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

Input:
Title: THE CHALLENGE OF MANAGING THE COMMERCIAL HARVESTING OF THE SEA URCHIN PARACENTROTUS LIVIDUS: ADVANCED APPROACHES ARE REQUIRED
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript presents an interesting dataset on field observations carried out in shallow subtidal rocky reefs of Western Sardinia (Italy) with the goal of analyzing patterns and processes in abundance and population structure of the edible sea urchin Paracentrotus lividus in relation to biological and physical properties of the studied areas before a major decline in local sea urchin population. Results are discussed in the context of the management of the sea urchin fishery, and how knowledge of key ecological processes at relevant scales should be integrated with urchin stock management and conservation strategies to promote sustainability.
In general, the manuscript is well written and comprehensible. Nevertheless, I found it hard to follow some of the contents of the text, some graphs and tables require further description and labelling, and the raw data and code should be curated before making it publicly available.
At present, I see authors need to accommodate/clarify some issues before the paper is accepted for publication (please see specific comments to the manuscript in the general comments section below). In my opinion there are several points in each section of the paper that require further clarification in order to improve the interpretation and readability of the manuscript. The authors should also consider having their manuscript proofread by a native speaker before resubmitting.
Experimental design: The main goals and hypotheses being tested in the present study are not clearly identified.
Methods described with sufficient detail, but some aspect could be improved (please see specific comments to the manuscript in the general comments section below)
Validity of the findings: The findings are interesting, certainly contribute to increase the knowledge regarding local sea urchin populations and may have direct application on resource management and conservation strategies. However, there is room for improvement of the discussion and conclusion sections (please see specific comments to the manuscript in the general comments section below).
Additional comments: Specific comments to the manuscript:

Title

Consider mentioning in the title that the target species in this study is a sea urchin, as I’m sure some PeerJ reader will not be familiar with the species Paracentrotus lividus.

Abstract

In my opinion, the abstract doesn’t mention important background for the pertinence of the study, like the importance of this commercial activity in the study region and the management strategies in place (MPA, harvesting zones, etc..) and the severe decline observed in sea urchin abundance from 2007 onwards that motived this study.

Consider mentioning the species Paracentrotus lividus is a sea urchin the first time it is mentioned. You only refer to sea urchins about halfway through the abstract.

Line 20: ”…. functional species Paracentrotus lividus…”. Please describe the functional species concept in this context. It is not clear to me what authors are aiming.

Line 22-26:  “Sea urchin density and …. “. Consider rephrasing this sentence where a brief description of the methodology is intended. There is no mention to which environmental constrains were addressed, which types of habitats were studied and what is meant by spatial organization in the context of the study. Also, the “inside/outside the local MPA” issue, which refers to the different sectors assessed in this study, lacks some contextualization in the abstract. Authors do not mention when the observations were carried out.

Introduction

Line 41: “…and reducing…”. Change to “reduce”.

Line 48: “…”boom-and-bust” patter…” . Missing an “n” in pattern.

Line 52: “Thus… ”. Consider rephrasing. “Thus” does not read well.

Line 59-62: Please develop this paragraph, briefly describing the main management strategies developed in the regions that are mentioned.

Line 67-69: “… seems to be much more important than…”. Please further describe what you mean by “more important”, in what sense?

Line 72: “… since decades”. Since when? Please provide a more specific time-frame.

Line 72-76. Authors should describe in further detail the harvesting/conservation regulation in the Peninsula de Sinis region in time and space. This should be explicit in the introduction section as it is important for the contextualization of the study. Later they provide more info on this issue in the methods section, but in my opinion, this should be included in the intro section.

Line 77: “Despite these restrictions… “. What restrictions? Please refer to the previous comment.

Line 79-81: “From 2015 to….”. Consider rephrasing, does not read well.

Line 81-83: I believe that some commas “,” are missing between “enforcement, further” and “actions, and…”.

Line 87: “Ouréns, Naya and Freire 2015” is a study carried out in Galicia (Spain) and seems not to be well referenced in this context, because you mention “…in this area”. Perhaps adding an “e.g.” when citing Ouréns et al 2015 is enough to solve this issue.

Line 89: “…. of biology…” . Change to “… of the biology….”

Lines 91-96: It is not clear how the example of New Zealand management plans (with number of licences and fishing limitations ) relates to the previous paragraph were you mention that some sustainable sea urchin fisheries rely on a good overview of the biology and population dynamics. Consider further development of this paragraph, providing the reader info on how scientific knowledge was incorporated into the management strategies there.

Line 94: “…fishing gears etc.)”. Add “,” between “fishing gears” and “etc.”

Line 129-139. Consider reviewing this paragraph as it is not easy to follow. Include references for the monitoring studies mentioned in lines 129-131. Authors refer to Pieraccini et al 2016 to state that the period between 2004 and 2007 can be considered the “pre-crisis era”. Yet, Pieraccini et al 2016 suggest that sea urchin stock contraction occurred between 2004-2005. As so, the period 2004-2007 encompasses that contraction in bigger individuals, and might not represent a reference environmental status as authors suggest in this manuscript.

Line 140-142. It is not clear the pertinence of this statement. The authors should consider rewriting this paragraph and clearly state the main goals and hypotheses being tested in the present study.

Material & Methods

Lines 147-153. Authors refer to Fig.2 and Fig. S1. Fig2 does not provide a general view of the MPA limits making it hard to interpret the spatial distribution of the study area.  In my opinion Fig.2 should merge both figures (e.g. using fig s1 as the inset in fig2), so that the reader could follow this paragraph by looking at just one figure.

Line 154-162. This paragraph provides relevant background information on the sea urchin fishery regulations that could have been included in the introduction section for contextualization of the problem. Here, the intention seems to be a description of the harvesting pressure variability along the study area (inside/outside the MPA), and could be made more explicit and using comparable units of maximum allowed harvesting effort for ease of interpretation.

Line 164. “… (Fig.S1)…”. Why refer to a supplement figure? See previous comment on merging Fig.2 and Fig.S1.

Line 180. Typo. Remove “,”.

Line 189-196. This paragraph provides relevant background information on the sea urchin fishery regulations that could have been included in the introduction section for contextualization of the problem.

Line 200-201. “… size class ranges…”. For ease of interpretation, please provide numerical ranges for each size class considered.

Line 219. “… how are structured.” Change to “… how are they structured.”

Line 220. Typo. “suiTable”

Line 222-223. “These types of habitat…”. Does not read well.

Line 231-246. Why didn’t you run the model for the period 2004-2007 which is the period of interest in the present study? You mention that you only consider data between January and June, because it corresponds to the period active recruitment. Why this time frame and what do you mean by active recruitment? Don’t you mean settlement? In lines 264-265 you define recruits as individuals <2cm that survived ~2 years after settlement. It is not clear to me why the characterization of average bottom water speed only take into account this six months period.

Line 251-254. “In the pre-crisis…”. This should be described in the results section.

Line 280. “All the analysis…”. Change to “analyses”.

Line 284. “…no-parametric…”. Change to “non-parametric”.

Results

Line 306-317. Consider rephrasing. It is not easy to follow the description of these results.

Line 320-325. No easy to follow. Consider showing results in a graph instead of referring to supplement tables.

Lines 327. “Table S3”. Change to S2.

Line 332. “Fig. 4…”. Check unit labels in y axis of fig.4.

Line 331-336. Not easy to follow these results by looking at figure 4 and table S4. Table S4 lacks title and legend.

Line 348-351. “…General Linear Model…”. I would expect to see some model selection procedure that would eventually lead to a more parsimonious GLM model being fitted to the data without the non-significant explanatory variables being included in the final model. Can you please comment on this.

Line 350. “…density distribution of on the future stock….”. It is not clear what authors mean.

Discussion

Line 359-360. “…CR-1 was 1.5 and 1.6 times higher than…”. But according to the results not significantly different.

Line 367. “Surprisingly…”. Why surprising? Please expand on this discussion.

Line 374. “… when recruitment…” Do you mean settlement? Please make sure the concepts are coherent with what is described in the manuscript. In this study you don’t provide any data on settlement intensity, and the relationship established between recruitment (ind. <2 year old) and current speed may be the result of variability in both pre and post-settlement processes.

Line 384-385. “…1.2 higher than in sector 1…”. Are these differences significant?

Line 406-410. It is not clear to me what is meant in this paragraph. Consider rephrasing.

Line 412.”…then…”. Change to “in”.

Line 419. “…. Population dynamic…” . Change to “dynamics”

Line 423.”… …shed new light that might help management plans…”. How? Please develop.

Line 443-446. Doesn’t read well. Consider rephrasing

Conclusion.

Authors should also consider mentioning the importance of sound fishery related data (besides ecological studies) and active management tools that could allow to timely responses/adjustments to harvesting pressure in the region in order to promote sustainability of the resource and of this fishery.

References

Please thoroughly check references as there are several typos and inconsistencies in the format.

Figures and tables (main text and supplement)

Please thoroughly check all titles, legends, axis labels and units. All suplement material lack descriptions.

Raw data and code

Please check all raw data provided. It is a hard dataset to deal with. Should be curated before submitting.


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

Input:
Title: THE CHALLENGE OF MANAGING THE COMMERCIAL HARVESTING OF THE SEA URCHIN PARACENTROTUS LIVIDUS: ADVANCED APPROACHES ARE REQUIRED
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript in the present form is of difficult reading, mostly due to the English language, that should be improved in order to allow a better flow of the manuscript and to ensure that the audience can clearly understand the text. Not clear  and unambiguous, professional English has been used throughout. The litterature references are sufficient, some suggestion of additional references is provided to authors.
Experimental design: The main aim of the study, providing an ecological based approach for the management of P. lividus commercial harvesting, is a particularly interesting and current topic. Additionally, the amount of data gathered and variables considered are definitely relevant.
In my view, several clarifications in the different section of the manuscript are needed in order to clarify the different steps of the work done, the rationale behind some analyses and better explain results obtained. Some suggestion about clarifications needed are provided.
Validity of the findings: I think that authors should make more explicit which are the specific indications for fishery management plans provided by the present study. The conclusions should be more focused on main findings and possible use of the evidences from this study in management plans.
Additional comments: ABSTRACT: The abstract section in general is very confused and the aim of the study is not very clear from it. I think it should be entirely revised, in view also of following comments on the article sections. For example, the very first sentence (line 16) is very general… add a geographical reference (in the Mediterranean? Worldwide?). Lines 16-17: Which “crisis” are authors talking about? Make it clear for the readers. Line 22: replace “the environment” with “environmental factors/features”
INTRODUCTION: Line 40: add reference about “fish-sea urchins-macroalgae” trophic interaction. Line 44: remove “ecosystem” (barrens is sufficient). Line 54: this is valid also in the Mediterranean Sea? Please add references. Line 63: I think it could be useful mentioning that P. livisud is an “edible” species (instead of functional, given that its ecological role is explained in the following sentence). Line 70: “P. lividus” instead of this species. Lines 79-81: it is not clear the period of time to which authors are referring here. Lines 81-83: this sentence is not clear at all. Line 83: “preservation” instead of preserve. Lines 91-93: get rid of the first “management system” after “quota”. Line 102: “varies” instead of “vary” Line 134: pre-crisis era is a term present in literature or created by the authors?  Probably it could be useful to explain better the concept and clarify why it should be used as a reference period. Lines 140-142: authors have not yet presented results, so it is not probably the case of concluding the introduction mentioning the “not negligible results”. Instead, it could be useful to state an aim of the study, e.g. “We used pre-crisis data to provide a reference framework to be used for planning future management strategies…”
MATERIALS & METHODS: Line 151: provide information about the year of establishment of the MPA. Line 154: add the temporal information about the pre-crisis era. Line 157: “the” before years should be removed. Lines 158-160: the regional decree is dated 2009, but the studied period is 2004-2007. The regulation outside the MPA was the same also before 2007? Line 199: add the information that the “types of habitat” will be described later in the following paragraph. Lines 200-201: define the size class ranges. Lines 204-207: this sentence is very difficult to follow, pease rewrite improving clarity. Additionally, explain the rationale behind the comparison between bottom current speed and recruit density and between predatory fish density and middle-size urchin density. Lines 240-242: explain why 2009-2010 water circulation data are appropriate for the 2004-2207 period. Lines 264—269: why the size of urchins is provided with letters and not with the actual numbers? It would be clearer. Additionally, I suggest to present data in fig. 4 presenting data grouped per size classes, or at least add this information with rectangles or different colors in the graphs. Lines 271-272: the anova on whole sea urchins’ density has been performed separately for the two factors “sector” and “habitat”? Lines 272-274: why analyses on the different size classes has not been performed considering the same factors? Please define “population”. Lines 284-287: as before, please explain the rationale behind these comparisons. Lines 288-289: Please describe the model: response variable, predictors….
RESULTS: Lines 343-345: the correlation is significant given the p-value, but it is very low (Spearman coeff. = -0.3). Lines 348-351: the model results should be described more in details. Assessment of goodness of fit of the model should be reported (e.g. % of variance explained; adjusted R2).
DISCUSSION: Lines 358-360: in the discussion session in my opinion authors should be more explicit and avoid acronyms in order to improve and facilitate the understanding of the readers. Here authors should for example refers to differences among the same type of habitat in the different sectors, with higher densities in calcareous rock in Sector one compared to the same habitat in sector 2 and 3. Lines 365-367: results of the model should be better explained. It has been presented as a predicting model: which predictions are derived from it? Line 376: the correlation is very weak.  Line 400: how far larvae can spread? How much of the studied area is affected by larval dispersion? Lines 422-423: which are the indications for management provided by the present study?
CONCLUSIONS: I think that conclusions should be more focused on main findings and possible use of the evidences from this study in management plans. “Strong natural relationships” are mentioned: to which relationships authors are referring to?


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

Input:
Title: THE CHALLENGE OF MANAGING THE COMMERCIAL HARVESTING OF THE SEA URCHIN PARACENTROTUS LIVIDUS: ADVANCED APPROACHES ARE REQUIRED
Review round: 2
Reviewer: 1

Output:
Basic reporting: The present version of the manuscript is definitely improved compared to the first submission. Authors clarifiied some ambiguous aspects and explicited hypotheses.
Literature references are properly reported. The article structure is in line with PeerJ requirements and figures and tables are correctly provided. Raw data are shared.
Introduction and Discussion sections are quite long, partially because of some repetition and not perfect organization of the narrative buildup.
I still think that the manuscript would greatly benefit from a proofreading by a native speaker, in order to better organise the different sentences/sections, have a better flow and make it more easily readable.
Experimental design: The main aim of the study, providing an ecological based approach for the management of P. lividus commercial harvesting, is a relevant and current topic.
Research questions are in the present version better defined and several clarification about the experimental design were provided, improving the manuscript.
Rigorous statistical analyses have been performed and methos are described in full details.
Validity of the findings: Results are now better discussed in the management perspective, which is the aim of the study. Relevant insight about management recommendations are provided in the discussion.
Additional comments: The manuscript in the present version is definitely improved, but sometimes is still hard to read, in particular concerning Introduction and Discussion. I suggest to the authors to re-read the manuscript trying to avoid repetition and redundancy. A proofreading by a native speaker would be a great help in further improving the manuscript, which is relevant and plenty of useful information, sometimes hard to understand/catch because of the writing (I had to re-read some sentences several times to get the correct meaning).
Some specific comments on the text:
ABSTRACT:
Line 20: get rid of "On this island of... ". It is sufficient "In Sardinia..."
Line 33: get rid of "Also" (in general, do not use also at the beginning of a sentence!)
Lines 34-35:  change "An abundance of different size classes..." with "Size-frequency distribution of sea urchins..."
Posidonia should be in italics

INTRODUCTION:
Line 61: after "target species" add examples: "target species SUCH AS...."
Line 65: I would change "However" with "On the other hand..."
Line 82-83: I would report as "area-specific"
Line 95: get rid of "locally" or move it before "considered"
Lines 95-98: merge the to sentences in order to make it clear that the regional decree refers to Sardinia; you may simply add "where" before "populations" in line 95, and change the full point with a comma in line 97.
Lines 101-102: I would change and simplify the sentence as "More restrictive regulation were enforced within marine protected areas (e.g....)
Line 106: get rid of "of it"
Line 173: "from when they were still undamaged" it is not clear to me, please explain better
Lines 194-195: but even larger size were sampled, even if not studied in relation to environmental factors. I suggest to clarify this point here and in M&M section
Line 196: change "in according to" with "for each"

MATERIAL & METHODS
Lines 242-245: move at the beginning of the following paragraph "Environmental constraints"
Lines 260-265: define better patches of what...
Lines 281-284: Move this sentence before the previous one (after the sentence ending with Marra et al 2016, line 279)
Lines 298-299: "...commercial stock and this size-class range is not considered in the analysis..." please explain better how you decided to use data, because those data about commercial size urchins are reposted as frequency distribution in describing population structure, and are correctly reported also in the result (are relevant data at is a pity to state that tey have been neglected, when they actually were not).
Lines 321-327: I am a little bit worried about auto-correlation of predictor variables. maybe a thouth about it should be useful

RESULTS:
Line 343: change "was" with "were"
Paragraph "Sea urchin population structure": data about the commercial size class (>5cm) are reported (and they are relevant!): please correct the description in M&M

DISCUSSION:
The discussion is improved from the previous version, but is very repetitive. I suggest to shorten it.
Line 399-400: change "OF Posidonia oceanica" with "CHARACTERISED BY Posidonia oceanica"
Line 404-405: get rid of "the high values of patch density and mean patch size seem to further increase the..." and change the sentence as "larger and more dense patches seems to further enhance sea urchin density...". Get rid of "sector 1" at the end of the sentence.
Line 406: start the sentence with "In fact, ..."
Lines 409- 414: these sentences are very repetitive and difficult to follow: try to summarise, merge sentences and expain better the concepts
Line 488: "their" instead of "its"
Line 495: some thing is missing between "unknown" and "the period"
Line 510: I would change "information and data" with "knowlwdge"


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: ISOLATION, GROWTH, AND NITROGEN FIXATION RATES OF THE HEMIAULUS-RICHELIA (DIATOM-CYANOBACTERIUM) SYMBIOSIS IN CULTURE
Review round: 1
Reviewer: 1

Output:
Basic reporting: In general, the manuscript is written in clear, unambiguous, professional English. The intro and background show sufficient context and motivation for the experiments. This area of research is of great interest to the DDA community as there is very limited laboratory experimentation or published isolation methods for these important organisms. The figures were relevant although I note some comments regarding apparent inconsistencies (see line-by-line general comments). Raw data underlying individual figures was provided in a spreadsheet. It would be nice to know which strains were used with which raw data points, where applicable. I think this is mentioned in the text and figure legends but was hard to keep straight. Having it labeled in the figures and supplemental would help with clarity. Some data appears to be missing, for instance on Line 125 it is indicated that growth rates were measured at 7 different light levels, but much of this data is missing from the results and supplemental data. Perhaps this data was lost due to Hurricane Harvey?
Experimental design: The research conducted herein is very relevant to the field and provides much-needed information on culturing, growth, n-fixation rates, and ecology of DDA’s formed by Hemiaulus-Richelia. In general, the methods were well written and relevant to the goals of the study. In some cases, however, the methods were a little hard to follow as written and some suggestions are listed below in the General Comments. In particular, a statement to the reason different strains were used would help clarify this point to readers. Also, more clarity as to which strains were used in which figure would help. A more detailed description of statistical methods to infer significance is needed.
Validity of the findings: As mentioned elsewhere, all underlying data for the figures are provided, but not all underlying experimental results mentioned in the methods. These data would be of interest to the readers I feel, assuming they were not lost in the hurricane. The discussion of results and intercomparing with prior field and laboratory observations, as well as cross genera comparisons, were robust and very well written. I truly enjoyed reading this manuscript. The manuscript will have a large impact on the field and is very relevant to our ecological understanding of these keystone organisms.
Additional comments: Lines 12 to 13 – looks like a space was between citations
Line 35 – “Richealia” is spelled wrong
Line 56 – there is a “cc” after “medium”
Line 110 and 112 – it would be helpful to reviewers if a note is given as to why the use of different strains. In addition, rather than requiring the reader to go look at the figure legends or further in the text for environmental conditions of the experiments, it would be helpful if they were spelled out here as the N and light experiments are done. But maybe its just that sentence which is confusing.
Line 134 – add ‘nm’ after 565
Line 154 - italicize “a” in chlorophyll, here and elsewhere, also, how much volume for chl measurements?
Line 159 – what is the 10 ml sample medium used for? I assume the nuts, but since it follows the method it reads as though it will be used for a different analysis. consider moving up.
Line 160 – what stats were conducted with Excel? and on which data sets?
Line 191 – I think a period is supposed to be here after the volume range
Line 197 – Perhaps heterotrophic bacteria? Was any observations made on their numbers?
Line 205 – chlorophyll is now abbreviated where on line 204 it was not
Line 236 – what stat?
Line 242 – were these cultures axenic? If not, how might heterotrophic remineralization impact measured N+N values?
Figure 4 – How are the individual isolates represented in the figure? Are these the black squares? Not sure I see diamonds. If so, consider either using different colors to designate strain or additional shapes.  If red squares are a 4 h incubation, how do the points span from 8am to 8pm?
Figure 6 – Where is the 3rd N source? Or is this the -N? No closed squares are visible. If strain #9 is here, it should be called out in the figure as well as mentioned in the methods for this experiment. Also, legend says only 1 um of NH4 was added whereas methods and results state 10um.


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: ISOLATION, GROWTH, AND NITROGEN FIXATION RATES OF THE HEMIAULUS-RICHELIA (DIATOM-CYANOBACTERIUM) SYMBIOSIS IN CULTURE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The language in this manuscript is clearly written.  Take a closer look at the use of hyphens for compound adjectives.  Because of the spotty use of some strains, communicating the experimental design sometimes became muddled.  Comparisons to Rhizosolenia early in the manuscript led me to believe that there would be data presented from this strain, so I was left confused.  The figures are clear, but in some cases require a legend or the captions need review (see specific comments below). Data are provided and I was hoping it could clarify questions I had regarding the N2 fixation calculations, but I now have more questions.
Experimental design: Isolating diatom-diazotroph associations from the field requires a valiant effort and I applaud you. The ephemeral nature of these cultures and the loss of cultures to Hurricane Harvey prevented the authors from conducting these experiments as thoroughly as they might want.  The experimental design was unfortunately patchy, but there are important results here nonetheless.  I have concerns about how the acetylene reduction assay was sampled and calculated.  Unfortunately, the data provided did not answer them.
Validity of the findings: So many strains have been lost that there is no opportunity to replicate the experiments.  The nitrogen addition experiments seem robust.  If there are any DNA or RNA samples stashed away, they could be helpful in confirming some conclusions by determining whether the cultures were affected by viruses or whether host and asymbiotic strains had diverged.

I am trying to understand how the N2 fixation numbers were calculated and reported.  It is a well-documented assay so I don't think the methods need to be extended, but perhaps an explanation to the editors how the data were handled might be helpful.
Additional comments: General comments:

It’s difficult to keep track of which species and strains were used in what experiment even though they are enumerated in the text.  Please add a table listing the strains used and the measurements performed on each.

The culture isolation techniques described in the Results were very interesting, but I think they would be better served in the methods section.  State what you learned, but leave the details in methods.

One confusing aspect of the paper is that Rhizosolenia is mentioned occasionally, but wasn’t used in the experiments.  If there are no results to report, perhaps this paper should be written more clearly as a Hemiaulus paper.

Italicize “a” in chlorophyll a.

Specific comments:

Abstract:
- No hyphen is required for “nitrogen fixers” or “nitrogen fixation.”  Do use a hyphen for the compound adjective “nitrogen-fixing,” however. Check the use of hyphens here and throughout.
- The phrase “a cyanobacteria-diatom symbiosis” sounds as if you only isolated one DDA but then you report two Hemiaulus species.
- The first time you report a species, spell out the entire genus, even if you’ve already used the genus for another species.
- Hypenate “symbiont-free.”

Introduction:

-Line 35:  “Richelia” is misspelled.
-Line 56:  Delete “cc” from “medium.”
-Line 64:  This entire paragraph is about Hemiaulus, so it’s better if you don’t bring in new information about Rhizosolenia.

Methods and Materials:

Lines 86 - 87:  Use plural for “strains” when you list multiple strains.

Lines 114 - 115:  Were the cultures acclimated to the 2-L flasks or did you transfer them straight from the 50-mL tubes to the flasks?

Lines 125 - 126:  It is not clear to me which light levels ere used in the spectrophotometer and which light levels were used to grow the cultures.  Some of my comments elsewhere reflect that confusion.  In addition, did you determine an optimal light level for each strain?

Line 139:  Why the hyperbolic tangent?  Is it the model that fit your data best?  The hyperbolic tangent model is purely empirical with no theoretical biology supporting it.  If it suits your data best, then use it.  But if you haven’t tried one of the exponential functions, they can be derived from first principals. See Aalderink and Jovin, 1997, Journal of Plankton Research 19:  1713 - 1742.

Line 148:  How many different start times did you use in a 24-hour period?

Line 155:  “Nucleopore” is a brand.  I presume these are polycarbonate?  PC has a dye that extracts in methanol.  Did you use a filter in your blank measurements?

Line 160:  What kind of filter?  What material was it made from?

Results:

Lines 174 - 175:  Do you know what kinds of contaminants you had in your isolates?  Were there phytoplankton other than the target diatom and symbiont cyanobacteria?

Lines 190 - 191:  Did you measure cell height?

Line 206:  “Both” light conditions?  You listed 7 levels in your methods.

Lines 208 - 210:  In the graph it seems that HL symbioses had more chl a than LL.  This seems counter-intuitive to me when considering photoacclimation.  What do you think is going on?

Line 209:  Define “symbiosis” earlier at the first use.

Lines 219 - 220: Could another curve work?

Discussion:

Lines 258 - 261:  This is a very interesting result and is in contrast to cyanobacteria, which fare better in natural oligotrophic seawater.

Lines 263 - 272:  Did you take any samples for DNA or RNA analysis?  Perhaps a colleague could help you look for evidence of viruses.

Lines 335 - 337:  Do you have any genetic material preserved to determine whether these host and asymbiotic strains did diverge?

Lines 345 - 346:  What environment light levels are relevant?  Did you go high enough?

Figures:

Fig. 1:  Excellent micrographs.  If possible, please include a higher magnification to better see the structure of the cyanobacteria.

Fig 3:  Legend, please!  And you used 7 light levels.  Why are only three shown?

Fig 4: This is an astonishing number of points.  How many aliquots did you take throughout the day to add acetylene?  What do the errorbars represent? Each point on the graph should represent multiple measurements in duplicate for one aliquot amended with acetylene.  Multiply each point by 6 - 10, and it becomes an inhuman number of jabs in the GC.  I’m not sure if the 5-point mean is meant to be a moving mean or something else.    And the 4-h incubation is unclear to me.

Figure 6:  What was fit to a hyperbolic tangent function?  There’s no P-E curve here.


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: ISOLATION, GROWTH, AND NITROGEN FIXATION RATES OF THE HEMIAULUS-RICHELIA (DIATOM-CYANOBACTERIUM) SYMBIOSIS IN CULTURE
Review round: 1
Reviewer: 3

Output:
Basic reporting: Overall the text reads fine. I have suggested a few revisions.

Citations and discussion are heavily focusing on a narrow list of authors. An example of a relevant early reference that could be additionally included and discussed:

John F. Heinbokel 1986 OCCURRENCE OF RICHELIA INTACELLULARIS (CYANOPHYTA)WITHIN THE DIATOMS HEMIAULUS HAUKII ADN H. MEMBRANACEUS OFF HAWAII  J Phycol
https://doi.org/10.1111/j.1529-8817.1986.tb00043.x

The figures are ok. I have suggested a few edits to axes. One curve fit appears missing (see detail under General comments).

The manuscript is self-contained, but some additional detail should be provided. See below.
Experimental design: The manuscript reports important data on methods of isolation of a marine diazotroph symbiosis. There are not many known cultures of these associations, and very few laboratory observations.

I have some comments and suggestions regarding clarity of the data presentation. Several methods should be explained in more detail.

I suggest the authors include a table including key information about the specific strains they used in the experiments, to help reference these strains and experiments in any future studies.
Validity of the findings: Overall the data should be useful to the N2 fixation research community
Replication is not always clear. Was there biological replication in the experiments or are the means measurements from a single culture over time?
‘The validity of using a ‘5-point running average’ for determining N2 fixation rates is not possible to assess without sufficient detail about the AR assay setup (see comments below).
One or two anecdotal statements without sufficient evidence are included in the discussion and should be removed.
Additional comments: Title: The title reads a bit cumbersome and unclear. I suggest re-wording such as:
Isolation, growth, and nitrogen fixation rates of the oceanic Hemiaulus-Richelia (diatom-cyanobacterium) symbiosis in culture

Abstract:

The abstract should add a sentence to clarify whether Hemiaulus was cultured in a monoculture or whether the stated growth and N2 fixation rates were done while it was kept in the symbiosis with the host.
Diazotrophic symbiosis>reword. The symbiosis likely involves not just diazotrophy
Maximum growth rates of H. hauckii symbioses> remove the word ‘symbioses’
the H. membranaceus symbiosis> remove the words ‘the’ and ‘symbiosis’

Narrative:
L2 prokaryotic and eukaryotic
L4 replace ‘these oligotrophic seas’ by ‘in the open ocean’
L7 Here and elsewhere: check the journal requirements about ordering citations. Typically you would mention the earliest citation first (here Villareal 1992).
L55-56 The sentence about Calothrix is not well tied to the rest of the paragraph.
L64 this may also BE true for…
L70 revise the sentence for clarity/remove redundancy regarding ‘growth rate’ and ‘growth rate response’.
L72 ‘Modeling blooms’ is a bit ambiguous. Modeling bloom formation and fate?
L84 uM > um. The former is a unit for micromoles.
L87-90 State the purity grade of the chemicals.
L93 Unclear what you mean by ‘if required’.
L97 delete ‘quanta’ from the unit
L106 Remove the weblink. Replace with a citation to a permanent source for the information (such as an external data repository) or remove entirely.
L110 Unclear what you mean by ‘given experiment’. Specify which experiment or state more clearly.
L113 The wording “symbioses growth” is ambiguous as the symbiosis is composed of two organisms. State more specifically. Do you mean, growth of Hemiaulus and Richelia and N2 fixation of the host-symbiont association?
L114-115 Were there experimental replicates?
L115 Erlenmeyer
L116-117 Is there a reason why nitrate concentration was 4x the time that of ammonium?
L117 Was the salinity periodically checked? Evaporation is likely to have increased it over time.
L123-124 ‘Gradient’ here is misleading. Each treatment is a distinct light level, thus the experiment did not test the influence of a light gradient. The experiment tested the influence of distinct, constant light intensities.
L129 > “In addition, H. hauckii strain #92…”
L132 Cell counts were performed > Cells were counted
L140-141 Do you mean the N2 fixation curves were forced to zero? Why was this different from the growth curves?
L140 This term>The y-intercept term
L142 More information is needed on how the assay was conducted. What was your vial size, liquid volume, gas volume, gas injection volume? What acetylene was used? How long did you incubate each vial and under what conditions? How much gas was removed from the vial per time point? How many times was each vial sampled?
L144 ethylene conversion ratio 4:1> this needs to be stated more clearly. What does the 4:1 stand for?
L151 ‘center point of the time differential’> reword for clarity
L151 ‘5 point mean of the multiple time series...’ Are you referring to replicate vials of the same treatment? Reword for clarity.
L152-153 A citation to a MS thesis is not sufficient here to explain how the rates were calculated. Include the information in the methods and include a supplement if need be.
L153 How did you calibrate the GC? Did you run calibrations during the day?
L154 Do not underline a in Chlorophyll a – it should be italized. Abbreviate consistently through the text as chl a (or Chl a).
L158-159 Nutrient methods need to be explained with appropriate citations, including detection limits.
L170 what is sterile filtered seawater – explain how it was made
L175 >The use of these techniques resulted in…
L177 Delete ‘as well’
L179-180 Have been found by whom? Is this your personal experience? If so, revise sentence to indicate it.
L181 Laboratory structure? What is this? A partially covered wetlab building? A device placed on top of the bin?
L181-182 Unclear what ‘this site’ is referring to.
L190-191 Diameter? The cells are not spherical. Please state all the dimensions.
L204 time>times
L205 ‘chl per symbiosis’ is confusing. Do you mean per cell of Hemiaulus?
L208-209 the definition of chl a per symbiosis should be stated earlier. In the Figure 2 you use chl a per cell though. This is confusing.
L228 scotophase?
L230-231 photophase or photoperiod? Would help the reader to stick to consistent terminology.
L249-253 The information about the symbiont free Hemiaulus is anecdotal with no other evidence shown except a statement in the narrative. Are there any observations of this strain that could be shared such as morphology, growth rates etc. Without such data the paragraph should probably be removed.
L283 Website citation here needs to be changed into a citation to a permanent source or company name, city, state, country.
L285 What does ‘their rates’ mean? Did you convert their cell numbers to rates? Or did you take rates from the paper? If using actual rates from the paper it is unclear what the digitizing was used for.
L287 With ‘these rates’ are you referring to rates in Carpenter et al. 1999?
L299 nifH gene expression?
L306 >at least two mechanisms that..
L305-309 I suggest breaking this sentence into two to clarify the meaning.
L314 utilized > used
L316 utilize > use
L331 The statement about origin of the organisms is unclear. The origin of the cyanobacterium is likely earlier than the host.
L339 ‘symbioses blooms’  This wording is awkward > reword.

Fig. 1. These are beautiful images. What filters were used for the fluorescence images? Are these images overlays of images with two filter sets? State in the figure legend. Remove ‘image credit’ – no need to credit yourself.
Fig. 2. Delete ‘quanta’. Chlorophyll > Chlorophyll a.
B: Specify whether you are showing chla per Hemiaulus or chla per Richelia cell?
Fig. 3. The strain with x- symbols does not appear to have a Growth-I curve associated with it. The light affinity appears substantially different for this strain. This result does not seem to be discussed in the text as you only refer to two G-I curves and a very similar alpha for the two.
Fig.4. ‘average… calculated from the slope’ > reword to clarify. How long were the incubations for the black symbols?
Fig.5. ‘slope of successive measurements over a 4 h period’: This is confusing. Explain what you mean by a SLOPE of successive measurements. Are you referring to measurements representing a different cumulative incubation time from the same vial? If so, isn’t the comparison simply the rate/time from each of these measurements. This is not a slope.
Fig. 6. Should the y-axis be ‘% symbiotic Hemiaulus’? If not, the results do not correspond with the text narrative.


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: ISOLATION, GROWTH, AND NITROGEN FIXATION RATES OF THE HEMIAULUS-RICHELIA (DIATOM-CYANOBACTERIUM) SYMBIOSIS IN CULTURE
Review round: 2
Reviewer: 1

Output:
Basic reporting: As mentioned in my prior read of this manuscript, the authors have written a clear, unambiguous, professional paper with sufficient literature background and context, figures, tables, and raw data. The manuscript is self-contained and after the minor revision, has met all the issues brought up previously.
Experimental design: The authors have done an excellent job of addressing all comments, adding clarity where needed, and describing statistical methods as necessary. Methods are very well written and will spark new investigations into these important associations.
Validity of the findings: As mentioned earlier, this manuscript is an important contribution to the state of knowledge for these keystone species.
Additional comments: The authors have addressed all of my noted issues in the revision.


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: ISOLATION, GROWTH, AND NITROGEN FIXATION RATES OF THE HEMIAULUS-RICHELIA (DIATOM-CYANOBACTERIUM) SYMBIOSIS IN CULTURE
Review round: 2
Reviewer: 2

Output:
Basic reporting: Description of the experimental design, especially for N2 fixation measurements, is much more clear now.  Thank you.

Specific comments:

L17:  Hyphenate compound adjectives (“nitrogen-fixing”) but not nouns (“nitrogen fixation” or “nitrogen fixers”).

L55:  Insert a space between “fixation” and “(.”

L56: Diazotrophy has been reported in the Arctic Ocean as well.  See Harding et al, 2018, PNAS 115 (52) and other publications  regarding UCYN-A.

L111:  N2 abounds, so perhaps you should specify “fixed N.”

L122: Either use a comma on both sides of “where examined” or leave them out completely.

L140:  Insert degree sign after “27” and a comma between lat and lon.

L156:  I think rinsing implies discarding, so you don’t need to state it.

L252 - 266:  Put the acetylene reduction assay description with the rest of the N2-fixation description.

L329 - 330:  “Light-saturated growth” should be hyphenated while “light saturation” should not be.

L363:  “Symbiont-free”

L366:  Insert “microscopy.”

L393 - 395:  Be consistent in using “fmol N2.”

Table 1:  State the strains used.

Figure 2:  Put the strain name in context somewhere in a sentence rather than a free-standing phrase.  And capitalize “chlorophyll” after “B.”

Figure 3:  Be more explicit about the circles.  From the graph it looks as if they extend the experiment with the closed squares rather than the open squares.

Figure 4: State what the error bars represent.
Experimental design: With the clearer description of the methods, I no longer have reservations about the experimental design.

L164:  That’s a good success rate!  I applaud you.

L171 - 175:  Any hypotheses on why YBCII worked better?
Validity of the findings: L319 - 321:  I find it interesting that the chl per symbiosis was the same.  Is photoacclimation not generally a trait for these diatoms?

L346 - 353:  Is light or growth affecting N2 fixation rates? If you normalize N2 fixation rates to growth rates instead of volume or biomass, do you see a difference among the treatments?

L396 - 399:  Maybe it’s light directly, but maybe all metabolic processes are ramped up.  Again, I encourage you to normalize N2 fixation rates to growth rates to compare. Or do you have a proposed mechanism for how light directly affects N2 fixation?

L407 - 408.  Goodness, yes!
Additional comments: I’m looking forward to seeing more DDA isolates in the future.


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: ISOLATION, GROWTH, AND NITROGEN FIXATION RATES OF THE HEMIAULUS-RICHELIA (DIATOM-CYANOBACTERIUM) SYMBIOSIS IN CULTURE
Review round: 2
Reviewer: 3

Output:
Basic reporting: x
Experimental design: x
Validity of the findings: x
Additional comments: x


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: OVERGRAZING-INDUCED LEGACY EFFECTS MAY PERMIT LEYMUS CHINENSIS TO COPE WITH HERBIVORY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Review of Guo et al. “Overgrazing-induced legacy effects on phenotypes prepare Leymus chinensis to cope with herbivory” for PeerJ MS# 49307 June 2020
In this manuscript, Guo et al. seek to demonstrate if evolutionary adaptations/herbivory-induced legacy effects alter the phenotypes of populations of a highly palatable rhizomatous clonal grass species, Leymus chinesis, that have experienced significantly different grazing histories. Collecting samples from two pastures located in Inner Mongolian steppe grasslands that have experienced either high intensity, long-term livestock grazing or been protected from mammalian grazing they grew potted transplants in a greenhouse and subjected them to different simulated herbivory/clipping “grazing” intensities.  The plants were then assessed after 90 days for differential demographic population responses based on the plants origin (legacy) and herbivory intensity treatments.
Utilizing plant material from only two different small pastures and comparing them could be considered problematic, both ecologically and statistically (pseudoreplication).  Concerns regarding the latter are somewhat mitigated since the authors are primarily interested in how plant populations from these different “legacies” differentially respond to the different “grazing” intensity treatments that they experimentally implement in controlled and randomized conditions, but the former concern regarding the broader generality and applicability of these sampling areas remains.
There are a number of interesting demographic/population results reported herein, but unfortunately, the manuscript suffers from numerous shortcomings including significant weaknesses in the study design, awkward structural organization of the text, confusing conceptual development of the central themes, and issues with the interpretation and presentation of the data. Additionally, I encourage the authors to seek language editing assistance as the current grammatical state of their manuscript exceeds the reasonable editing expectations of external referees. I have made a few suggestions below throughout the manuscript, but the overall revisions needed are extensive and will require a committed editorial service. Finally, simulated herbivory studies in controlled greenhouse conditions are not novel in the ecological literature. The authors claim they are contributing information that fills a critical knowledge gap, but I, at least as presented here (and they, to an extent), am not convinced (see below).  As such, I do not believe most of these concerns could be easily addressed without the revision constituting an entirely new submission.
My major concerns include:
Introduction - The introduction is poorly organized.  It fails to both identify and justify the central thesis the authors seek to examine and adequately support it with previously published literature. The large paragraph describing resistance, avoidance, and tolerance is overly general and can be shortened considerably. The Methods sections suffers from numerous inadequacies and insufficient descriptions of critical procedures.  Section 2.1.2. entitled “The materials sampling and cultivating” is particularly confusing and unclear.
Methods - There is little to provide confidence that the arbitrary heights of 4cm and 8cm aboveground selected to differentiate morphological adaptations to grazing have a sound ecological justification.  Are there specific sheep grazing behaviors that affect suitable forage heights?  Would 3cm and 7cm or 5cm and 9cm yield different results? And given that the authors merely measured the heights based on their growth in pots in artificial greenhouse conditions how would these be affected by exposure to the real-world conditions of climatic elements and animal trampling?  Both here and in the Introduction, the authors fail to develop a convincing case why these results will potentially translate into ecologically meaningful patterns. Moreover, since these leaf angle and height data end up being some of the most significant results reported and strongly influence the SEM interpretation I remain underwhelmed and skeptical. There are some interesting population demography data reported, particularly the root biomass responses, but these fail to rescue the overall deficiencies noted above.
Results – The results are very dense and could be improved by adding more description with less dependence on referencing figures and tables.  Indeed, based on my downstream suggestion to eliminate and/or reduce some of the numerous figures and tables, converting some of the key findings into more comprehensive or detailed text may improve the readability of this section.
Discussion – The most parsimonious explanation for L. chinensis in the OG plot pre-allocating less biomass to rhizomes, is not so much a tolerance strategy where the plant benefits from shorter rhizomes and more tiller ramets, but a cost or tradeoff associated with biomass losses and altered source-sink relationships within the plant.  The authors describe this type of biomass reallocation in their Introduction and somewhat revisit it in the Discussion, but the text takes on a meandering explanation that merely leaves the reader confused. Indeed, I believe these are some of the strongest and most interesting results in the study and they merit a thoughtful and thorough interpretation in the Discussion.
Lines 254-279 – This entire section of the Discussion needs to be restructured, better clarified, and edited with improved grammar.  I reread it multiple times and am still not entirely certain what takeaway message the authors are attempting to convey.
Section beginning at line 280 – there is some interesting speculation here that would be of interest to a broader base of ecological readers.  Unfortunately, the authors really need field data, which they do not have, on the frequency and abundance of asexual vs sexual reproduction of the different genotypes in the different pastures to answer these questions in a satisfactory manner.
It is discouraging to read the authors state “more detailed research is needed to understand the responses of plant ramet numbers to herbivore defoliation, especially in combination with grazing-induced legacy effects on plants” at the end of their manuscript when the primary intention of their study was to make exactly this contribution!  I appreciate that no study is wholly comprehensive and there are always moving goalposts on opportunities to gain more knowledge and insights, but regret that (and as the authors confess above) the current iteration of this submission falls considerably short of adding sufficiently to existing published ecological literature.
Figures & Tables - While I appreciate that supplemental electronic figures and data eliminate physical journal space restrictions and provide authors an opportunity to be more comprehensive, the cartoon drawings for Supplemental Figures 1-4 are overly simplistic and unnecessary. In their place, I recommend that several of the main figures and tables be moved to the supplemental category.  Eight multi-panel figures and three data intensive tables are excessive for a study as simple and straightforward as presented here.  I recommend considerably reducing the primary visual data presented to only the essential results that best tell the story the authors are crafting from their study.
A non-comprehensive sampling of other grammatical issues:
Line 12 - conditions
Line 21 - “against our prospection” is unclear.  Do the authors mean “Contrary to our prediction…”?
Lines 26-27 - Awkwardly phrased sentence.  Requires grammatical clarity.  I also submit that the definition is either incomplete or insufficient
Line 31 - extra space after bracket
Line 32 – above the authors define legacy effects as effects on ecosystem structure and/or function, but here they refer to plant defense strategies.  Plant defense strategies are conventionally thought of as plant population processes, not ecosystem process.  This may seem semantic, but it is a rather critical point and influences not only the accurate usage of the legacy effect terminology, but the entire context and interpretation of it both here and as applied to other species in other ecosystems.
Lines 37-39 - You emphasize the majority of published herbivory induced legacy effects research over recent decades has focused on short-term insect damage but then fail to provide any references to support this assertion.
Line 84 – precariously?
The term “past decades” is overused in the Introduction.
Line 140 – replace “weighted with a weighing balance” with “weighed”
Line 154 – mesh bag
Line 255 – increasing trend of what?
Figure 5 legend – light
Experimental design: see above
Validity of the findings: see above
Additional comments: see above


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

Input:
Title: OVERGRAZING-INDUCED LEGACY EFFECTS MAY PERMIT LEYMUS CHINENSIS TO COPE WITH HERBIVORY
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript reported a novel experiment aiming to declare how the performance of Leymus chinensis in response to simulated grazing is influenced by the overgrazing-induced legacy, and what phenotypic traits contribute to the changes of the performance. The novelty of the study lies in that it not only declared the legacy effect of overgrazing by livestock rather than by insects, but declared its consequences for future coping strategies in responses to grazing. In my view, the experiment was nicely designed, the introduction section has given sufficient context of the study, the results presented are necessary and clearly stated, and the discussion section is cohesive and in depth. However, I still have some concerns as follows.

Major concerns
1.	L217, when you calculated the aboveground, either for every vertical layer or the total, did you pool together all the three harvest?
2.	L214-215, Why simulated grazing reduce, but not increase the aboveground biomass distribution close to the ground? As expected, the aboveground biomass distribution should be more towards ground surface in response to grazing. I cannot see the explanation for this in the Discussion section.
3.	L243-245, I think this result is very interesting, and worth more deeply exploited in the Discussion section. For example, the OG plants is more conservative compared to NG plants, which are more exploitative, as shown by its greater rhizome biomass than OG plants when cultivated in CK.

Minor concerns
4.	L179, “not both responded differently…” is grammatically wrong.
5.	L183, there is nothing in the parentheses in the formula.
6.	L207, “NG had smaller….”, can this sentence be written otherwise like: “OG had greater…..”, because intuitively, the readers will take NG as a control to OG, so you’d better use OG as the subject of the sentence, and compare with NG. The same problem with the sentence in L209-210.
7.	L218, here the authors mentioned Fig.3, but in my view, this figure is of little significance, because genet is composed of ramets, genet and ramet should have the same biomass distribution.
8.	L269, “……may not be ascribed to defoliation by livestock.” I don’t agree. Over-grazing induced legacy is formed during a long-term period through many generations. It is a bit arbitrary to conclude that “the over-grazing induced legacies distribution may not be ascribed to defoliation by livestock” only based on one-off and short-term test.
9.	L298-301, this explanation is a bit far-fetching. In addition, I am afraid that the vegetative tillers sprouted from the rhizome nodes cannot termed as seedlings, as they were not originated from seeds.
10.	Since you have define the codes like OG and NG, you should use them when necessary, to avoid redundancy. See Line 308-310 in the annotated manuscript. Please make the similar changes where else when necessary throughout the entire manuscript.
11.	I would term the OG plot overgrazed plot instead of overgrazing plot, and term the NG plot ungrazed plot instead of no grazing plot.
12.	Figure 2, I would like the two panels combine into one panel (of course you should first do the same data transformation for below 4 cm distribution as for below 8 cm distribution).
13.	Figure 7, GZ in the parentheses should be OG?

Please see attached annotated manuscript for other very minor corrections


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: INVESTIGATION FOR A MULTI-SILIQUE TRAIT IN BRASSICA NAPUS BY ALTERNATIVE SPLICING ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In this manuscript, Chai et al reported an interesting investigation in genome-wide gene alternative splicing in rapeseed for the muti-silique trait. The authors totally identified about 220168 AS which could be sorted into 12 AS types.  Eleven differently expressed AS genes were identified, and their annotations implied that five of these genes including BnFUL were directly associated with the multi-silique trait. Additionally, the 205 stably expressed AS genes were analyzed, of which 187 could be annotated, and three candidates including BnaC06g32640D, BnaC07g25280D, and  BnaC01g16410D were considered to be related to the multi-silique trait. These findings may be very helpful for our understanding of muti-silique formation in rapeseed.

Some comments:

In the manuscript, authors mentioned that AS is a pathway by which the environment could regulate plant physiology, therefore in this study, we analyzed AS events in order to investigate the mechanism by which plants perceive temperature fluctuations. However, I could not see it from their data because they did RNA-seq using NILs zws-217 and zws-ms growing in the normal environmental conditions . Low temperatures can switch off the multi-silique trait in zws-ms rapeseed, I suggest authors should also do the RNA-seq at different temperature condition using zws-ms rapeseed.


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: INVESTIGATION FOR A MULTI-SILIQUE TRAIT IN BRASSICA NAPUS BY ALTERNATIVE SPLICING ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript is to investigate how alternative splicing may contribute to a multi-silique trait in Brassica napus by comparing the transcriptome difference between two NIL lines: one has such a trait, while the other does not. The authors previous found the genomic region and candidate genes associated with this trait. In this manuscript, the authors performed an RNAseq experiment and reported their data analysis in regards to all the transcripts that exhibited alternative splicing in this experiment, differentially expressed genes between the two NIL lines that also exhibited alternative splicing in this experiments, and line-specific alternatively spliced genes which the author defined as “genes with stable AS events”. The introduction provided an adequate overview of prior work that support the rationale of this study.
Experimental design: The RNAseq experiment was performed with three biological replicates of each comparison group, which is standard. However, there is a substantial lack of information and experimental confirmation as listed below:
1.	Currently the manuscript does not include sufficient AS analysis results to support the conclusion. Supplementary tables need better title and column headers to provide more detailed information. For example, in Table S3, column G has a header of “event pattern”, while the actual cells only listed the coordinates of each splicing event. This does not provide any information as to the read count of each AS event. Information from all six samples should be analyzed and aggregated into one table to show a comparison across samples that directly support descriptions of AS distributions in Result section.  The fact that the authors only described any genes that exhibit AS in any samples without their abundance and analysis on how much changes there may be between the two lines does not provide much insight in how AS may contribute to the multi-silique trait. Authors did mention eleven differentially expressed AS genes. However, this diminishes the value of alternative splicing study because changes in ratios of differently spliced isoforms of the same gene may be important in regulating protein abundance and function without changing total transcript level of that gene.
2.	In addition, the authors reported 205 “stably expressed AS genes” which was later explained as “with a particular AS events that occurred only in zws-ms or in zws-217”. The term “Stably expressed AS genes” is not self-explanatory. This group of events are basically line-specific AS events according to this description. They could be interesting to have a deeper look and validated by rtPCR. Unfortunately, the authors did not validate any of them. Table S9 only provided their annotations without their abundance or any statistical measures.
3.	The description in “AS Event Analysis” and “Annotation of Genes” lacks details regarding statistical parameters, statistical tests used and FDR used, etc.
4.	There was no qPCR validation of differentially expressed AS genes. In table 2, the eleven differentially expressed AS genes are only reported as upregulated or downregulated without any fold change and statistics in there. These should be added.
5.	Classes of alternative splicing events have been long established in the past (Reddy. Annu. Rev. Plant Biol. 2007. 58:267-94). The authors should use the existing classification and integrate theirs as subclasses.
Validity of the findings: Without enough quantitative and validated results and appropriate analyses on these AS events, the conclusions of this manuscript are weak.
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: INVESTIGATION FOR A MULTI-SILIQUE TRAIT IN BRASSICA NAPUS BY ALTERNATIVE SPLICING ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The newly added RNA-seq data from a colder condition was not analyzed and discussed.
Experimental design: no comments
Validity of the findings: The authors did not validate RNA-seq results.
Additional comments: 1.	It is good that the authors included a different set of data from a colder condition. But this set of data needs to analyzed and discussed in this manuscript. It can’t be just there without any contribution to the manuscript.
2.	The authors completely ignored the question on validation of RNA-seq data. Table 2 provides logFC from RNA-seq. That is reporting the same information, not validation.
3.	Lines 119-122: Please specify annual average temperatures in both Xindu and Ma’erkang.
4.	Line 155. Although there is a statement that default parameters were used. It is very cryptic and does not offer any specific information. Please specific statistical tests used and FDR used as asked earlier.
5.	There is no Table 5, while it was referred to on line 190. Is it supposed to be Table S5?
6.	Line 220: It is differentially expressed, not differently expressed. This mistake appeared again later in the manuscript (Line 315).
7.	Lines 263-273: Since new classifications of AS events are created here, there need to be some sort of illustration to visualize the difference between the establish classification and this new one. Since Table S7 is mentioned here. What is the FDR for in Table S7? Column headers need explanations for what they represent in all tables.
8.	Lines 275-276: There is a statement saying that 10 genes were selected for further study. But there is no information on what this further study is and what the results of the further study are.


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: INVESTIGATION FOR A MULTI-SILIQUE TRAIT IN BRASSICA NAPUS BY ALTERNATIVE SPLICING ANALYSIS
Review round: 3
Reviewer: 1

Output:
Basic reporting: The authors have made significant improvement on the manuscript. I am fine with the manuscript with two minor revisions:

1. the manuscript still has grammar errors, such as line 79 "did not showed". The authors should do read the entire manuscript thoroughly and correct all language errors.

2. For the added qPCR results in Figure S1, there is no mentioning of basic experimental conditions, such as the use of biological replicates and how many? the description of methods in manuscript should have sufficient information to allow readers to understand the design of the experiments. It should not be assumed that every paper uses the same conditions.
Experimental design: ok.
Validity of the findings: ok.
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: ADEQUACY OF CLINICAL INFORMATION IN X-RAY REFERRALS FOR TRAUMATIC ANKLE INJURY WITH REFERENCE TO THE OTTAWA ANKLE RULES—A RETROSPECTIVE CLINICAL AUDIT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article presents the clinical problem, methods used and results of the study clearly. Professional English is used throughout the text. Authors provided abundant data regarding the background with corresponding references. Article is well-structured with appropriate use of figures and tables. Raw data was available for the review.
Results of the study are clearly presented and discussed in context of previous knowledge and the goal of the study.
Experimental design: Clinical problem of the article is well known in scientific community, but lack of data regarding local, Southern Australian region, can justify the research. It is important for both quality control on a local level as well as for comparison of results on a global scale to gather and publish information even regarding previously researched clinical problems. Experimental design is of a sufficient research integrity and reproducible.
Validity of the findings: The Authors admit that this is a novel research on a local level only, clearly underlying that there are some weaknesses of the study. Results seem statistically sound and are interesting to a medical professional. Discussion is well-written summarizing the results and possible impacts of the study.
Additional comments: The article offers a new insight to an old problem, offering possible reasons why Ottawa Ankle Rules application in Emergency department setting differs between medical professionals who refer patients for radiography. While not assessing the accuracy of the test itself, but rather in a context of different medical professions, this is still important information for scientific community.


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: ADEQUACY OF CLINICAL INFORMATION IN X-RAY REFERRALS FOR TRAUMATIC ANKLE INJURY WITH REFERENCE TO THE OTTAWA ANKLE RULES—A RETROSPECTIVE CLINICAL AUDIT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is clear and well written. References, figure, and tables are adequate. Results, although not entirely new, are relevant.
Experimental design: The study is in accordance with the scope of the journal. Research question and hypothesis were adequately addressed, following all the required issues in terms of ethical standard. Methods were thoroughly described.
Validity of the findings: Although the findings are not entirely new, the results are interesting and worthy of disclosure. The manuscript results are adequately presented and compared with  previous similar studies.
Additional comments: Dear authors, I appreciate the opportunity to review this interesting manuscript.
The topic is relevant in terms of cost-effectiveness regarding ankle trauma.
The article is well written and the methodology is adequate and thoroughly presented.
Ethical standards were also adequately addressed.
However, I have two minor suggestions aiming to improve the manuscript quality:
1-Background section is excessively long. Make it more straightforward and compare the  previously published studies with your findings in the discussion section.
2- Discuss about the relationship between Ottawa Ankle Rules and subjective examinador perception to evaluate radiograph necessity following ankle sprain. Furthermore, some discussion should be presented in terms of the most important item of OAR to predict the presence of a foot/ankle fracture.


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: COUPLED EXUVIAE OF THE ORDOVICIAN OVALOCEPHALUS (PLIOMERIDAE, TRILOBITA) IN SOUTH CHINA AND ITS BEHAVIORAL IMPLICATIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Review of “Coupled exuviae of the Ordovician Ovalocephalus (Pliomeridae, Trilobita) in south China and its behavioral implications (#48931)”

By: Ruiwen Zong

Reviewer: Nigel Hughes

The paper presents data that in the associations of trilobites studied there is a clear tendency for alignment of long axes in clusters of differently sized mature individuals that include several specimens, but rarely more than 4. Many of these individuals show patterns of partial disarticulation, mostly either at the “neck suture” or at the facial sutures. As far as I am aware this particular style of trilobite sclerite association is new and therefore of some interest: it joins a growing literature of case study descriptions that collectively build the descriptive basis of trilobite sclerite disposition. So, in those terms it certainly has merit as a study.

Where I am less keen is the interpretative part. PeerJ is a short format journal, and the paper does not appear to indicate a store of supplementary information that documents and supports the contentions made herein. Such information needs to be available. The paper does refer to some other studies, but it’s unclear whether these provide the specific documentation needed. For example:

1. More information of sedimentology (perhaps on an online supplement?) is necessary. Sedimentary evidence is insufficient to assess represent events taking place in burrows either with or without a roof) or on the seafloor at sediment water interface, and no data is presented on the range of directions that the various clusters pointed towards – only that of the internal variance in orientation within clusters. The sedimentary log provides is fine for orientating us stratigraphically but tells us almost nothing of the details of the depositional regime. The bed yielding the clusters is ~1.5 m thick. What is it comprised of? Are there multiple separate depositional events? What is the relationship between episodes of deposition and the clusters? Here are they located specifically within the bed? Do different clusters on the same bedding surface align? The paper speculates that clusters accumulated on the sediment surface over a period of time. Is there any evidence from sedimentology to corroborate this?

2. As Whittington (1990) pointed out, making the case for trilobite sclerite associations being the product of ecdysis is a challenge. In my view, there is a recent tendency towards tolerating relatively lax criteria for the confident recognition of exuvae. For example, this paper treats the study of Brandt (1993) as an accepted authority – I view that study as flawed in several ways. In my view it’s possible to argue that almost any association of sclerites derived from the same individual is the result of ecdysis – but that doesn’t mean that all actually are the result of that process. As Whittington (1990) made clear, there have to be specific arguments from the disposition of sclerites that make not only ecdysis a candidate process for the disposition, but also one that is more likely than the sum of alternative explanations.

What’s novel (and thus genuinely interesting) here is the association of sclerites derived from individuals and the alignment of multiple individuals, because these particular associations place limits on the processes that can reasonably explain them. What I was hoping for in this paper was, as in Whittington 1990, constraints provided by the alignment pattern itself that independently supports ecdysis as the explanation for the arrangement of the sclerites. I don’t see that here. What if these were animals clustered in burrows that then decayed or were scavenged (see for example Hughes and Cooper, 1999 Journal of Paleontology for an example of scavenging of carcasses)? The pattern of sclerite disposition reported are quite diverse – not the stereotyped associations whose posture can only be reasonably be explained by movements associated with ecdysis. For example, Fig 2 shows a whole range of associations, including a partly enrolled specimen. Of course, the diversity of postures could be because Brandt (and Daley and followers) are right and trilobites did show an unusual diversity of ecdysial strategies atypical compared to living arthropods. But other explanations – such as the disturbance of carcasses (or exuvae for that matter) by various agents (physical processes or biological agents) are possible. How can it be argued that the weight of evidence favors a diversity of modes of ecdysis?

What to say overall? I am very not familiar with PeerJ, but I certainly see a useful publication emerging from this work. It will need careful documentation of sedimentology and taphonomy of the associations, and I’d like to see at least a good portion of that work in the main text of the publication, rather than relegated to the supplementary materials because I think the documentation is critical to interpretation. If PeerJ supports that format then I think a revised version should be encouraged. If not, I would suggest a longer manuscript submitted to a discipline specific journal.
Experimental design: see above
Validity of the findings: see above
Additional comments: see above


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

Input:
Title: COUPLED EXUVIAE OF THE ORDOVICIAN OVALOCEPHALUS (PLIOMERIDAE, TRILOBITA) IN SOUTH CHINA AND ITS BEHAVIORAL IMPLICATIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Review of “Coupled exuviae of the Ordovician Include”  (Pliomeridae, Trilobita) in South China and its behavioural implications by Ruin Song.

1) English language, while clearly not written by an anglophone, is on the whole easy to follow (but see detailed suggested improvements below).
2) The author has done a fairly thorough job of researching the topic, and that is shown by his knowledge of the relevant literature.
3) The figures are of reasonable quality and necessary for the paper.
4) I have a number of suggestions that I hope would serve to improve the paper slightly, and perhaps change the conclusions slightly.
5) The paper is interesting and worthy of publication.
6) Raw data are supplied.
7) The paper includes original research, and improves our understanding of ancient events and biology.
8) Methods are described with sufficient detail and information.
9) On whole, findings are clear, original, but I would suggest that some minor but significant changes should be made to them (see detailed comments below).
10) Manuscript provides interesting new data and ideas.

Detailed comments on MS:
Title: Ovalocaphalus should be in a different font to rest of title to show that it is a binomial name.
Line 16: change “reflecting” to “preserving”.
Lines 21-22: remove “,combined with the overlying phenomenon,” as it is confusing.
Line 24: change “the herding behavior of trilobites”  to “the postulated herding behavior of some trilobites”.
Line 25: I do not think that this work has demonstrated what is staled here: “representing a behavioral response of the trilobites to choose a nearby safe zone in a crisis situation”.  See more detailed comments below.
Line 27, trilobites do not have an exoskeleton of just chitin, it is mainly calcite with various amounts of Mg, probably with a chitinous outer layer (that is true ion many other arthropods (crabs, lobsters, etc.), so perhaps
Line 35: the thoracopygon is the term used for the thorax and the pygidium (see “morphological terms” in the latest Treatise on trilobites, Whittington et al., 1997, p. 329).
Lines 45-49. Not really relevant to the argument, could be left out.
Line 52: change “literatures” to ‘literature’.  Literature is usually used as a collective noun.
Liner 58: change “”related to the herding behavior of trilobites” to :’herding behavior of some trilobites” - since we do not know that all trilobites exhibited this behavior (some probably did not).
Lines 59-60” Change to read “They provide new material for understanding exuvial techniques of trilobites, and the behavior of trilobites when melting”.
Line 70:  change “trilobites with the high abundance” to “ an abundant and diverse trilobite fauna”

Line 74: change “machaerids” to “machaeridians”
Line 78-80: Change to read “Ovalocephalus tetrasulcatus (Kielan, 1960) is the only pliomerid trilobite in the Linhsiang Formation. Ovalocephalus is largely restricted to peri-Gondwana, but is widely distributed in the Ordovician of China (Zhou et al., 2010).
Line 87 and 88: Change “together” to “one another”.
Line 89: Change “same horizon” to “ same interval”.
Line 94:  Change “In the above materials” to “In the present work”, and change “included” to “include”
Line 95: change “corpses” to “carapaces” or “exoskeletons”. If they are molts it is not appropriate to call them “corpses” as if they were carcasses or cadavers.
Line 102: change “specimens has” to “specimens have”
Line 113” change “thoracopygidia” to “thoracopyga”  (see reference above).
Line 124-125: “Remove first “together” and change to read “Preservation of two Ovalocephalus tetrasulcatus exoskeletons preserved together is similar to the preservation of an arthropod in …”
Line 133: either “are no burrows” or “is no burrow”. How about “no evidence of burrows”, as burrows tree often very difficult to see or not preserved in strata that are of a single composition (particularly pure mudstones).
Line 143: change “thus, so, it” to “thus it”
Lines 145-147. I agree that it makes no sense to regard them as the products of a single animal returning to the same place to molt. We do not know how many times trilobites were able to reproduce or whether larger, older trilobites were able to reproduce with younger animals (as occurs in many species today). We do not think that they were like insects and copulated once and then died (although that is a possible scenario for some forms). We also know that trilobites, like any other species probably had a bell curve relative to size (with large and small individuals of the same age and degree of sexual maturity - we certainly have some examples that show a size range in most molt clusters/instars - see my chapter in the treatise and a number of later papers, often with Catherine Cronier as an author).
Line 153: change “latter: to “later”
Lines 156-157: I wonder why you have not considered sea floor currents as a reason for the trilobite molts pointing in the same direction (your effective rose diagram). It makes much more sense that the trilobites were orienting themselves relative to prevailing sea floor currents than a need to invoke linear depressions in an environment where the currents were not strong enough to move the exuviae. The currents could still have been strong enough that a trilobite which was about to lose its exoskeleton (so it would have been slow and weak, and subject to predation) might wish to minimize its exposure to the current (by facing into it to reduce drag forces). I know that at the substrate surface (the base of the hydrodynamic boundary layer) there was practically no current, but unless the trilobite burrowed mostly into the sediment before molting it must have been high enough to have been affected by sea floor currents. We also know that sea floor currents strong enough to form ripple marks can occur at depths of thousands of meters in some areas of the ocean floor (probably not in regions that deposit mudstones?).
Line 163. I suggested that phacopids probably selected nautiloid shells as melting sites as far back as 1971 in Palaeontographica A (for the Devonian of Australia).
Line 167: change “nautilus: to “nautiloid” (could not be Nautilus in the Ordovician!)
Line 186: Why do you think that the trilobites reproduced before molting. If it was at all an active behavior, it would be easier to accomplish while they still had an exoskeleton, and then they could have melted afterwards. Limulus and related forms certainly moulted with the exoskeleton in place, as shown in modern studies and Jurassic trace fossil studies.
Lines 188-190. I am not sure that I like these closing comments. You have not demonstrated that there were any “ crisis situations” unless of course you are referring to attacks by nautiloids. Also, getting together in twos or threes (quite possibly for reproduction” does not really, to my mind, mean “herding behavior”
I have not read through the references carefully, but would point out that on line 249, it is Radwanski and Radwanska, 2009) - I looked at the original paper.
I enjoyed reading this paper.
Experimental design: Ok
Validity of the findings: Slight changes suggested
Additional comments: I enjoyed reading this. I hope that you take my suggested improvements seriously. They are meant to be constructive and to improve the work.


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: COUPLED EXUVIAE OF THE ORDOVICIAN OVALOCEPHALUS (PLIOMERIDAE, TRILOBITA) IN SOUTH CHINA AND ITS BEHAVIORAL IMPLICATIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: The author has done a good job making most of the changes suggested. There are some improvements needed to the English language in the MS (see suggestions in the revised MS sent back. It would have been easier to have made a list of changes that need to be made had there been line numbers in the revised MS.
    I am still not convinced why sex could not or would not have been accomplished before molting (which seems to me to be more likely).
   The only other general problem that I have with this work is that the author seems to think that only one male is or can be involved in impregnating or fertilizing the eggs of a female, whereas there are numerous examples in the animal kingdom, involving many classes and orders, of more than one male attempting to do this job (ranging from our own species to fishes, squids, bees and probably many other invertebrates, if one was to research this area thoroughly). So the fact that there are three moult carapaces in some of the specimens described in this paper, if they are directly associated with mating behaviour, could simply be the results of there being 2 males and one female in the group.
Just a note that it is the Katian Stage, not the Kaitian.
Additional comments: Please check minor changes to the MS to improve the English language and note the comments above under validity of the findings. Otherwise, I am pleased that you have clearly carefully considered the improvements suggested by the reviewers.


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

Input:
Title: A TAXONOMIC REVIEW OF THE GENUS ASTROCLADUS (ECHINODERMATA, OPHIUROIDEA, EURYALIDA, GORGONOCEPHALIDAE) FROM JAPANESE COASTAL WATERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: See attached files. review below.  MS is basically good. But requires editing.
Experimental design: Acceptable and proficient.
Validity of the findings: Yes.
Additional comments: Overview:  Okanishi et al. address the species of Astrocladus present in Japan. Three are addressed, but morphological and molecular data show that there are in fact four species.

A solid, well-researched and meticulously crafted piece of work.

******I thought a key to Japanese species OR a table summarizing characters between the four Japanese species would be a distinct improvement. ********

Other comments:

*The MS displays some difficulty in summarizing results and explaining the results of their work.

One specific issue: the abstract and the introduction assumes that Astrocladus coniferus  has two synonyms: A. pardalis and A. dolfleini.. and yet, WoRMS lists both of these species as “accepted”.   This confused me until I read further… While this could simply be a shortcoming of the WoRMS editor, the authors cannot ignore this..and must cite the proper citation to support their argument.

English is not the primary language of the authors, and although they are clearly proficient in the taxonomic descriptions and technical sections there are places where the language regarding synonymies and designation of lectotypes is a bit difficult to follow.

Edits are made directly to the pdf.


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

Input:
Title: A TAXONOMIC REVIEW OF THE GENUS ASTROCLADUS (ECHINODERMATA, OPHIUROIDEA, EURYALIDA, GORGONOCEPHALIDAE) FROM JAPANESE COASTAL WATERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article has been proof read by an English speaking taxonomist and so in general is well written and professional. I have made some small alterations in the attached pdf.

The illustrations are numerous, clear and useful.

The cited literature needs a little attention, there is a mixture of abbreviated and full journal titles, some missing data, etc.
Experimental design: Not really relevant. The authors have accessed all the specimens they could of the genus from Japanese waters. The species are relatively rare.

I have checked the COI sequences provided and blasted them against my own reference database. I agree with the phylogeny provided in the manuscript.
Validity of the findings: I agree with the taxonomic decisions made in the manuscript. They reestablish an old species that had been previously synonymsied.
Additional comments: I have only two general comments and some minor editorial suggestions that are in the attached annotated pdf.

1. Some of the material examined have a voucher code starting with MO. What is the collection status of these specimens? Will they be placed in a museum (eg in the NSMT) after the study is published? If so it would be better if you put NSMT registration numbers in the manuscript.

2. I am a little confused by the distal hooklet-bearing plates that you illustrate (pl 6A, 9J, 14H). Are these really lateral arm plates with arm spines and hooklets, or are they distal hooklet bearing plates that lie on top of the lateral arm plates. Your descriptions and figure captions do not make this clear. In fact you say that the lateral arm plates are completely covered in granules/plates (eg lines 388-9). If they are completely covered, how do the arm spines stick out? This needs to be made more clear.