibliographic work.
Additional comments: - In my opinion, by his thorough revision, the author gives due credit to the previous key dissorophid studies. Instead of simply dismissing or underrating them due to what he thinks is the lack of best practices, he tries to unravel the differences among their results. In this regard, he explicitly states that he is not advocating against propagating an existing matrix (l. 843-844) and shows (Fig. 3) that even his previous works were also derived from Schoch’s matrices. However, I think the manuscript would be improved by including a paragraph explicitly explaining the relevance of the key works the author revises.

- The general tone in which the author criticizes previous studies is appropriate. However:
1. In l. 959-977, where it is stated that inferred character states are not valid, references are needed, or it needs to be made clear that this is the author's opinion. It would also be worthwhile to acknowledge that it was common practice to use “assumed” states, and was not something just done by Schoch.
2. Abstract: “others relate to best practices (or the lack thereof)” may sound pretentious. I would suggest rewording it (e.g., “others relate to discrepancies with respect to what are currently considered best practices”).

- l. 193. A more detailed explanation is needed about why some multi-state characters were ordered. Ordering character states following an evolutionary hypothesis is problematic because it implies circular reasoning. As it is explained in l.962 “While a phylogenetic analysis may be a test of inferences, it is not a test that can be based on inferences”.


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: RETURNING TO THE ROOTS: RESOLUTION, REPRODUCIBILITY, AND ROBUSTICITY IN THE PHYLOGENETIC INFERENCE OF DISSOROPHIDAE (AMPHIBIA: TEMNOSPONDYLI)
Review round: 2
Reviewer: 1

Output:
Basic reporting: The paper is now mostly fine. I found very minor problems that could be fixed at the page proof stage (see below, section 4, Additional comments).
Experimental design: Fine.
Validity of the findings: Good.
Additional comments: I think that the author has done a good job of addressing my comments, so I feel that the paper is ready for publication, or could be subject to very minor changes, which I list below.

In this report, I refer to the line numbers in the pdf file.

Line 46: Replace “essentiality » by « great importance” because some migh confuse the former with relating to essentialism.

Line 58: I would replace “agree” in “and therein agree is well-designed » by “ assume” or both: “agree or assume” because as this draft shows, this may be more frequently an assumption than an explicit agreement made after checking data quality.

Lines 71-72: Thank you for specifying what you mean by “amphibian”, but a slight rewording is required. Please change:
Temnospondyli, often referred to as ‘amphibians’ (nonamniote tetrapods in a broad historical sense and as the putative amphibian stem-group in more recent work),
Into:
Temnospondyli, often referred to as ‘amphibians’ (nonamniote tetrapods in a broad historical sense and as the putative amphibian stem-group in several recent works),

The current wording has two problems: the use of the singular implies a single work supports the TH, which even the author surely does not believe, and second, the “more” suggests that alternatives (PH, LH) have been abandoned, which is also wrong.

These very minor changes could be made in the page proofs.


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: RETURNING TO THE ROOTS: RESOLUTION, REPRODUCIBILITY, AND ROBUSTICITY IN THE PHYLOGENETIC INFERENCE OF DISSOROPHIDAE (AMPHIBIA: TEMNOSPONDYLI)
Review round: 2
Reviewer: 2

Output:
Basic reporting: Ready for publication.
Experimental design: Almost ready for publication: TNT analyses should be repeated with partial uncertainty changed to polymorphism (if that is even necessary) rather than to full uncertainty, which has apparently distorted the results a little.
Validity of the findings: Almost ready for publication.
Additional comments: The manuscript is approaching perfection! Here are my remaining comments, of which only the first three, arguably four, concern substance. I ask for "major revisions" only because I would like to take one last brief look at the manuscript before it is accepted; the revisions I suggest below are quite minor.

In lines 267–270 and 1251–1254, you state that because TNT cannot apparently handle partial uncertainty, you changed those scores to full uncertainty for the TNT analyses. That must be the reason why branch-and-bound in PAUP* and implicit enumeration in TNT didn’t always give the same results: the matrices were different! TNT can handle polymorphism, and polymorphism is computationally exactly the same as partial uncertainty (because ancestors cannot be reconstructed as polymorphic); the only difference is that every polymorphism adds one step to the tree after its topology has been calculated, while partial uncertainty doesn’t (i.e., polymorphism is treated as requiring a step inside the OTUs that have it, while OTUs with partial uncertainty are simply optimized as having one particular state). That is why PAUP* lets you choose whether to treat both polymorphism and partial uncertainty as polymorphism (pset mstaxa=polymorph) or as partial uncertainty (pset mstaxa=uncertain) or to treat them differently (pset mstaxa=variable). I must ask you to repeat all TNT analyses; I predict the resulting topologies and numbers of MPTs will be identical to those found by PAUP*. (I suppose it’s possible, however, that Dilkes [2020] really didn’t let his PAUP* analyses run for long enough to find any MPTs.) If TNT gives you an error message, change all partial uncertainty to polymorphism by hand, but I think TNT will read the partial uncertainties just fine and simply treat them the same way as polymorphism.
1250: Does that mean implicit enumeration only works for binary characters???
1701–1702: Is stemward slippage of small/immature specimens really expected when the clade found next to Dissorophidae consists almost only of large-bodied trematopids? Shouldn’t the small/immature dissorophids instead cluster as far away from the large trematopids as possible?
Reply to reviews: I’m not happy with the approach by Brazeau, Guillerme & Smith (2017), as I wrote in the 2019 paper (p. 21): “Too recently for us to use, Brazeau, Guillerme & Smith (2017) published a new approach to dealing with reductively coded inapplicability in phylogenetics software. We are looking forward to further developments of its implementation. However, we strongly disagree that so-called “neomorphic characters” should be scored as having the presumedly plesiomorphic state when they are inapplicable. This requires identifying the plesiomorphic state in advance, which increases the danger that the phylogenetic analysis will conform to one’s preconceptions just as much as an all-zero outgroup would. It is also much less easy than Brazeau, Guillerme & Smith (2017: 23) implied when they stated that in their analysis “every inapplicable token in each neomorphic character was replaced with the token corresponding to the presumed non-derived condition (typically ‘absent’)”—for example, our matrix contains many characters for the presence or absence of bones that are, in our taxon sample, plesiomorphically present and are apomorphically lost several times, while different taxon samples (e.g. vertebrates generally, or actinopterygians) would support the opposite polarization or none at all. Further, this method creates redundancy just like absence coding does: in the example by Brazeau, Guillerme & Smith (2017: table 1), absence of the tail unfailingly predicts absence of eyespots on the tail.”

Then there is the matter that I only just noticed Hystrix (Old World porcupines) is feminine (with species names like H. cristata Linnaeus, 1758). Therefore, so is Platyhystrix, so its species name is automatically P. rugosa. No nomenclatural act is necessary. ICZN Article 34.2 has already taken care of it: “The ending of a Latin or latinized adjectival or participial species-group name must agree in gender with the generic name with which it is at any time combined [Art. 31.2]; if the gender ending is incorrect it must be changed accordingly (the author and date of the name remain unchanged [Art. 50.3.2]).” In other words, as soon as Williston (1911) transferred Ctenosaurus rugosus Case, 1910, to his new genus Platyhystrix, it became Platyhystrix rugosa (Case, 1910), regardless of whether Williston was aware of that fact and regardless of whether that form has ever actually appeared in print.

Lines 89–90: “in phylogenetics”, as an insertion into the sentence, needs either to be surrounded by commas at both ends or none at all.
208: The coauthors and year of Zaragüeta i Bagils are missing.
250: A citation for this overestimation would be good. The phenomenon is widely known, but I don’t personally know where it has been studied.
401: Remove the apostrophe.
429: Replace “Cacops” by “Cacopus”, and replace the parenthesis by “(which is ‘Cacopod’, not ‘Cacop’)”.
430: “per the same article”, as an insertion into the sentence, needs either to be surrounded by commas at both ends or none at all. “1994”, as an insertion into the sentence, likewise needs to be surrounded by commas on both sides.
484: Replace “for” by “form”.
536: Remove the comma after “lucius”.
538: “earliest-diverging” needs its hyphen because Conjunctio is the cacopine that diverges earliest, not the earliest one of diverging cacopines.
556–557 and fig. 10A, B: No, Xerodromes has a node-based definition (as indicated by “least inclusive clade” in the original wording; the external specifiers are part of a qualifying clause), so Perryella, not Micromelerpeton, is the sister-group of Xerodromes in these trees.
609–610, fig. 6A and fig. 13A: Olsoniformes is misplaced; having a node-based definition (as for Xerodromes, the external specifiers are part of a qualifying clause), its first member is one node more highly nested, so that Iratusaurus, Palodromeus and Reiszerpeton are not members in fig. 6A, and Reiszerpeton is the sister-group of Olsoniformes in lines 609–610 and in fig. 13A. In fig. 6A it might also be a good idea, as a matter of visual presentation, to move Kamacops to the other side of Dissorophidae, so that all the single-OTU members of the dissorophid polytomy are shown on the same side.
640: Remove “were”, or replace it by “that were” or “which were”.
649-650: see 538.
698–699, fig. 18C: That’s true for nominal dissorophids, so inserting “the nominal” at the beginning of line 699 would repair the text. As defined, however, Dissorophidae would include Ecolsonia, Mordex, Rotaryus, Tambachia, Anconastes and Actiobates, so the dot that marks the basal node of Dissorophidae in fig. 18C must wander rootward accordingly. Conversely, Mattauschia is the sister-group of Olsoniformes in that strange tree.
732–733, 819, 833, 837: see 538.
829: Replace “slightly” by “slight”.
1100: “thereby”, as an insertion into the sentence, needs either to be surrounded by commas at both ends (for emphasis) or none at all (which I would personally prefer here).
1227: Uppercase R in PAUPRat, which I recently used for the first time (currently in review); it is not part of PAUP*, not widely known, and appears not to have been used ever before in phylogenetic analyses of early tetrapodomorphs. (Ruta, Coates & Quicke [2003] and Ruta & Coates [2007] did use the parsimony ratchet, but through Quicke’s unpublished, undocumented implementation rather than through PAUPRat.)
1252: Insert “not” before “others”, or remove the entire parenthesis.
1319: Replace “poor” by “poorly”.
1361: Replace “the paraphyletic Olsoniformes of” by “why Olsoniformes was not found in” – Olsoniformes has a definition with a qualifying clause, so it is either a clade or does not exist; it can’t be paraphyletic.
1379: Similarly, Trematopidae is a clade by definition, so what that tree really does is to restrict Trematopidae to Acheloma + Phonerpeton; I suggest replacing “Trematopidae” by “the nominal trematopids” or similar.
1396–1397: Likewise, Kamacopini is a clade by definition, the question is just whether it contains anything other than Kamacops. I would simply delete “and whether it is even a clade”, because the problem of its composition is already mentioned.
1526: “however”, as an insertion into the sentence, needs to be surrounded by commas from both sides.
1544: “Therein” doesn’t seem to make sense here (it triggers the question “in what?”); I recommend “Therefore”.
1551: I actually recommend “morphospecies” rather than “species” to avoid all issues with species concepts.
1581–1582: “illustrations” or “represents”?
1773: Replace “of” by “after”.
Fig. 6B, 22G: Replace “neglecta” by “neglectus”.
Fig. 14A: Remove the dot for Xerodromes (as the text correctly states).

Finally, let me strenuously disagree with Reviewer 4 on one point: I really hope it was never common practice to score assumed/inferred character states in phylogenetic analyses of any taxon. In the fields I know, it has not been usual in about 30 years, if ever. In the mid-late 90s, Paul Sereno once said (or so I was told) about his analyses of the phylogeny of various dinosaur clades that once you’re experienced enough, “you know what the tree is going to look like, and then you code accordingly.” Indeed, his analyses, unlike everyone else’s, had CIs above 0.8, sometimes above 0.9; given the size of his matrices, such CIs are simply impossible to reach in any honest way. He was at least cherry-picking his data and thought that was a good thing. Even his own slightly later analyses, however, had much more realistic CIs (and different topologies, too…). He had evidently learned that if you score your OTUs after their presumed close relatives, you’re committing circular logic. The analysis is supposed to tell you if they really are close relatives. It is not supposed to merely illustrate your opinions (as was done in some works in the late 80s and early 90s), it’s supposed to give you an independent result based on an independent assessment of the evidence – independent of you, that is. If you assume a tree a priori and invent scores for missing data to fit the tree, your analysis will give the tree you assumed back to you, and you’ll have no way of telling if that’s accurate – which it may well be! – or just another case of “garbage in, garbage out”. Do not score states that aren’t preserved in the actual material, no matter how reasonably you assume them. Phylogenetic analysis is supposed to be science, not circular logic. Or as the manuscript says in lines 1001–1002: “a phylogenetic analysis should inform taxonomy, rather than vice versa.” – The manuscript already makes clear, e.g. in lines 353–355, that the malpractice wasn’t limited to Schoch for dissorophids, but that e.g. Holmes, Berman & Anderson (2013) committed it as well. – Too bad the archives of the Dinosaur Mailing List are currently down. Some 20 to 25 years ago, the question actually came up if Tyrannosaurus should be scored as laying eggs in a phylogenetic analysis. None less than Thomas Holtz, a pioneer of theropod phylogeny, spoke up and said no: there is no fossil evidence on the reproduction of Tyrannosaurus specifically, so the correct score for Tyrannosaurus is a question mark. As a matter of fact, we don’t know if Tyrannosaurus had any unexpected autapomorphies in this respect, no matter how unlikely it may seem; and before the analysis is done, we also don’t really know if reproduction evolved in a more complex way than we think. That post might have been citable, should a citation really be felt to be necessary.

On ordering, I agree with Reviewer 4 that ordering based on an evolutionary hypothesis runs the risk of circularity and should accordingly be avoided; but that is not the same as ordering characters that represent a morphocline, because such characters can be recognized in the absence of any phylogenetic/evolutionary hypothesis. Moreover, the assumption that it is easier to change from one state to a close state than to a distant state has already been used to divide the character into discrete states in the first place, so it would be inconsistent not to use it again. Citations for this are in the manuscript and in my & Laurin’s 2019 paper.

David Marjanović


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: RETURNING TO THE ROOTS: RESOLUTION, REPRODUCIBILITY, AND ROBUSTICITY IN THE PHYLOGENETIC INFERENCE OF DISSOROPHIDAE (AMPHIBIA: TEMNOSPONDYLI)
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The finding that implicit enumeration in TNT and branch-and-bound in PAUP* produce different results seems very important, but also beyond the scope of the manuscript. Maybe add something, after line 1269 of the tracked-changes manuscript, to the effect that their behavior in phylogenetic inference should be studied further.

209: Replace “Rineua” by “Rineau”.
276: Close the parenthesis at the end of the sentence.
438: Insert a comma after “‘Cacopod’”.
446: I’m not sure how best to word this, but “should have been” is not the best option. Maybe “was in fact” or “was, as a matter of legal fiction,”…

That’s it – I recommend acceptance after the minor issues above are resolved.

David Marjanović


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 FULL-LENGTH TRANSCRIPTOME AND METABOLOME OF DICHOCARPUM (RANUNCULACEAE): IMPLICATIONS IN EVOLUTION OF SPECIALIZED METABOLISM OF RANUNCULALES MEDICINAL PLANTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The submitted manuscript entitled as "Dissection of full-length transcriptome and metabolome of Dichocarpum (Ranunculaceae): Implications in evolution of specialized metabolism of Ranunculales medicinal plants" by Dr. Hao and colleagues. This manuscript deciphered the full-length transcriptomes and metabolomes of five Dichocarpum species endemic in China through the third-generation high-throughput sequencing and metabolomic techniques. They obtained 71,598 non-redundant full-length transcripts, and twenty-seven orthologs extracted from trancriptome datasets were concatenated to reconstruct the phylogenetic tree and found that the extensive correlations between metabolite content and gene expression.
This work is meaningful, which will promote the insights into the transcriptomic and metabolomic landscapes of Dichocarpum, which will be usefully for further studies on genomics and application of Ranunculales plants.
Experimental design: The authors are also recommended to examine the expression of some representative genes by conduct Quantitative Real-time PCR (qPCR) to validate the transcriptomic results.
Validity of the findings: About transcriptomic and  metabolomic analysis, the authors are recommended to give more comparison with previous studies in the results and (or) discussions.
Additional comments: The abstract of this manuscript should be improved to make it more concise. There are a number of grammatical errors in the manuscript and should be proof-read by a native English speaker or a professional language editing service.


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 FULL-LENGTH TRANSCRIPTOME AND METABOLOME OF DICHOCARPUM (RANUNCULACEAE): IMPLICATIONS IN EVOLUTION OF SPECIALIZED METABOLISM OF RANUNCULALES MEDICINAL PLANTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors compared the transcriptome and metabolome among the five Dichocarpum species, and proposed biosynthetic pathways of BIAs in Dichocarpum.
Experimental design: Major points:
1.	A better phylogenic tree with more species is needed to replace Figure 3. How do the five species fit into a previous published the phylogenic tree of the Dichocarpum family? Please refer to:  https://www.sciencedirect.com/science/article/abs/pii/S1055790316304493
2.	Please clarify the experimental materials – What tissues or organs at what developmental stages were used in the study. Gene expression and metabolism are very different in different tissues/organs at different developmental stages.
3.	Please compare the transcriptome and metabolome results with other model organisms, eg. Arabidopsis and Chlamydomonas.
4.	What is the significance of the BIA biosynthetic pathway? Are there candidate compounds that have potential or identified medical effects in the BIA biosynthetic pathway?
Validity of the findings: Minor points:
1.	The authors stated that CDS length distributions and transcriptomes are similar in the five species. If so, why there are so many unique transcriptome sequences in the five species?
2.	Which tissues or organs are known to be enriched with annotated KEGG pathways such as “Phenylpropanoid biosynthesis” and “Glutathione metabolism?”
3.	Line 70-73 Lack of citation.
4.	Line 84-85 The meaning of the sentence is not clear. What does the “language” mean?
5.	Figure 1D. What are the outliner incRNAs? Do those outliers share some similarities in sequences or locations in the genomes?
6.	Figure 2A. Why does SCJY have very different interval distribution pattern than the other four species?
7.	Figure 2C. Why do gene expression patterns seem to have poor correlations among the five species?
8.	Figure 2D. Please make clear annotations on the gene clusters.
9.	Figure 4 & Figure 5 – a lot of the information in the figure legends belong to the method section. Please rewrite the legends to be more concise and clear.
10.	Line 474 – 498 The hypothesis on the whole genome duplication is a big stretch. Please provide clear explanation with more detailed information.
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 FULL-LENGTH TRANSCRIPTOME AND METABOLOME OF DICHOCARPUM (RANUNCULACEAE): IMPLICATIONS IN EVOLUTION OF SPECIALIZED METABOLISM OF RANUNCULALES MEDICINAL PLANTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors clearly clarified the questions that I raised. Basically, I am satisfied with the current version.
Experimental design: The authors clearly clarified the questions that I raised. Basically, I am satisfied with the current version.
Validity of the findings: The authors clearly clarified the questions that I raised. Basically, I am satisfied with the current version.
Additional comments: I have no more 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 FULL-LENGTH TRANSCRIPTOME AND METABOLOME OF DICHOCARPUM (RANUNCULACEAE): IMPLICATIONS IN EVOLUTION OF SPECIALIZED METABOLISM OF RANUNCULALES MEDICINAL PLANTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript was well edited and written. The authors answered all the questions with plenty of details. I support to accept the article.
Experimental design: NA
Validity of the findings: NA
Additional comments: Two minor things:
1. The world “defense” were misspelled as “defence” -  line 121 and 132.
2. The color scheme could be improved. The same species could use the same color in all the figures.


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: EVOLUTIONARY HISTORY OF THE VERTEBRATE PIWI GENE FAMILY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper provides a detailed phylogenetic analysis of PIWI Argonaute proteins across vertebrates. The analyses reveal interesting evolutionary turnover of PIWIs across vertebrates, significantly expanding on previous work.

In general, the authors had good language and a sufficiently detailed introduction, background and discussion. However, I suggest that authors elaborate/clarify evolutionary jargon to make the work more easy to read and accessible to all scientists. For example, it would be useful to define different clades (e.g. gnathostomes vs cyclostomes) at least once in the text or to use a species tree as a supplemental figure to label different clades.

I would also recommend the following changes that could improve ease of understanding the data:
1. Figure 1 is a bit overwhelming and difficult to read. The use of too many colors, especially since some colors overlap (for example, piwil1 and cartilaginous fish) make the figure more tedious to understand. I would suggest simplifying the figure by using only the color scheme for piwis or the major vertebrate clade. My preference as a reader was for the former (piwis colored). Alternatively, the authors could use box brackets next to the tree to indicate PIWIs without using colors. If using color for PIWIs, I would recommend a uniform color scheme across the paper. In addition, I would have appreciated an additional main or supplemental figure to summarize the presence or absence of a piwi in each vertebrate clade to complement Figure 1 (a simplified schematized version of Supplemental Table S1) similar to the last figure.

2. More descriptive figure legends would help the reader understand all aspects of the figure better. For example, in Figure 2, empty spaces for piwi that are meant to represent lack of evidence for presence or absence of the piwi. While this was described in the text, it would be useful to also include this in the legend.

Minor comments:
1. Simplify bootstrap representation in Figure 1 to just a % number and explain this in the legend as well.

2. The raw data provided was sufficient. My only recommendation would be to make labels in trees and alignments match that of the figures in the paper to allow for easy comparison.
Experimental design: The research covered in this manuscript are within the scope of the journal and addresses an open question in the field. The phylogenetic analyses span many vertebrate organisms revealing diversification of PIWIs. The methods section and supplemental data are largely sufficient, but some additional clarification and inclusion of some additional data would improve the presented work.

1. The phylogenetic trees constructed (Figure 1 and Supplementary Figure 1) use the entire sequence of PIWIs. However, N terminal residues are poorly conserved based on the alignment provided in the raw data for each tree. Poor alignment could lead artificial groups in the tree; therefore, I suggest using the most well aligned regions of the sequences to make a tree. The alignment submitted suggests these might be the PAZ and PIWI domains but not the N-terminal regions. A tree made only on these well aligned regions could be included as a supplementary figure.

2. The positive selection analyses presented in the paper span larger vertebrate clades. Since these species (for example, mammals) are very diverged, their dS tend towards saturation and can distort the omega calculation for these analyses. I would suggest also including a more closely related set of mammals (for example simian primates) in their analyses in Figure 3. Similar analyses have been previously done in Wynant et al., 2017 (https://doi.org/10.1038/s41598-017-08043-5), where they also suggest Piwil3 shows signatures of positive selection. However, with more available primate genomes now, a more comprehensive analysis (with more simian primates) would be informative. I would also suggest that authors reference Wynant et al., 2017 in their manuscript.

3. A nice addition to the current analyses might be to show the sequence divergence/ identity between PIWIs. A LOGOs plot spanning a fixed set of species (particularly in the PIWI and PAZ domains) or even a schematic showing differences in protein length with % identity labelled will be informative.

4. Similarly, it would be useful to actually see the positively selected residues (with BEB>0.9) across a group of species for Figure 3C. For example, a figure or table that shows each positively selected residue and how they have changed across a group of species to see the range of residues that can be found at each position.

5. From the descriptions in the text and the methods, it is not clear if the authors did a thorough analyses to identify all pseudogenes of PIWIs (other than for some species in 2B). Regardless, it would be informative to add details about if and how pseudogenes were identified in the sequence acquisition section in methods similar to their explanation in lines 204-208.
Validity of the findings: 1. Since PIWIs appear to be expressed in ovaries (even if at lower levels than in testes), I suggest including a paragraph in the discussion on PIWI functions in the ovaries.

2. In the discussion section, a possibility that is not addressed is one where PIWIs can have redundant functions. Despite the divergence in sequence, I wonder if it is possible that in cases where a PIWI is pseudogenized or missing, it is because an alternate PIWI is functionally redundant even if this is species-specific. I suggest that the authors raise this possibility in the discussion and cite existing literature (if any) to support/oppose this hypothesis.
Additional comments: I recommend that the authors ask colleagues in both the evolution and PIWI field to read and comment on their manuscript to improve its readability for a broader audience. I would also like to commend the authors on their analyses during the difficult circumstances of the COVID-19 pandemic.


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: EVOLUTIONARY HISTORY OF THE VERTEBRATE PIWI GENE FAMILY
Review round: 1
Reviewer: 2

Output:
Basic reporting: See general comments
Experimental design: See general comments
Validity of the findings: See general comments
Additional comments: Guiterez et al employ phylogenetics, synteny, evolution rate and expression to explore the evolution of vertebrate Piwi genes. I am in agreement with their findings and support publication. I appreciate the authors expertise does not primarily reside in the piRNA pathway and as such some of the statements regarding PIWI function require revision to incorporate more recent findings.

Major concerns
I think the evolution of Piwil2 should be incorporated into Figure 5.

PIWI Literature
1.	The following statement requires revision: ‘Piwis are primarily expressed in gonads and protect the germline against the mobilization and propagation of transposable elements (TEs) through transcript cleavage and genome methylation.’. Not all animals utilise DNA methylation to transcriptionally silence TEs. Maybe one could utilise the term ‘transcriptional gene silencing’ and explain the role of DNA methylation in mammals.

2.	The following statement requires revision: ‘PIWI proteins are generally restricted to the germline, bind their own class of small RNAs, PIWI interacting RNAs (piRNAs), and regulate TEs and viruses through transcript cleavage and genome methylation (Girard et al 2006; Lau et al. 2006; Brennecke et al. 2007).’. Same criticism as point 1.

3.	The following statement requires revision: ‘PIWIs will then cleave the newly bound transcript and repeat the cycle. These piRNAs can also be used to guide PIWI proteins to TE loci in the genome and initiate methylation (Aravin et al. 2008; Kuramochi-Miyagawa et al. 2008; Rozhkov et al. 2013).’ The above sentence is not accurate. PIWIL4 is not an endonuclease (PMID: 22020280) and does not engage in ping-pong cycle. Phase piRNA biogenesis should be incorporated into the sentence (reviewed in PMID: 30446728 and original refs found within).

4.	The following statement requires revision: ‘From these experiments, we understand the expression of Piwis varies temporally during embryonic and testis development.’. The following sentence would be more accurate: ‘From these experiments, we understand the expression of Piwis varies during germ cell development and spermatogenesis.’.

5.	The following statement requires revision: ‘Piwil4 (Miwi2) is expressed for a short period of time between E14.5 and post-natal  day 3 (P3), complements PIWIL2 in the ping-pong cycle, and is linked to the de novo  establishment of methylation marks among TE loci in primordial germ cells (Carmell et al. 2007;  Aravin et al. 2008; Molaro et al. 2014).’ As point 3.  De novo methylation occurs in foetal gonocytes not PGCs. PIWIL4 is also expressed in a population of undifferentiated spermatogonia in the adult mouse testis (PMID: 28461596 and PMID: 29728652). I would add the following reference (PMID: 32674113) to above sentence which shows a detailed timeline moue PIWIL4 expression during germ cell development.

6.	The following statement requires revision: ‘Piwil4 was detected in the testes of all species examined except the mouse (Fig. 4B). Aravin et al. (2008) reported that Piwil4 is only expressed for a short period of time during testis development in mice, but that does not appear to be universal among vertebrates.’. As point 5.

7.	The following statement requires revision: ‘Because PIWIL4 localizes to the nucleus and PIWIL2 localizes to the cytoplasm, it was thought that PIWIL2 was responsible for loading PIWIL4 with piRNAs and PIWIL4 “marks” TE loci for subsequent methylation (Carmell et al. 2007; Aravin et al. 2008).  However, it was later shown that both PIWIL2 and PIWIL4 function independently in methylation  pathways and PIWIL2 initiates the methylation of more TE loci than PIWIL4 (Manakov et al. 2015).’. The second sentence should be revised in the light of a more recent paper (PMID: 28530707). I suggest eliminating the second sentence, how can a cytoplasmic protein function in the nucleus. The discrepancy arises between the purity of germ cells isolated for DNA methylation analysis.

8.	The following statement requires revision: ‘For example, in mice, the expression of Piwil4 coincides with primordial germ cell methylation  erasure and re-establishment (Aravin et al. 2008; Carmell 2008; Molaro et al. 2014), which led authors to suggest Piwil4 is essential to the de novo re-establishment of TE methylation in  primordial germ cells.’. PIWIL4 is only expressed during the period of de novo methylation during germ cell development (see & also cite PMID: 32674113), this occurs in gonocytes not PGCs. The other important evidence is that PIWIL4 is a nuclear protein.

9.	The following statement requires revision: ‘In contrast, our expression analyses suggest Piwil4 is also expressed in mature, adult testes of all other gnathostomes (Fig. 4B). It is currently unclear what the function  of PIWIL4 exactly is in adult testes since this protein is no longer synthesized three days after  birth in mice.’. PIWIL4 is expressed in a tiny population of undifferentiated spermatongonia (PMID: 28461596 and PMID: 29728652) but its function is dispensable for homeostatic and regenerative spermatogenesis (PMID: 29728652). Also PIWIL4 is expressed in the lung (PMID: 28920925).

10.	In my opinion the following statement is not supported: ‘Therefore, the expression of Piwil4 in mouse primordial germ cells during methylation re-establishment could be coincidental, lineage specific, or a temporal function.’. The literature suggests that the primary function of PIWIL4 is during germ cell development and to instruct piRNA-directed DNA methylation.


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: EVOLUTIONARY HISTORY OF THE VERTEBRATE PIWI GENE FAMILY
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is well written, and enjoyable to read. The central message(s) is (are) simple and clear, and the manuscript makes a useful addition to the literature. The structure seems reasonable, the figures are excellent (I particularly like figure 5), and the raw data are all available as supporting material.

The one major issue I have with the writing is the lack of reference to the evolutionary literature; a casual reader is given no context by the present study. There are many papers on the evolution of RNAi pathways (including the piwi genes, e.g. https://doi.org/10.1093/gbe/evw018, https://doi.org/10.3389/fcimb.2021.653695, https://doi.org/10.1371/journal.pntd.0007919), and none of this literature is mentioned. The present manuscript is certainly novel - it is not duplicating previous work, which is primarily focussed on invertebrate taxa and plants - but it is odd that this large body of literature is not even mentioned.

This is even more striking given the analysis of evolutionary rates, as that has been a major focus of research on the evolution of RNAi, including in piwi-family genes (e.g. https://doi.org/10.1534/genetics.117.300567, and many references therein). For example, lines 314-316 note that rapid evolution is often associated with defence and reproduction, but this has been discussed in the context of piwis on many occasions (starting with https://doi.org/10.1098/rstb.2008.0168).

I understand that perhaps the authors wanted to keep things brief by not mentioning the invertebrate literature. However, this omission causes the introduction to become misleading in places. For example, the abstract and introduction state more than once that piwi expression is germline- and testis-biased. But this is an idiosyncrasy of vertebrates (and Drosophila!). It is not true of most animal phyla (e.g. https://doi.org/10.1038/s41559-017-0403-4,  https://doi.org/10.1371/journal.pgen.1007533). Similarly, where the authors say that the “majority of animal genomes encode at least two copies of the Piwi family, Piwil1 (-iwi) and Piwil2 (-ili),”, this is not technically correct. Most animals (however you count) are probably nematodes, and most of the others are beetles. Most nematodes do not have these homologs, and the manuscript is very unclear on the homology with insect piwis. I don’t think it would even be true of most animal phyla? Are these two vertebrate piwi clades orthologous with Piwi/Aub and Ago3 (of insects)? I think they are not, but no literature is cited (e.g. https://doi.org/10.3389/fcimb.2021.653695, and references therein). If the intention was to ignore the invertebrate literature (which I think would be a mistake), it is very odd that the authors mention an antiviral role for piwis, as this is only known for a recent duplication in Aedes mosquitoes (https://doi.org/10.1371/journal.pntd.0007919)
Experimental design: With two exceptions (see ‘3. Validity’), the approaches seem reasonable, although the methods are a little light on explanation in places (did the Bayesian analysis not have any rate heterogeneity? Will all readers know what GTR+F+R7 is? From memory, GTR with estimated frequencies and an 7-category discrete rate variation?).

-	How were the invertebrate sequences chosen? (I think it would be really useful to identify homology with the non-bilaterian piwis and ecdysozoan piwis.)

-	I think (e.g.) line 125 “reverse translated” is the wrong terminology, since the degeneracy of the genetic code means that there is not a 1:1 reverse translation.

-	I would also suggest PAML model 8a (max category fixed at 1) as the null for model 8, rather than model 7 (one fewer categories), although this is a minor issue

-	The actual method of inference for the time and location of duplications in the tree is not made explicit. It is essentially manually-inferred / informal parsimony, which is fine in this simple case, but more sophisticated methods of gene-tree species tree resolution are available (e.g. https://doi.org/10.1093/molbev/mst100, and the literature that derives from it)
Validity of the findings: There are a couple of things that I think need attention.

The first, and most serious, is the Nei-Gojobori pairwise analysis. First, at this level of divergence such a simple counting approach is likely to be saturated. Was any saturation seen? If so, how was it dealt with? Second, the pairwise distances were then analysed with ANOVA. This is not a legitimate use of ANOVA, as it would assume independence of observations and observations are not independent because regions of the tree will appear in the analysis on multiple occasions. The appropriate way to analyse these data would be to extend the PAML analysis, using a clade analysis and distinguishing between models using (e.g.) AIC or likelihood ratio tests. See, for example, https://doi.org/10.1093/gbe/evw018.

The second thing, which needs checking but is unlikely to be a serious problem, is the extent to which the underlying data have been ‘cleaned’, or at least manually checked to accuracy. For example, while skimming the supporting material I noticed:

-	The PIWIL3_Opossum has a long repetitive 5' region “GPAP ….”, which does not have a homologous repeat in other taxa, and this cannot be correctly aligned

-	The mam piwi4 codon.aln file has several internal stops in XM_023739852.1_PIWIL4_Manatee, which cannot be correct (unless this is thought to be a pseudogene? But then it should probably not be included in analyses of the rate of evolution)

-	At first glance, the mam.codon.fas for the site analysis has Piwil3 from a Megabat that is totally unaligned with the other sequences. I think there must be an error here?

-	For the branch site analysis file mam.codon.aln the armadillo Pwil1 is clearly mis-spliced in that it has a retained intron.

On a more minor note, in lines 320-322, I think the authors have misunderstood the issue with recombination. Recombination inflates rates when there is recombination among the sequences used to build the tree, because by forcing relationships to a single tree (when there would in reality be one more tree topologies than recombination breakpoints) would generate homoplasy. Recombination per se (or nearby) is not a problem, and would not inflate rates unless it actually affected these sequences in a way that meant their relationship cannot legitimately be captured by a tree
Additional comments: It would be really useful to make clear the homology with piwis in invertebrates, and to note which piwis are (or are not) likely to be involved in a ping-pong process.

L84 – I would mention retrogenes in here too, since one is later found.

L207 – Was the putative pseudogene expressed?


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: SPATIAL AND TEMPORAL DYNAMICS OF SAR11 MARINE BACTERIA ACROSS A NEARSHORE TO OFFSHORE TRANSECT IN THE TROPICAL PACIFIC OCEAN
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a very well written, straight-forward manuscript addressing SAR11 community structure in an interesting environmental gradient off of O'ahu, Hawai'i. The authors use a monthly time series of 16S amplicon sequencing and sophisticated statistical analyses of limited environmental metadata and SAR11 community structure. The data is present in very clear figures and the manuscript contains ample supplementary figures and tables.

I didnt find accession numbers for the raw data, and there needs to be a mention that  sequence information of the ASVs is presented in Table S3.
Experimental design: The experimental design is very straight forward and follows a high standard. The analyses offers additional information on SAR11 ecotype structure and its relation to environmental parameters. Methods are described well.
Validity of the findings: The data is discussed in the context of previous findings on ecotype structure in SAR11. All presented data is robust and conclusions are well stated. The data informs our understanding of ecotype diversity and its relation to environmental parameters. Additional data, both abiotic (e.g. nutrient concentrations, DOC characterization) as well as biotic (composition of eukaryotic phytoplankton) would have been helpful, but should not be required.
Additional comments: This is a well written paper and I only have very minor comments.

1) The speculation on marine picophytoplankton shaping SAR11 ecotype structure (lines 490 - 493) should also mention the possibility of terrestrial sources of DOC or eukaryotic primary producers having a strong impact here.

2) Selection of figures and tables for main manuscript:

Figures 4 and 5 are somewhat redundant and one can be moved to the supplement.

The results start with the discussion of the whole microbial community, and I would suggest moving Fig S4 into the main manuscript.

The tables 1 and 2 are not very informative and can be moved to the supplement. Table 1 is redundant with Fig 4.

Table S1 could be moved to the main text.

3) Title: I would drop 'sampled'


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: SPATIAL AND TEMPORAL DYNAMICS OF SAR11 MARINE BACTERIA ACROSS A NEARSHORE TO OFFSHORE TRANSECT IN THE TROPICAL PACIFIC OCEAN
Review round: 1
Reviewer: 2

Output:
Basic reporting: The study by Tucker et al. provides a 2-year time-line series analysis of the abundance of SAR11 (Pelagibacterales) in surface waters along a coast to offshore transect from a tropical region. Its unique location and high resolution (almost monthly sampling) make it an interesting comparison to other studies that investigate SAR11 or bacterioplankton in general. It is well written, and the illustrations are very helpful.

There are several points that should be addressed:

A major flaw is that the sequencing data are not made public available (not even for reviewers). The author should submit their data to a public resource and add a section at the end of the methods, where they indicate the resource (e.g., the SRA accession numbers).

DNA was extracted from a size fraction (85-0.1 um, line 123-125) that includes not only free-living bacteria (see comment in Experimental design). Given that the generated data is a relative abundance, the author should provide a complete ASV table and not just a table for SAR11 ASVs. This could be divided in three tables, one for each of the three habitats. Non-SAR11 taxa could be presented at phyla or kingdom level. This way, it would be possible to see how much the relative abundance of SAR11 is influenced by a potential uneven distribution of picophytoplankton (see line 231) or typically particle-attached bacteria between habitats.

SAR11 clade V should be removed from the analyses as it does not belong to the Pelagibacterales (see e.g., Haro-Moreno et al. 2020 EM or Viklund et al. 2013 PLoS One 8: e78858), which is the group that is analyzed here (e.g., line 26, line 43).  The authors appear aware of the problem as the current Silva annotation is marine AEGEAN group 169 in the order Rhodospirillales (line 192), but added it to the study. Is there any particular reason for this?
Experimental design: The authors collected the size fraction 85-0.1 um (line 123-125) to analyze the known free-living (line 47) object of interest, SAR11. Typically, eukaryotes and particle-associated bacteria are removed, when interested in free-living bacteria (e.g., the authors of the paper cited by the authors about their primer choice (Parada et al. 2016) used a 1 um pre-filter, other typical choices are 2.7 um or 5 um). The resulting DNA thus will contain also eukaryotic DNA and particle-associated bacteria. Given that 16S rRNA amplicon data are relative abundances (and the chosen primer will also amplify chloroplasts) this will lead to reduced SAR11 fractions even though the absolute numbers might be the same. While the authors remove chloroplasts and mitochondria data from their SAR11 analyses (line 188-190), a potential difference in particle-associated bacteria between habitats is not addressed. Differences in relative abundance could therefore reflect differences in the number of particle-attached bacteria (e.g., the coastal site might have more particles than the offshore sites). The authors could maybe add some information for their choice of pre-filtration.

The author compare ASV counts and assign frequencies on not subsampled data (e.g., test of difference in ASV numbers line 204-207, frequency assignment line 212-216, as well as results line 288-298, line 335-337, 340-346, 353-368). As the authors got large differences in sequencing depth ranging from 9,393 to 100,278 reads per sample (line 262), these comparison and numbers are hard to interpretate as they could be heavily influenced by differences in sequencing depth. I suggest providing a supplementary table with the sequencing depth for each sample or a summary for each habitat and test if there are significant differences between habitats and seasons within a habitat. Subsampling should be done if species diversity is compared to ensure the same kind of “sampling effort”.

In line 474, the authors refer to alpha diversity, do they refer to the comparison of ASV counts? Otherwise, it is not shown which diversity index they used, and no results are shown. This could be clarified. Again, samples should be subsampled/rarified for alpha diversity comparisons (and samples with extreme differences in reads should be removed).

The authors are interested in ecological differentiation (line 38) and individual (!) SAR11 amplicon sequence variants of SAR11 (line 112) and refer to ecotypes (line line 60). It is therefore not clear why the authors choose a 99.6% similarity threshold (1 mismatch over 245 bp) rather than exact sequence variants (no mismatch) -  especially, as the authors refer to the need of single nucleotide resolution in the introduction (line 71-74).
Validity of the findings: The authors hypothesize that differential abundance of SAR11 clades is linked to difference in the picocyanobacterial structure (line 425-428). This is certainly a valid hypothesis also in respect of recent findings about Prochlorococcus (e.g., Roth-Rosenberg et al. 2021 Limnology&Oceanography) and the observed a statistical difference in Prochlorococcus and Synechococcus distribution. However, the authors did not test for covariance with different SAR11 clades. Indeed, no analysis of the influence of environmental factors on the relative abundance of SAR11 clades is made. These analyses would certainly add some explanation for the observed differences and allow comparison to previous findings (line 422-424).

The authors describe that clades Ib and IIa have the most dramatic increase in the offshore and state that in other studies these increases have been associated with decreasing salinity and nutrients (line 415-417). The salinity difference is opposite in KByT, the coastal salinity is lower than offshore one (line 229-230). Therefore, the distribution of these two clades is opposite of the expectation if salinity is the driving factor.

The authors state that there is a sharp increase in SAR11 relative abundance toward the offshore waters (Line 410-413). Could this be due to less particle-associated or other bacteria (e.g., the bacterial counts were lower in offshore stations)? So that absolute numbers would be similar between offshore and coastal environment (and thus there would be an independent carrying capacity for SAR11).
Additional comments: The authors should avoid using the term ecotype/ecotopic for the difference between clades and rather use ecological differentiation. Ecotypes as the authors state (line 60-62) is about closely related bacteria. Each clade of SAR11 contains different genera-level groups (see e.g., Haro-Moreno et al. 2020 EM). For within subclade comparisons the term ecotype might be correct.

The authors should state (especially in the discussion) that they analyzed only the surface water (2m, line 119). SAR11 clades have been shown to have depth specific distribution as I am sure the authors are aware of (e.g., line 422).  Hence all the conclusions are only valid for the analyzed layer.

Some minor things:

Line 46-47: Morris et al. 2002 and the 25% are old numbers. Metagenomic analyses and use of recent primers indicate that as much as 40% and more of the microbial community can be SAR11 (e.g., Morris et al. 2012 EM, West et al. 2016 Frontiers in Microbiology, Dubinsky et al. 2017 EM).

Line 53: Please add here also the reference from Jimenez-Infante et al. 2017 that is given later. Maybe add of how many clades isolates are now available.

Line 88: Add references for the 16S-ITS studies.

Line 88-89: Move Jimenez-Infante et al. 2017 to references for cultivation of new strains.

Line 87-91: Add efforts from FISH based studies (e.g., Herlemann et al. 2014, or studies on SAR11 from the Western Mediterranean Sea).

Line 94: One steep gradient is salinity (e.g., in the Baltic Sea). Therefore, the Herlemann et al. 2014 study, cited later, should be mentioned in this paragraph, especially since salinity is mentioned in line 99.

Line 195: Add the used outgroup.

Line 227: The authors should add that the difference is small, but significant between transition and offshore (e.g., only 7.1% of the variability between them is explained due to this separation). The same can be seen in the Venn diagram of shared ASVs (Figure 3A), which is quite high between offshore and transition and low between the other comparisons, and the distribution of Prochlorococcus. Also, it should be added that only two of the eight analyzed factors were different, compared to 6 between coastal and transition.

Line 257-258: Add statistical test used.

Line 274-278: How was the chloroplast distribution along the three habitats?

Line 281-284: Add the statistical test and the test values.

Line 284: The authors could add a short paragraph for the order Pelagibacterales relative abundance before going into the different subclades (done in the same way as Pro/Synechococcus). (e.g., move lines 310-313 and 322-324).

Line 313: Give the test statistics.

Line 314-321: This paragraph seems to repeat descriptions made in the paragraph above (lines 299-313). The two paragraphs should be combined. I suggest removal of mentioning specific stations and rather focus on the three habitats.

Line 322-326: Give statistical test and test statistics when using the words “significant differences” or a p-value.

Line 341: Were in fall and summer 73 ASVs? Maybe clarify.

Line 378-394: To me this paragraph is not about biogeography as it is just a comparison to cultures and not for example to datasets like the TARA ocean. Furthermore, as only a small part of the 16S rRNA gene is analyzed, even a 100% match does not say much about the similarity of the strains. In addition, bacteria with 100% identical 16S rRNA gene similarity can have largely different genomes, which might be true for SAR11 as it the 16SrRNA gene might be a recombination hotspot (Haro-Moreno et al. 2020). When using the word “significantly”, please give test and test statistics. Change abundant (line 380 and 387) to dominant, as the data are about relative abundance. In absolute numbers a coastal ASV might be the highest given the higher number of heterotrophic bacteria.

Line 465: The salinity is only increased compared to the coastal stations at other times. It reaches the same level as offshore.

Line 470: Are there nutrient data available for the bay?

Line 484-493: This is a repetition of the hypothesis raised in line 425. This should be combined.

Line 503-522: The conclusion should be shortened. It seems more like a summary of the results and discussion.

Line 531: Replace the xxx.

Figure 2: A) ASV numbers are not comparable as they are from samples with different sampling depths.
	B) Which dataset was used? With chloroplast and mitochondria or without?
Please inverse the order of the legend to match the order in the diagram.

Figure 5 could be given as a panel of Figure 2.

Supplementary Tables: Include Supplementary Table S4 in the file with the other supplementary tables. Please provide for each a headline above the respective table (e.g., as if it is in the main text).

Supplementary Table S3: Please indicate significant values (e.g., make them bold or red) to ease reading.

Supplementary Figures: Please add the descriptions for each supplementary figure below the figure in the pdf file.

Supplementary Figure S3: How was the nMDS calculated (e.g., which software was used)? Give the value of k (e.g., k=2 or k=3). To ease reading, remove station names and use different symbols for the three different habitats. Color filling could be done according to season. Different stations could be indicated by different shades of the same color.

Supplementary Figure S4 and S5: Please give the legend in the same order as in the plot (e.g., inverse the order).

Supplementary Figure S6: Legend and Figure differ in A and B.

Supplementary Figure S10: This is a great figure. Consider giving this in the main manuscript.


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

Input:
Title: SPATIAL AND TEMPORAL DYNAMICS OF SAR11 MARINE BACTERIA ACROSS A NEARSHORE TO OFFSHORE TRANSECT IN THE TROPICAL PACIFIC OCEAN
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment, see my full review.
Experimental design: No comment, see my full review.
Validity of the findings: No comment, see my full review.
Additional comments: I found the manuscript “Spatial and temporal dynamics of SAR11 marine bacteria
sampled across a nearshore to offshore transect in the tropical Pacific Ocean” very interesting to read. The authors report their results from surveying the microbial community off O’ahu, HI, clearly a region of global relevance, especially the offshore stations. The nearshore sites provide interesting contrasts to these offshore sites, and indeed distributions and ecology, in particular, of SAR11. I appreciated the summary statistics provided for the nearshore, transition, and offshore sites for the different clades of SAR11 and other groups. I also appreciated the evaluation of the microdiversity of SAR11, showing how different ASVs vary between the sites. These strong gradients showed a surprising degree of variability in some groups of SAR11, though many were found cross all sites. In general, I am supportive of the paper (very interesting and valuable dataset!), but I have some comments and suggestions, see below.

The phylogenetic placements and taxonomy of SAR11:
I think more methodological details are needed for the phylogeny (Figure S2).
What sequences from SILVA (version?) were used (seems a low number)?
I’m unclear how ARB was used for the alignment and/or sequence selection.
What program within GAPPA and more details can be provided?
What options for EPA-NG?
What do the circles on the nodes represent (Figure S2)?

Is there any debate on whether or not the clades IV, Va and Vb are actually SAR11? Especially confusing since manuscript states “AEGEAN-169 within the order Rhodospirillales (for SAR11 subgroup V)”. I would appreciate a comment on this. And again at Line 54: These sequences that at 18% different: are we sure they are all “SAR11”. Do the phylogenetic reconstructions and current genome taxonomy support this (e.g., are they all order Pelagibacterales in GTDB)?

In general, I think the authors would perhaps find interesting this paper which describes SAR11 diversity using high throughput sequencing of the ITS gene: https://www.nature.com/articles/ismej201729. In particular, I think it would be insightful to consider the results from 16S presented in the present MS, with the results from the ITS from that paper. The ITS seems to show remarkably more variability (much less dominance by any single ASV), with an average of about ~0.5% for the dominant ITS-based ASVs vs ~10% for 16S-based ASVs (which seems indeed comparable to the present study). This suggests (unsurprisingly) that 16S is masking potentially important variation, and might be considered at, e.g., Line 443 where a discussion about intra-specific and inter-specific variation occurs.

For Line 443, also might consider calling it intra-clade and inter-clade, as specific would suggest these are species.

I find this sentence in the abstract hard to follow: “On average, 77% of the SAR11 community was compromised of a small number of ASVs (7 of 106 in total) spanning subclades Ia, Ib, IIa, IV, and Va, which were ubiquitously distributed across all samples collected from one or both of the end-member environments sampled in this study (coastal or offshore).” I think the confusing part is “ubiquitously distributed across all sampled collected from one or both”

Line 123: With use of YSI for pH etc, these aren’t really oceanographic grade measurements, I would say, right? Maybe can note these measurements were made with YSI in the figure legends

Line 175: At what stage were Illumina adapters, indexes etc added?

Line 181, can authors report how it was determined that the quality was too low to go forward with the two sequences? Of course, now that only one end is used there has to be lower confidence in that single read. Might be nice to cite other work that has used this single-end approach for Illumina amplicons. Also might increase confidence if a sentence could be inserted to say something like, “Analysis of mock communities using this approach demonstrated accurate denoising of sequences despite only using the single end.”

Line 187, in regards to how mitochondria were treated, unclear to me. I assume “community structure” refers to the multivariate statistics/ordinations like Figure S3?

Line 223, “KByT” here for the first time, but not defined?

Line 270, might consider if Synechococcus seems dominant because they probably tend to have 2 rRNA gene copies? How about a correlation to the flow cytometry?

Line 417, is this cited result (Quéméneur et al) opposite for salinity as shown here?

Line 497, wouldn’t consider Chow et al. 2013 oligotrophic ocean gyre. Coastal, subtropical, maybe.

Figure S8, it would be nice to label these y-axes to say “Average relative abundance as proportion of all SAR11”, or something like that, especially if legends will not be with the figures themselves. Note, I think it would be helpful to label the Supplementary Figures with figure identifiers (Figure S1, S2, etc


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: SPATIAL AND TEMPORAL DYNAMICS OF SAR11 MARINE BACTERIA ACROSS A NEARSHORE TO OFFSHORE TRANSECT IN THE TROPICAL PACIFIC OCEAN
Review round: 2
Reviewer: 1

Output:
Basic reporting: The submitted manuscript has been very nicely revised.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Minor suggestions
(Note line numbers refer to the track changes file.)

Quote from letter of the submitting author:
"As the reviewer points out, we do indicate at the appropriate place in the Materials and Methods that we sampled surface seawater (line 119). We also do not find any occasions where we are misleading regarding this point, or over-extending our observations to other layers of the water column. If we missed something in the text, please do point it out."

I think this should be clarified in two places Line 555-556 and Line 576-577.
Line 555-556: "We observed a fundamental change in the picocyanobacteria that dominate coastal versus offshore environments across the KByT system,...". The change was only observed in the surface water and not the whole KByT system (which in my opinion would include depth).

Line 576-577 “Across the three environments of KByT, the relative abundance of SAR11 was dominated by seven ASVs” Only the surface layer of each environment was analyzed. I am pretty sure that different SAR11 ASVs will dominate deeper layers of these environments (e.g. at a potential DCM or in non-photic parts). So worth adding here “Across surface waters of the …”.

Other small points

Line 59-60 Consider adding a sentence regarding the uncertainty of clade V in the introduction. Maybe something like “clade V (also known as AEGEAN-169 group)”.

Line 194-196 The data of the mock communities are not shown anywhere, right?

Line 250: Replace “false discovery rate” with “corrected p-value” to avoid confusion with FDR concept, which is a different multiple test correction.

Line 338: Please specify “Euryarchaeota Marine Group II” (also in Table S6).

Line 488-493: State which dominant ASVs, which subclades, and which environmental factors are part of cluster 1 and which of cluster 2.

Line 493-495. To me the sentence is misleading as only 3 of 7 dominant ASVs had strong positive correlations. The four mentioned exceptions are the majority. Could the weaker correlation in these four ASVs be because they belong to clades known to be environmental more variable (e.g. Ia)? Any other possible explanation?

Line 591-599: You could add that ASV16 and ASV18 & 47 are in different clusters (Figure S9) and the latter two in two different subclusters further supporting their ecological differences.

Line 506 to 523: I still do not understand the importance of this paragraph for the article as a whole and suggest deleting it. As the authors point out themselves the 16S rRNA gene in SAR11 is highly conserved (see discussion 583-590). Given that the sequence of only one read is used for the comparison (only 245 bp) for me the comparison is almost meaningless (and again not relevant for this article). It is certainly very interesting if someone is looking at a cultured isolate and wants to know its natural abundance, but again a comparison of only 245 bp is hardly sufficient for this in SAR11. I leave the decision whether to keep it or not to the authors.

Table S6: Could you add columns with relative abundances (or the normalized abundance)? Otherwise, it takes time to find out which order is more abundant in the comparison.

Some typos
Line 337: Pelagibacterales
Line 350 two-fold

Other things
Line 609: Good 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: SPATIAL AND TEMPORAL DYNAMICS OF SAR11 MARINE BACTERIA ACROSS A NEARSHORE TO OFFSHORE TRANSECT IN THE TROPICAL PACIFIC OCEAN
Review round: 2
Reviewer: 2

Output:
Basic reporting: I approve
Experimental design: I approve
Validity of the findings: I approve
Additional comments: The only remaining issue I have is that the SAR11 clade V issue was not resolved in a convincing way. I suggest adding a comment stating that careful, thorough phylogenomics needs to be done to clarify their relationship to the rest of the SAR11. I started to do my own phylogenomics for the group, ran out of time to do a non-fasttree (there are now a lot of SAR11 genomes, thanks to single cells!), but clearly they are _very different_ than the rest, as are a couple of novel groups: o__TMED127, o__GCA-002720895 (SAR11 groups 6 and 7, perhaps?).

Otherwise, the authors addressed all my concerns, thank you!


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: GLOBAL GAP-ANALYSIS OF AMPHIPOD BARCODE LIBRARY
Review round: 1
Reviewer: 1

Output:
Basic reporting: I enjoyed reading this paper, and I believe it makes a timely and useful contribution to biodiversity, species recognition and DNA barcoding. The paper conforms with all journal criteria in this category. However, I strongly feel that the paper could be much more concisely written. As a random example, Line 357:

Ratnasingham & Hebert (2013) when presenting the BIN system indicated that overall 12% of the sequences available in BOLD at that time were lacking a family name, 19% a genus name and 40% a species name.

This sentence can be written much more concisely as follows: Ratnasingham & Hebert (2013) indicated that 12% of the amphipod sequences in BOLD lacked a family name, 19% a genus name, and 40% a species name

I believe that a careful revision of the paper along the lines above would make it much easier to read, and substantially reduce it's length
Experimental design: The paper meets the journal criteria outlined in this section
Validity of the findings: The data presented in the paper are straight forward, and the conclusions are supported.
Additional comments: Your paper is quite timely, and accurately highlights gaps in the amphipod barcode library. I suspect that  many other taxa likely share the same geographic patterns of deficiency. Your paper would strongly benefit from revisions aimed a making the text much more concise.


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: GLOBAL GAP-ANALYSIS OF AMPHIPOD BARCODE LIBRARY
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this work, the authors compile a very detailed gap-analysis for an important invertebrate group. This type of work is very important in an era where DNA-based tools are being used for monitoring programs.

The paper is solid and good. The language is clear. The references are appropriate. The authors made a good job with clear goals, analysis and conclusions. I do not have major suggestions. The paper was very easy to read and review.

I think three minor issues may be clarified.

Line 216-219-> BAGS allows the user to choose. The user can remove records without data on country of origin or latitude or the user can download everything and do not apply this filter. Correct the phrase and explanation.

Line 232-> These 44 records were not removed? Supp S4 and lines 198-199 indicate that these records were removed.

Fig.1-> In the last box, “studies” in my opinion is confusing. Should be “records” no?

I also found some minor typos in lines 372,378, 407,499,501,539

(Without self-promoting) Two papers were recently published and may be interesting to add, if the authors wish so.

Line 121 and 407 -> Vieira et al 2021 could be a good citation if the authors wish
Vieira, PE, Lavrador, AS, Parente, MI, et al. Gaps in DNA sequence libraries for Macaronesian marine macroinvertebrates imply decades till completion and robust monitoring. Divers Distrib. 2021; 00: 1– 13. https://doi.org/10.1111/ddi.13305

Line 553-558-> Some initiatives for BOLD data curation are already happening
Radulovici AE, Vieira PE, Duarte S, Teixeira MAL, Borges LMS, Deagle B, Majaneva S, Redmond N, Schultz JA, Costa FO (2021). Revision and annotation of DNA barcode records for marine invertebrates: report of the 8th iBOL conference hackathon. bioRxiv 2021.03.07.434272. https://doi.org/10.1101/2021.03.07.434272
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Congrats on the good work. It is rare to review "a final product" in the first stage of the reviewing process.


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: GLOBAL GAP-ANALYSIS OF AMPHIPOD BARCODE LIBRARY
Review round: 1
Reviewer: 3

Output:
Basic reporting: This manuscript by Jazdzewska et al. provides a descriptive analysis of barcode records for Amphipoda, Their results show that the barcoding coverage of amphipods is only 10%.  Marine taxa had lower representation than freshwater taxa despite being more abundant. They also showed that some regions are less well studied than others (Africa and South America vs Europe and North America) and that several species rich families are not well represented.
This manuscript is straightforward and clearly written. The authors have carefully examined records of the bold database for analyses.
Sufficient background is provided but some relevant barcoding references were missing (ex. Tempestini et al. 2018, Duarte & Costa 2020).
The tables and figures are of professional quality.
Experimental design: This study is a descriptive one. The authors use a public database to provide a portrait of barcode gaps in Amphipoda. The authors claim that their objective was to provide a detailed understanding of strength and limitations of barcodes. I found that the manuscript had more to do with basic reporting of where most of the barcodes of Amphipoda came from and families that needed more coverage. I would have liked to see information on intraspecific divergence vs interspecific divergence in species from different habitats (marine, freshwater, terrestrial). Also, it would have been interesting to report amphipod records for different ocean basins.

Minor details
L250 something missing in the sentence
The authors often use the term 'environmental information' (for example l258). Collection locations would be more appropriate.
Validity of the findings: The underlying data are robust and sound. Statistical treatment does not apply here.
The discussion was focused mostly on amphipods. I suspect that similar results would be found in other orders of marine invertebrates. The conclusions are well stated and linked to the original question.
Additional comments: No additional comments