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: LAND-USE HISTORY IMPACTS SPATIAL PATTERNS AND COMPOSITION OF WOODY PLANT SPECIES ACROSS A 35-HECTARE TEMPERATE FOREST PLOT
Review round: 1
Reviewer: 1

Output:
Basic reporting: General comments
The manuscript entitled “Land-use history impacts spatial patterns and composition of woody plant species across a 35-hectare temperate forest plot” aims at evaluating the historical drivers that shaped the structure and composition of a 35 ha permanent plot temperate forest. Several researches around the world have demonstrated that 1-ha or less permanent plots are often not big enough to describe the structure and dynamics of forest stands.
Experimental design: This manuscript presents an impressive amount of information of fully mapped trees (> 108 thousands), but in my opinion lacks in the analyses part. I find the ms well written and I suggest transforming it in a “Data paper”. The point pattern analysis and other methodological issues need to be better explained in details.
Validity of the findings: The manuscript in its present form is a mere description of the forest inventory, without a clear ecological research question. For these reasons I suggest to reconsider the ms after minor revisions.

Specific comments
See the attached PDF “peerj-reviewing-64305-v0_rev.pdf”
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: LAND-USE HISTORY IMPACTS SPATIAL PATTERNS AND COMPOSITION OF WOODY PLANT SPECIES ACROSS A 35-HECTARE TEMPERATE FOREST PLOT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The language is good, then references, structure and tables are good.
Experimental design: No comments.
Validity of the findings: No comments.
Additional comments: In this manuscript, the authors gave a good and comprehensive introduction of community composition and structure in the 35 ha Harvard Forest dynamics plot. They also assessed the intraspecific and interspecific spatial relationships of some dominated species. Especially, using good historical data in their plot, they evaluated the legacy effects of the land-use history on current species abundance and distribution, and interestingly they found land-use history showed a significant effect at a relatively local spatial scale (35 ha). In general, this manuscript has a good narrative and using appropriate methods to generate their results. I have no specific comments on this manuscript, and I think it can be accepted on this journal because very few studies has evaluated the effect on the past history events on present community structure in forest dynamics plot at a local spatial scale.

Very minor comment:
Lines 28 and 29: please use the full names of these two genera names for the first time.
Line 227: please cite the R language.


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: LAND-USE HISTORY IMPACTS SPATIAL PATTERNS AND COMPOSITION OF WOODY PLANT SPECIES ACROSS A 35-HECTARE TEMPERATE FOREST PLOT
Review round: 1
Reviewer: 3

Output:
Basic reporting: Excellent.
Experimental design: Good execution of sampling and analysis.  Need to clarify some methodology.
Validity of the findings: Strong.  Need to clarify issue with foundational species.
Additional comments: Land use history is a critical legacy explaining the spatial structure of tree communities and large forest dynamics plots offer the opportunity to study this at appropriate scales to infer the relevant ecological processes.  This is a first look at these relationships from the Harvard Forest plot, a site that pioneered our understanding of land use effects on contemporary forests.  The perspective is mostly comprehensive and offer insights into the interplay between land use, environment, and intra- and interspecific competition on the spatial structure of the forest.
The statistics are top notch and demonstrate new ways of analyzing spatial point data to address ecological questions.  I have only a few concerns.

It is unclear to me how multi-stemmed individuals were handled in the point pattern analysis.  Did you use all the stems and, if not, how did you choose which stem to represent the individual tree?  Whatever the choice, it should be carefully justified.

Why were the most common species chosen as the top 7?  Why not the 13 with >1000 stems?  More?  Obviously, the choice is arbitrary (other than these 7 do dominate density and BA) but can we provide more justification here?

I understand that Tsuga canadensis has been demonstrated to be a foundational species, but I wonder if you wouldn’t get a better understanding of its uniqueness in this respect if you applied the same analyses to some of the species to see if they lack the same degree of effect?  Otherwise, the argument seems circular to me.

I have a few other marginal notes in the attached file.  There were some typos in Table S2: check capitization of species names.


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: LAND-USE HISTORY IMPACTS SPATIAL PATTERNS AND COMPOSITION OF WOODY PLANT SPECIES ACROSS A 35-HECTARE TEMPERATE FOREST PLOT
Review round: 2
Reviewer: 1

Output:
Basic reporting: Dear authors,
I am completely satisfied by the changes adopted by the authors of this interesting manuscript.
Experimental design: Dear authors,
I am completely satisfied by the changes adopted by the authors of this interesting manuscript.
Validity of the findings: Dear authors,
I am completely satisfied by the changes adopted by the authors of this interesting manuscript.
Additional comments: Dear editors,
I am completely satisfied by the changes adopted by the authors of this interesting manuscript.


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

Input:
Title: LAND-USE HISTORY IMPACTS SPATIAL PATTERNS AND COMPOSITION OF WOODY PLANT SPECIES ACROSS A 35-HECTARE TEMPERATE FOREST PLOT
Review round: 2
Reviewer: 2

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: The authors have addressed all the reviewers 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: IDENTIFICATION OF THE OCCURRENCE AND POTENTIAL MECHANISMS OF HETEROTOPIC OSSIFICATION ASSOCIATED WITH 17-BETA-ESTRADIOL TARGETING MKX BY BIOINFORMATICS ANALYSIS AND CELLULAR EXPERIMENTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.	Please alphabetically re-order your keywords.
2.	Problem is well stated but the aim of the study should be better presented. We should be seeing your hyphotesis and why you made this study in the introduction section.
3.	The used databases are being presented in the materials & methods section. Those informations should be given in the introduction.
4.	The websites should be placed in the references section as internet references.
5.	Please make sure you used latin alphabet symbols. There are too many round shaped symbols instead of “.” in the manuscript.
6.	Please check all the spacings. There are too many spacing errors between words.
7.	The presentation of databeses should be moved into the introduction section.
8.	Fatal bovine serum should be corrected as FETAL BS.
9.	While giving concentrations please place numbers first and the agent afterwards (for ex: %7  FBS, 1% penicillin streptomycin instead of FBS %7, penicillin-streptomycin %1).
10.   Authors are suggested to use terms of real-time PCR or RT-qPCR instead of RT-PCR as it commonly refers to reverse transcriptase PCR.
11. A statistics section should be prepared.
Experimental design: I evaluated your manuscript for your in vitro experiments as other studies are not in my expertise. I have some minor inquiries about those studies but I also come up with some serious problems in your PCR analysis.
1.	Please mention which method is used to quantify total RNA amounts.
2.	Please mention the real time PCR device.
3.	Please mention how many cycles were performed in real-time PCR.
4.	Please make a table for your primer sequences and give information for base pairs and Tm values for the primers.
5.	The RUNX2 primer is also binding with Cbfa1 gene in humans. So it’s not specific to RUNX2. Cbfa is also being effected by 17ß-estradiol and also an important gene in osteogenesis. So this is a major problem.
https://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Get&RID=MBM9GR00013 (Link expires on 19.09.2021, 19:44 pm)
6.	Your GAPDH primer is not specific for human GAPDH. It unfortunately makes your PCR results unusable.
https://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Get&RID=MBMADSTF013 (Link expires on 19.09.2021, 19:45 pm)
7.	Please mention cell seeding densities for MTT assay.
Validity of the findings: As the GAPDH primer is not showing specifity for human GAPDH gene and RUNX2 primer is also binds with Cbfa1 gene the PCR results are unfortunately unusable.

Cbfa1 gene expression is also being effected by 17ß-estradiol and have a role in osteogenesis as well. If the PCR primer binds with both RUNX2 and Cbfa1 we are actually given Ct values for both genes.

Housekeeping gene is being used for normalisation of the Ct values of other target genes. So if the housekeeping gene is not specific, both delta Ct values, delta delta Ct values and fold expression values will be wrong.

I have to stress that the manuscript is not publishable with these PCR results.
Additional comments: Dear Authors;
I had finished reviewing your manuscript on behalf of PeerJ. I unfortunately should inform you that I decided to suggest rejection to your manuscript due to non-specific PCR primers. Those results are unfortunately unusable as both your primer for the housekeeping gene is not specific and RUNX2 is also matches with Cbfa1 gene. Authors suggested to repeat PCR analysis with different PCR primers. My other comments are also visible in my detailed report at the end of the conclusions section.
Best Regards.


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

Input:
Title: IDENTIFICATION OF THE OCCURRENCE AND POTENTIAL MECHANISMS OF HETEROTOPIC OSSIFICATION ASSOCIATED WITH 17-BETA-ESTRADIOL TARGETING MKX BY BIOINFORMATICS ANALYSIS AND CELLULAR EXPERIMENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors have done considerable research to explore the mechanism of heterotopic ossification and initially elaborated that 17-beta-estradiol may bind MKX and affect its interaction with SIN3A/HDAC1.However, the article contains many grammatical errors and repeated words, which may confuse the reader.I suggest the article be revised and published.
Experimental design: The article is scientific and innovative.
Validity of the findings: Conclusions are well stated, linked to original research question & limited to supporting results.
Additional comments: First, I recommend that authors carefully review their articles, remove repetitive words, and consult with native English speakers to embellish the article. Please avoid wordiness in academic writing and maintain concise description.All aspects of the study that have already been completed should be mentioned in past tense.
Below I list in detail the entries in the article that need to be changed for the author's reference.
①　“Passaged BM-MSCs (n=1,000) were placed in each well of a 96-well plate, divided into three groups of 250 cells each, and were exposed to different doses of 17-beta-estradiol (0, 0.001, 0.01 nmol/L). ”This adds to 750, whereas the total number is 1,000. Were the remaining 250 considered the control group? You will have to state this clearly.
②　“the expression of genes of cytokine RUNX2” RUNX2 is not a cytokine. ① Please rewrite this sentence to correct the description.
③　“RUNX2 primer forward: GGTGAAACTCTTGCCTCGTC, reversed: AGTCCCAACTTCCTGTGCT。GAPDH primer forward: GGCTGCCCAGAACATCAT, reversed: ATGATGTTCTGGGCAGCC。” The description and punctuations used are confusion. Please revise it.
④　“the method described in previous studies (Jiang et al., 2020; Zhao et al., 2021, p. 5)” You have mentioned the page number here, whereas you have not in other citations. Please review and delete this.
⑤　“A multifunctional microplate reader was used to measure OD values.” Such abbreviations need to be explained specifically as the reader—even if he/she is an expert in the field would expect clarity.
⑥　Since the structure of MKX is simulated, the conclusion lacks credibility. Please provide specific data for the prediction information.
⑦　Please elaborate on the relationship between the 5 substances ( Pro-Tyr, 17-beta-Estradiol, MKX, SIN3A/HDAC1).


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

Input:
Title: IDENTIFICATION OF THE OCCURRENCE AND POTENTIAL MECHANISMS OF HETEROTOPIC OSSIFICATION ASSOCIATED WITH 17-BETA-ESTRADIOL TARGETING MKX BY BIOINFORMATICS ANALYSIS AND CELLULAR EXPERIMENTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: This study is properly planned but needs to be improved in several ways, such as-

Grammatical issues.

experimental planining
Experimental design: In the experimental planning, some of the things overlooked such as-
no information is provided about the homology modeling i.e.,
1. Template chosen for modeling studies.
2. Before docking study, the structure was validated or not and the structure minimization.
3. Docking studies were performed by AutoDock Vina Software, but not validated by redocking analysis.
4. The results obtained from the Docking analysis should be included in the table format.
4. The complex obtained by molecular docking was further needed to be analyzed by MD simulation studies and the results should be included in the paper.
Validity of the findings: the above mentioned queries needs to be addressed to further enrich the manuscript.
Additional comments: NA


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

Input:
Title: IDENTIFICATION OF THE OCCURRENCE AND POTENTIAL MECHANISMS OF HETEROTOPIC OSSIFICATION ASSOCIATED WITH 17-BETA-ESTRADIOL TARGETING MKX BY BIOINFORMATICS ANALYSIS AND CELLULAR EXPERIMENTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: These additions and corrections have been made. With these modifications, I recommend publication.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: IDENTIFICATION OF THE OCCURRENCE AND POTENTIAL MECHANISMS OF HETEROTOPIC OSSIFICATION ASSOCIATED WITH 17-BETA-ESTRADIOL TARGETING MKX BY BIOINFORMATICS ANALYSIS AND CELLULAR EXPERIMENTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: All the queries answered by the authors
Experimental design: N/A
Validity of the findings: N/A
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: FIRST REPORT OF FATTY ACIDS IN MIMOSADIPLOTRICHA BEE POLLEN WITH IN VITRO LIPASE INHIBITORY ACTIVITY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The present study entitled “First report of fatty acids in Mimosa diplotricha bee pollen with in vitro lipase inhibitory activity” by Phanthiwa Khongkarat and colleagues described the chemical composition of different floral bee pollen and the biological activities also been shown. Furthermore, naringenin was identified as a compound marker for Mimosa BP. Despite authors did some interesting work on these pollens, there still have some limitations for this study.
The presentation on the article is not sound and clear. There are too many redundant words in your abstract and introduction, make me sleepy. Only simple and concise writing for an audience lead to a clear understanding on the key information on your study.
Figures and tables must be self-explained, you used too many abbreviations in your tables, figures without necessary explanation. This also makes me puzzling for understanding your findings. I suggest you might take some photos of these plants, and showed them in your background, them give them explanation, and then define the abbreviations. These abbreviations also need to be explained as table note/figure legend.
Figure quality is low. You might chose professional software, like sigmaplot/prism, rather than excel to show the line chart. Some figure can be merged into a big figure, like figure 2,3. They are palynological analysis of pollens. Figure 7, figure 8 just showed chemical structure of known compound, there is no need to present them as separate figure. I suggest authors delete them or list as supporting information.
Authors indicated that “Thus, it can be concluded that naringenin was compound marker for Mimosa BP.” This is incorrect and makes no senses to me. As far as I know, naringenin is a common flavanone which has been widely reported in different types of bee pollen. FYI, I’ve listed some pervious publications, “Nano-liquid chromatography in nutraceutical analysis: Determination of polyphenols in bee pollen; Antioxidant activity of Sonoran Desert bee pollen; Impact of SchisandraChinensis Bee Pollen on Nonalcoholic Fatty Liver Disease and Gut Microbiota in HighFat Diet Induced Obese Mice.”
Other suggestions,
Abstract should be complete rewritten. You might concise it, make it up to 200 words.
Introduction, you need mention the biological activities of bee pollen, and explain the biochemical index you tested means what.
Methods, LN 139- Each BP was stored at room temperature (25 ℃) until used.
Please note that this storage of bee pollen is easily getting spoiled and fatty acids will be oxidized.
The morphology and 152 characteristics of the BPs were recorded and compared to the reported publications.-relative references is missing.
Results,
LN372 In order to confirm the plant origin of the BP, all samples were additionally identified by molecular analysis-suggest delete.
LN286 Thus, the six BPs were confirmed to be essentially monofloral, matching the principal flowers around the hives, and with the morphological and molecular analyses congruent with each other. -Such words can be moved to discussion part.
LN400- In this study, the inhibitory activity against.. DELETE
LN489- This is the first report that fatty acids in BP have a PPLI activity. In addition, naringenin can be used as a chemical marker for Mimosa bee pollen. DELETE. Also, such words should be toned town.
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: FIRST REPORT OF FATTY ACIDS IN MIMOSADIPLOTRICHA BEE POLLEN WITH IN VITRO LIPASE INHIBITORY ACTIVITY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English language used was intelligible, but the paper requires a major reconstruction in a way that the facts are logically presented. The background/context is not really stated properly and the research aim is not backed up by research gaps. Spelling errors were also observed as well as some grammatical errors. The author can easily address this by reconstructuring the introduction part and then clearly indicate the gaps of research.
Experimental design: The research question was not defined properly. a lot of information are lacking particularly the reagents and chemicals section, the model and brands for the equipment used. The authors had only performed one replicate extraction for all analysis. The authors has to include the said information and the justification on why they only performed one replicate for the extraction.
Validity of the findings: The authors had only performed one replicate extraction for all the assay analysis. the authors had also not indicated if whether the assays used were validated based on the ICH guidelines.
The result of the TLC analysis for naringenin has to be improved. include the Rf values of the standard and the unknown and if possible run it in a solvent of higher polarity so that the compounds will be sitting in the middle of the plate. the NMR analysis of the compounds are insufficient to prove that the compounds analysed were actually fatty acids. the fatty acid determination using the GC-FID is promising but i suggest that the authors should also add more verification for this. Quantification can also be done for the fatty acids so that the claims of detecting it in the be pollen samples can be more valid.
Additional comments: I commend the authors for using a bioassay guided isolation technique which is very helpful and useful to ensure that what ever secondary metabolite obtained is bioactive and of significance.
The abstract is very informative and had covered all the necessary information for a summary, however, it is too long. For the abstract, you do not need to introduce if what bee pollen is and can leave it to the introduction. You can start with the aim of the study instead. Also, you may not need to mention the other species of bee pollen floral source and focus only on Mimosa diplotricia.
The introduction is informative and have covered most of the information required but it has to be improved by presenting the facts more logically. Each paragraph must focus only on a certain topic until it builds up into the research gap and research aim. Furthermore, the introduction should add more information to justify if why the research needs to be performed (for example, the extent of research done on the bee pollen used in the study both in Thailand and abroad). You may also remove the details about what was done in the study and just focus on the discussion of the research gaps that prompted you to perform the research.
For the materials and methods, you need to include a section that indicates the chemicals and reagents (supplier, city and country) that were used in the study. Also, you needed to specify the name of all the equipment, model, city, and country where it was manufactured.
I have a question about the bee pollen, was it dried prior to storage at room temperature? And for ow long was it stored prior to use?
Have you performed an optimisation study on the solvent parameters used to extract the bee pollen? If not, you need to cite a reference for the ectraction method that you used.
How many replicates of bee pollen were extracted?
What were the concentrations used for the assay? this must be included in the methodology and not in the results.
Clarify the solvent used for the TLC analysis of naringenin. How much is the concentration of DCm and Methanol?


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

Input:
Title: FIRST REPORT OF FATTY ACIDS IN MIMOSADIPLOTRICHA BEE POLLEN WITH IN VITRO LIPASE INHIBITORY ACTIVITY
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript "First report of fatty acids in Mimosa diplotricha bee pollen with in vitro lipase inhibitory activity" is written in a clear and orderly way.  Supported by references on the subject. The information presented in the tables is clear and sufficient to show the results and the figures are of the necessary quality for publication.
Experimental design: The materials and methods presented are clear and adequate and presented in detail to be reproducuble by anothers.
Validity of the findings: The results are validate with the information generated and showed in the supplementary material.

The conclusions are connected to research and are limited and supported by results and experiments.
Additional comments: Line 90- change to "activities in"
Line 341 It is suggested to use statistical software for the determination of means and standard deviations.
Line 426. What does "reasonable" mean. Explain.


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

Input:
Title: FIRST REPORT OF FATTY ACIDS IN MIMOSADIPLOTRICHA BEE POLLEN WITH IN VITRO LIPASE INHIBITORY ACTIVITY
Review round: 2
Reviewer: 1

Output:
Basic reporting: English editing has to be done in order to have a more understandable and coherent paper.
Experimental design: The researchers has to indicate that the pollen samples used were dried using a specific process.
Validity of the findings: see above
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: FIRST REPORT OF FATTY ACIDS IN MIMOSADIPLOTRICHA BEE POLLEN WITH IN VITRO LIPASE INHIBITORY ACTIVITY
Review round: 2
Reviewer: 2

Output:
Basic reporting: Comments were answered
Experimental design: Comments were answered
Validity of the findings: Comments were answered
Only
Line 426. What does "reasonable" mean. Explain.

Authors We changed it to be “satisfying” instead. It means that this concentration was acceptable to use for the primary screening.

This "reasonable" or "satisfying" concentration  I suggest expressing in some kind of unit.
Additional comments: The authors of the article: "First report of fatty acids in Mimosa diplotricha bee pollen with in vitro lipase inhibitory activity", comprehensively answered the comments made 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: INVESTIGATION OF LIPOLYTIC ACTIVITY OF THE RED KING CRAB HEPATOPANCREAS HOMOGENATE BY NMR SPECTROSCOPY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article clearly describes the procedure to be followed to determine the hydrolysis of ester groups by NMR. However, I consider that it is necessary for you to describe more in the document, the excel sheet developed for the calculations and the methodology followed that they present in the supplementary material.
Include the article: https://doi.org/10.1016/j.rechem.2020.100063
On lines 48 and 49, cite the same reference, correct this.
Experimental design: Correct throughout the document ml by mL
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: INVESTIGATION OF LIPOLYTIC ACTIVITY OF THE RED KING CRAB HEPATOPANCREAS HOMOGENATE BY NMR SPECTROSCOPY
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript by Timchenko et al., discusses the use of NMR spectroscopy to analyze the lipolytic activity of hepatopancreas homogenate from the red king crab Paralithodes camtschaticus. The results are original and well presented and illustrated. However, some comments are made below.

-The authors state that little is known about crustacean lipases. However, although most were done in different Orders that do not include crabs, there are many reports on this subject. This should be clarified in the manuscript.

- Triacetin and caprylic/capric triglyceride are not commonly stored lipids in crustaceans or animals in general. Why did the authors select them for these experiments?

-Because medium-chain triacylglycerides are more abundant than short-chain triacylglycerides. What do the authors think is the biological significance of HPC homogenate showing high lipolytic activity against triacetin and moderate activity against caprylic/capric triglyceride?

-In the last paragraph of the Introduction section the full name of the species used as a study model should be written.

-The manuscript includes sufficient literature references, but many of them are  old and should be updated with more current citations. Some examples are:

Reference number 3 can be updated by:
Reis et al., 2009. Lipases at interfaces: A review. Advances in Colloid and Interface Science 147–148, 237–250.

References 6-10 can be updated  by:
Zaitsev et al., 2019. Biochemical aspects of lipase immobilization at polysaccharides for biotechnology. Advances in Colloid and Interface Science 272, 102016.

Navvabi et al., 2018. Novel lipases discovery specifically from marine organisms for industrial production and practical applications. Process Biochemistry 70, 61–70.

Kyung-Min et al., 2011. Lipase-catalysed synthesis of erythorbyl laurate in acetonitrile. Food Chemistry 129, 59–63.

-References number 4 and 5 should be relocated to the end of the following sentence: "Commercially significant lipases are usually obtained from micro-organisms that produce a wide range of extracellular lipases [4,5].

-Line 71: other reports on crab lipases should be considered

MichieIs et al., 2013. Effect of environmental salinity and dopamine injections on key digestive enzymes in hepatopáncreas of the euryhaline crab Cyrtograpsus angulatus (Decapoda: Brachyura: Varunidae). Scientia Marina 77(1). doi: 10.3989/scimar.03687.09D.

MichieIs et al., 2015. Lipase activity sensitive to dopamine, glucagon and cyclic amp in the hepatopancreas of the euryhaline burrowing crab Neohelice granulata (Dana, 1851) (Decapoda, Grapsidae).

-Lines 97, 118, 188, 212, 297, 314, 328, 337, 339 replace “red king crab hepatopáncreas” by it abbreviation (HPC).

-Line 145: add a point after .....”15 µl of HPC homogenate was added”

-Line 189: the abbreviation for triacetin (TA) appears for the first time. If this abbreviation is to be used, it should be clarified on line 79 of the Introduction section; otherwise, it should be removed.

-Lines 221-221: "followed by stopping the reaction by heating to 95 °C". This step of the protocol should be clarified in the Materials and Methods section.

-Line 226: replace "enzyme" by "homogenate".

-Lines 238-243 and 282-283 correspond to the Materials and Methods section.

-Line 244 and 261: the abbreviations for 1-monoacetin, 2-monoacetin and 1,2-diacetin have already been defined in lines 189-190. Use them on these lines and line 266.

- When the species is mentioned for the first time, the full name should be given. In the following mentions the name should be abbreviated. See lines 313 and 360.

-The captions of the supplementary figures are missing. In figure S4 it should be clarified which sample corresponds to which lane of the gel.

The format of the listed references needs to be revised and unified. Not all references are presented in the same format.

In captions of figures 2,3,4,5 and 7 the sign ° (°C) should be corrected.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No comment


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

Input:
Title: INVESTIGATION OF LIPOLYTIC ACTIVITY OF THE RED KING CRAB HEPATOPANCREAS HOMOGENATE BY NMR SPECTROSCOPY
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors described the activity of lipase present at the hepatopancreas of the red king crab Paralithodes camtschaticus. The use of NMR as the assay detection strategy is an increment in comparison to other methodologies previously described. The red king crab hepatopancreas is a waste of the crab food industry and could be a source of lipases with commercial value and industry application.  The results also suggest a difference of substrate specificty in comparison to other lipases. Although sequence analysis is not the aim of the manuscript, two literature articles present Paralithodes camtschatichus transcriptome data which could be useful to authors in order to evaluate lipase gene organization and some enzyme properties like molecular mass, pI, terciary structure prediction.

1-Transcriptome profiling and in silico detection of the antimicrobial peptides of red king crab Paralithodes camtschaticus.
Yakovlev IA, Lysøe E, Heldal I, Steen H, Hagen SB, Clarke JL.
Sci Rep. 2020 Jul 29;10(1):12679. doi: 10.1038/s41598-020-69126-4.
2-A crustacean annotated transcriptome (CAT) database.
Nong W, Chai ZYH, Jiang X, Qin J, Ma KY, Chan KM, Chan TF, Chow BKC, Kwan HS, Wong CKC, Qiu JW, Hui JHL, Chu KH.
BMC Genomics. 2020 Jan 9;21(1):32. doi: 10.1186/s12864-019-6433-3.
Experimental design: The experimental design in order to test NMR as an alternative methodology to lipase activity determination was addequate and the supplement material about activity measure was quite informative.
As a suggestion, the analysis of other substrates, including long fatty acid containing substrates and phospholipids. These data would allow the comparison of lipase data with lipases from other Arthropoda species and also could increase the potential industrial application of these lipase.
Validity of the findings: Despite the suggestions on experimental design and on the use of transcriptome data to increment the manuscript, the findings on Paralithodes camtschaticus hepatopancreas lipase and the improvement in lipase measurement activity using NMR contribute to lipase studies and waste potential application.
Additional comments: Although classified as minor revision (due to suggestions previously mentioned) and some English review   which could improve text fluidity, the article should be accepted by PeerJ.


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 OF LIPOLYTIC ACTIVITY OF THE RED KING CRAB HEPATOPANCREAS HOMOGENATE BY NMR SPECTROSCOPY
Review round: 1
Reviewer: 4

Output:
Basic reporting: The results reported are clear and unambiguous. However, I like to recommend a few corrections in English used.
In line numbers, 103, 126-127, 239, and 280, the authors have tried to add the citation as part of the sentence. That's not the right way. Please complete those sentences without the citation (keep the citation as a citation).
In line numbers, 130-131, it's not clear whether you recorded the NMR spectra or not.
In line number, 146, the sentence in parenthesis "the final concentration of triacetin was 70.7 mM" may be incorrect. Please check and correct it.
In line 159, it's not "To control". it is probably "As control"
In line 170, you stated that "the interval between scans was 10 s" Why such a long relaxation delay?
In line 178, the sentence "The delay time between the COSY pulses was 1 s" may be wrong. You might have meant to say that relaxation delay is 1 s. Change the sentence accordingly.
In line 185, what you meant by "at 10,000 g". Also, it's not "an NMR tube". It is "the NMR tube (in the same line).
In the following line (i.e 186, change "layer of a sample" to "layer of the sample".
Experimental design: no comments
Validity of the findings: no comments
Additional comments: No additional comments


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

Input:
Title: MOLECULAR GENETIC DIVERSITY OF SEAWEEDS MORPHOLOGICALLY RELATED TO ULVA RIGIDA AT THREE SITES ALONG THE FRENCH ATLANTIC COAST
Review round: 1
Reviewer: 1

Output:
Basic reporting: As correctly indicated by the tile this paper describes the molecular genetic diversity of seaweeds morphologically related with Ulva rigida at three sites along the French Atlantic coast.
The language needs improvement and I provide some (incomplete) suggestions in a file attachment.
The field background is correctly provided. A very recent relevant reference might be included (see below).
The article structure is correct and figures and tables as well. Raw data are shared, including even the R scripts that were used.
The results shown are relevant to support the conclusions made.
Experimental design: This paper presents and discusses original primary research data that are within the scope of PeerJ.
The introduction clearly describes knowledge gaps that are adressed and filled by this paper.
The methodology and the statistical treatment are rigorous and up to date, and described in detail to allow for replication by other researchers.
Validity of the findings: I have no doubt about the validity of the findings. Relatively large numbers of replicates have been analyzed at each of the sites that were investigated.
The underlying data are provided and their treatment is described with more detail than in many other similar studies.
The conclusions are clearly supported by the results and the authors take care to prevent overinterpretations.
.
Additional comments: Dear authors,
although this is not a criterion for PeerJ I wish to tell you that I found your study interesting...I feel the language needs some improvement and I provide a file attachment with some suggestions. However, please be aware that I am also not a native English speaker and thus not the best suited person to help in this respect.
A very recent publication that I think is relevant in the context of your work is Fort et al. (2020) in J Phycol (see  https://doi.org/10.1111/jpy.13079), who also studied (among others) samples from Brittany and found (limited) evidence for hybridization between U. rigida and U. laetevirens.
Florian Weinberger


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: MOLECULAR GENETIC DIVERSITY OF SEAWEEDS MORPHOLOGICALLY RELATED TO ULVA RIGIDA AT THREE SITES ALONG THE FRENCH ATLANTIC COAST
Review round: 1
Reviewer: 2

Output:
Basic reporting: This study examines the genetic diversity of green tide-forming Ulva along the Atlantic coast of France based on tufA sequence data from a large number of samples. The main conclusions are that these green tides are formed by four different taxa, including U. rigida, the introduced U. australis, U. fenestrate, and (possibly) U. pseudorotundata. In addition, some taxonomic issues are tackled, such as the synonymy of U. armoricana with U. rigida. The manuscript is generally well presented, although there is room for improvement.

The aim and main focus of the study should be stated more clearly. It is unclear if the study focusses on Ulva diversity in general, on diversity of green tides, or on the diversity within the morphotype U. rigida? If U. rigida is the focus of the paper, then the context of this taxon should be better explained in the Introduction. Now, U. rigida is only mentioned marginally in the Introduction. Along the same line,  the Material and methods section should state explicitly how sampling was performed: were U. rigida samples only identified in the field, or were identifications also based on microscopy in the lab? Were only free floating (green tide-forming) individuals sampled, or also individuals that were attached to the substrate?

The morphology and taxonomy of genus Ulva has been extensively studied in Europe in the 1960-ies to 1980’ies (papers by Bliding, Koeman, …). This should be mentioned in the Introduction. Now, only the statement “Taxonomic studies of this taxon are mainly based on molecular phylogenies” is made, which is clearly incomplete.
Experimental design: The authors should state more clearly how samples were identified (only in the field, or also based on microscopy) and sampled (only free floating/green tide forming Ulva individuals, or also attached individuals). Additional sampling parameters should be provided, such as intertidal/subtidal, coastal distance that have been sampled, etc.

Only a single marker is used in this study, tufA. In the Introduction, the authors argue that “most Ulva species have been identified on tufA”. However, many recent studies have used data from additional markers, including rbcL and ITS rDNA. In addition, sequencing studies of historic type material have mainly relied on the rbcL gene (Hughey et al 2020). It is therefore unfortunate that this study only includes tufA data. I believe a better argumentation should be given on why only tufA was sequenced, or preferably that additional markers are sequenced, at least for a selection of samples based on the different tufA haplotypes.

It is unclear how species clusters were delimited. In the Discussion it is briefly mentioned that some kind of threshold between intra- and inter-specific genetic distances was used, and the General Lineage Concept of species is mentioned, but details of these analyses are lacking. This should be clearly explained in the Methods and Results sections.
Validity of the findings: Details on species delimitation methods should be provided and reported in the Results section.
Additional comments: See 1. Basic reporting.
In addition, there are numerous language issues, so the entire manuscript should be carefully checked for spelling and grammatical errors.


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

Input:
Title: MOLECULAR GENETIC DIVERSITY OF SEAWEEDS MORPHOLOGICALLY RELATED TO ULVA RIGIDA AT THREE SITES ALONG THE FRENCH ATLANTIC COAST
Review round: 1
Reviewer: 3

Output:
Basic reporting: This study aims to provide a detailed picture on the  molecular diversity of leaf-like Ulva species associated with green tides along the French Atlantic coast. One of the main findings of this study is that five tufA haplotypes which were allocated to four different species are present at green tide hot-spots.
Furthermore, the authors including historic herbarium specimens to underpin the synonymity of Ulva armoricana and Ulva rigida. The manuscript is generally well written and illustrated. However, the main figure (figure 2) would benefit from larger fonts for the bootstrap values and from highlighting the respective discussed clades (haplotypes 1-5).
Experimental design: Ulva species are common and widely spread around the globe. In most of the cases they find their vegetation peak during the summer months. It is therefore unfortunate that only data from early spring (January/ February) is provided. However, the restricted sampling remains unexplained by the authors. In general the study is based on a solid sampling size (360 specimens) but excluding seasonality could be problematic due to green tides having their main abundance during summer (June-August).

One of my main concerns is that the methodologies for species delimitation are not explained in detail. The authors state that species delimitation is based on molecular data, but do not include distinct delimitation methods such as GMYC models to break down difficult relationships like within the Ulva rigida-laetevirens cluster.

Another concern is that only one marker (tufA) has been used. However, as correctly explained by the authors  in the introduction, data sets are in most of the cases either built on rbcL or tufA which makes comparisons difficult. Furthermore, the tufA gene is around 770+ bp long but the data set used in this study has been trimmed to 500 bp. The cut-off of 1/3 of the sequences is not explained by the authors.
Validity of the findings: As stated above, the study would have largely benefited from including a seasonal sampling in summer when green tides have their peaks. Additionally, the proposed synonymity of U. rigida and U. armoricana should be done in a formal way (naming homotypic and heterotypic synonyms, including morphological characters etc.). However, due to uncertainties of the taxonomic status of U. rigida and U. laetevirens I find it hard to base a synonymisation on tufA sequences only and would rather suggest to combine several markers or genome sequences/ cytoplasmic genome. The status of U. rigida/ U. laetevirens has been intensely discussed by Fort et al 2020 (https://onlinelibrary.wiley.com/doi/abs/10.1111/jpy.13079).
Additional comments: Introduction

Line 49: ” These species are considered as pioneer and opportunistic organisms”. Perhaps better: “some of their representatives….” since several Ulva species do not tend to proliferate.

Line 50: The genus in general has a global distribution, not all Ulva species.

Line 58: Be more specific here. The tragic deaths were not caused by the algal mats degradation itself but by its toxic gases that were released.

Line 80: I disagree. The largest database for genetic identification within the genus Ulva is still based on the rbcL gene. However, as explained below, tufA offers a more “reliable” marker.

Line 82: I agree. However, you are using a smaller fragment (~500bp) of the tufA gene in this study even though the gene itself contains 770+ bp. Why is the used fragment trimmed so much? You are losing nearly a third of information here.

Line 86: “This paper aims…” perhaps better “Our/This study aims…..”

Material and Methods:

Line 99: The sampling is restricted to spring only. Since it is known that the main green tides occur during the early to late summer months (June-August) it is doubtful that the sampling reflects the summer conditions (e.g. species appearance, amount, abundance). That the specimens were in early life-stages gets also reflected by their rather small size (line 104, 7cm).

Line 119: Can you explain why you designed new primers? Please state the length of the amplicon in comparison to Saunders and Kucera 2010. Furthermore, the table says the amplicon length is 800 bp why is the analysis performed on trimmed sequences of 500 bp lenth only?

Line 128: As stated in the introduction it is a pity that either tufA or rbcL sequences are available per data set. This study would have largely benefited from including rbcL sequences as well. By including another marker like rbcL this study would have had a more global impact and could also be connected to past results based on rbcL only.

Line 136: Please state the length of your amplicon in comparison to the significant longer reference sequences.

Line 144: How was the evolutionary model determined?

Line 148: Typo - Ulva compressa

Line 148: U. compressa as well as U. intestinalis have been found as leaf-like as well and are not obligatory tubular. Furthermore, both species have been found as essential parts of green tides.

Line 152. Why was no GMYC method applied. You are presenting the haplotypes here but it would be nice to have an estimation of the haplotypes per species which could be perfectly matched with a solid GMYC analysis. This would also give more insights into the species delimitation of the U. rigida/ U. laetevirens case.

Results:

In general I find it hard to follow the results. It would be easier if you would mark the respective clades which represent the haplotypes discussed in the text with a dashed box in figure 2. Although you are giving the numbers of sequences of one clade in brackets, it is not really clear if those sequences are the ones listed in table 1 and 2? Additionally, why is only one phylogenetic method used in the phylogenetic tree? I suggest to include at least the Bayesian method as well. The bootstrap values are hard to read.
Additionally, it would be great if morphological data on the sampled individuals would be made available. Is the longer alignment showing differences in the phylogenetic analysis compared to the trimmed (500bp) alignment?

Line 179: Typo – File

Discussion:

Line 205: The history of U. rigida/ U. laetevirens is nicely presented. However, Fort et al. 2020 (https://onlinelibrary.wiley.com/doi/abs/10.1111/jpy.13079) should be cited and discussed in detail. Your and above mentioned study are in a way contradictory when it comes to the species concept of U. rigida/ U. laetevirens. This could be problematic when suggesting the synonymy of U. rigida and U. armoricana.


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: MOLECULAR GENETIC DIVERSITY OF SEAWEEDS MORPHOLOGICALLY RELATED TO ULVA RIGIDA AT THREE SITES ALONG THE FRENCH ATLANTIC COAST
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have adequately revised their manuscript according to my previous comments. I don't have any further fundamental concerns. The text itself is still rather sloppy with numerous errors. Below is a non-exhaustive list. The authors are encouraged to carefully go through the entire text to correct spelling and grammatical errors.

Line 34 ‘genetic analysis at tufA’ -> of tufA
Line 108 ‘This intriguingly questions’ -> These intriguing questions
Line 112 ‘The conspecifity with U. rigida’ -> conspecificity
Line 185 ‘individual numbered plastic bag’ -> individually numbered plastic bags
Line 348 ‘and could not be further used to described’ -> to describe
Line 351 ‘However, these sequences should be temporary considered’ -> temporarily
Line 359 ‘As Areschoug did not designated’ -> designate
Line 469 ‘sequences of specimen collected at Roscoff’ -> specimens
Line 508 ‘on the basic of morphology’ -> on the basis of
Line 543 ‘U. stipiata’ -> stipitate?
Line 583 ‘Fort et al. (2020b) promoted the use cytoplasmic complete genome’ -> use of
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: MOLECULAR GENETIC DIVERSITY OF SEAWEEDS MORPHOLOGICALLY RELATED TO ULVA RIGIDA AT THREE SITES ALONG THE FRENCH ATLANTIC COAST
Review round: 2
Reviewer: 2

Output:
Basic reporting: As reported by the title, the manuscript aims to identify the molecular diversity and taxonomic relationships of foliose Ulva species associated with green tides in France. However, the results are based on 3 sampling sites only and therefore the title “Molecular genetic diversity of the macromorphological taxon Ulva cf. rigida along French Atlantic coasts” is somewhat mis-leading.
A special focus has been placed on the species Ulva cf. rigida. The study describes the molecular genetic diversity of seaweeds morphologically related with Ulva rigida at three sites along the French Atlantic coast.

As the main finding of the study, the authors highlight the presence of five tufa haplotypes which were delineated into four species by phylogenetic techniques. The manuscript appears correctly structured and figures and tables present the main results. The text and language has improved but still needs improvement and I suggest to have it revised and checked by a native English speaker.
Experimental design: Although, the authors give reasons why sampling was conducted in winter/spring only, it appears to me that an additional sampling during vegetation-maximum (summer/early fall) would have largely benefited the study. With the present dataset the authors can verify the samples as “present at green tide locations” but that the species found are involved in green tides can in my eyes not be validated by the present study.

Although, the authors now state that they used the ASAP method for delimitation purposes it appears unclear to me on which basis the clusters are defined? What is the cut-off to delimit a cluster/species? Why is U. rigida and U. laetevirens defined as separate species but as common cluster?

Although, the authors state that tufA is the marker of choice to apply for species delimitation and identification in green algae the available datasets for rbcL contain more typus sequences. The dataset would still largely benefit from including rbcL as well.
Validity of the findings: One of my main points is the application of names to clades and species clusters. The authors correctly highlight clades which were identified with holotypes/Lectotypes, however every single cluster or species that is not validated by the molecular validation of a lectotype should be stated as such and identified by cf.. E.g. Ulva cf. linza etc.. For this see also the latest literature of Hughey et al. (as cited in your manuscript).

The conspecificity/ non-conspecificity of U. rigida and U. laetevirens should be explained more precisely and in more detail. Although, you emphasize that those are two different species I suggest to include the reasons why they are often regarded as one species (see also Hughey et al. 2020; cited in your manuscript).There are several more literature evidences which regard both species as conspecific, especially due to the biological ability to form fertile hybrids.
Additional comments: Line 150: Why ca.? Give exact number

Line 160: Name the ID of the holotype and provide herbarium code

Line 167 pp.: When focusing on the Jan/Feb period, and previously mentioning that green tides are more prevalent in the summer/fall time, how could you make sure the individuals taken at these sites really tend to proliferate?

Line 181: Please explain. Do you mean you disregarded this other criteria?

Line 231: U. compressa is not obligat tubular, neither is U. linza, nor U. intestinalis (monostromatic occurrences in Baltic Sea tend to form green tides [Bäck, Saara, Annamaija Lehvo, and Jaanika Blomster. "Mass occurrence of unattached Enteromorpha intestinalis on the Finnish Baltic Sea coast." Annales Botanici Fennici. Finnish Zoological and Botanical Publishing Board, 2000.]. Rephrase.

Line 264: “private” rephrase. Perhaps something like solely found at…

Line 283: Rephrase “our haplotypes”

Line 283 pp.: Since the manuscript aims to clarify taxonomic relations I would suggest to include cf. in the species names where no “clear” identification of the clade is possible. Thus, I suggest to use this consistently in all entity names which were not validated by genetic examination of the type (Holotype, Lectotype …).
Additionally I would rephrase the presentation of clades in the results. I agree, that clades which only contain sequences from one “species” can be named accordingly (see the cf. situation above), however perhaps include “identified as”.E.g.: “The first one contains 68 sequences identified as U. laetevirens”. This leaves it open for the discussion to discuss the correct taxonomic affiliation.

Line 283: Why do you delimit 4 and not 5 clades?

Line 312: Why Ulva specific? There is no evidence they do not align with other green algae as well?

Figure 1: The first graph of France looks warped. Adjust the CRS layer. (If done in QGIS or GIS you can use the function”set layer from CRS” by right clicking on the map)

Figure 2: Caption  “…gene chloroplastic gene.” Delete one “gene”

In general it is hard to really identify the clades on names and I would adjust this (except for the clades containing a holotype sequence) by adding a cf.. For sure, there will be changes in the future when more type material gets sequences, thus it enables for keeping track.


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: SMALL-SCALE DRIVERS ON PLANT AND ANT DIVERSITY IN A GRASSLAND HABITAT THROUGH A MULTIFACETED APPROACH
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article documented differences in vegetation and ants between two closely placed sites, which differ considerably regarding environmental conditions (flat area with deep soil layer vs. southern slope with shallow soil). It was a good idea to examine two species groups: both ants and vascular plants. The examined habitat (semi-natural grasslands) are among the most important for biodiversity maintenance,  and simultaneously, strongly endangered habitats in agricultural landscapes of the European Union. The article is well written, the discussion is supported by results. The quality of figures and tables is appropriate
Experimental design: The topic is within the journal scope. The research question were well defined, the data were collected using up-to-date methodological approach, and the stastistical methods of analysis were correct.
Validity of the findings: The data are restricted to a single location only, and the significant effect of microrelief on vegetation structure and ants communities is already known. Because of examination of only one location, it is unclear to what extent the observed pattern is idiosyncratic. Moreover, there was no attempt to assess the magnitude of differences in ecological conditions (e.g. thermal conditions, moisture, soil parameters) between the two studied sites, except for soil deep. It caused that some of the conclusions, underlying effect of local differentiation within a habitat, are less useful. It is hard to assess how the contrast between microsites was strong, and to what extent the obtained results can be compared with studies from other sites/regions. Complementing the examination by measured or modeled environmental variables and including it in the analysis will increase the value of the research and allow to write a more specific discussion. Additionally, the observed pattern emerges not only from differences in landrelief but also from management intensity (Discussion: lines 307-310). It would be better to find study sites under similar management type and intensities.
Additional comments: Additional comments
•	Line 43 – it is not so easy; see Diekmann et al (2019)
Diekmann, M., Andres, C., Becker, T., Bennie, J., Blüml, V., Bullock, J. M., ... & Wesche, K. (2019). Patterns of long‐term vegetation change vary between different types of semi‐natural grasslands in Western and Central Europe. Journal of Vegetation Science, 30(2), 187-202.
•	Line 53. The management type is management, not an environmental factor. I suggest not confound it, especially that in management type its intensity, not only type, is important.
•	Line 83. How did You define ‘the ecological status of ecosystems’ here? This term is used rather in hydroecology. It sounds nice, but what it means exactly?

Materials & Methods
•	Line 137: ‘steeper slope’ –describe it using inclination in degrees, please.
•	Line 198. Does the data fulfill the ANOVA assumptions (e.g distribution, variance homogeneity)
•	Line 200. Provide a reference for the StrateFy tool

Results.
•	The map of local landrelief and sampling plots location should be provided
•	A brief description of the taxonomic diversity of studied communities will be helpful for the readers (e.g alfa and total diversity). Also, it will be good to add in the supplementary materials the rarefaction curves, for eco-sites and taxonomic groups.

Discussion
General comments. In the discussion, the effect of slope exposition is omitted. As is described in Materials & Methods, the Rocky area was places on southern slopes. It could be assumed that it considerably changed thermal conditions of compared sites, as well as influence the water conditions (higher evaporation on southern slopes). This issue should be discussed. Moreover, there is correlation between landrelief and management, thus its hart to divide the effect of the environment from management influence. This issue should be discussed.

•	 Lines 269-270. I guess that the differentiation between the eco-sites was also easy to indication using the digital terrain model and its derivatives
•	Line 304. What did You mean as ‘abiotic fraction’ of soil?
•	Lines 311 – 313. The second part of the sentence repeats information from sentence in lines 295-298
•	Lines 315 – 319. It seems that the sentence is contradictory to massage from lines 295-298. Can You explain it in more detail?

•	Lines 319-324 The plant height is a trait of vegetation (plants) and can not be shown as a variable describing the soil-plant interface. What do you mean by ‘habitat cover’? Moreover, I think that it will be better to not consider microrelief with soil. It could be considered as two different groups of factors

Conclusion.
The two first sentences, provides generally the same information. Thus, one from them is redundant and should be deleted.

Supplementary materials
The supplementary materials (excel file) should be prepared more carefully. There are no full names of ant species, only abbreviations (contrary to plants), lack of units in plant and ants traits, as well as environmental variables.
If the plant species cover from the ‘plant_species’ sheet is summed up, the value differs from the herb cover showed in the ‘environmental_var’ sheet. If you estimated the plant species cover with 0.1% accuracy, these values should be more or less similar. Is it OK?
Photographs documenting the general appearance of vegetation/habitats in bot studied eco-sites will be helpful.


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: SMALL-SCALE DRIVERS ON PLANT AND ANT DIVERSITY IN A GRASSLAND HABITAT THROUGH A MULTIFACETED APPROACH
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this work, author measured taxonomic and functional diversity of plant and ant communities at two semi natural dry grassland ecotypes, evaluating the differences among the two ecosites, how these differences are influenced by the environment and whether vegetation affects composition of the ant.
Experimental design: 156-157. How did you estimate species abundance? Visually, using a grid? I would change a bit this sentence since to estimate vegetation cover at the 0.1% is not an easy job.
160. On how many individuals per species did you measure SLA? Also specify here.
Validity of the findings: Despite the limited sampling strategy and the small area, the work is valid and well written and well designed, while statistical analyses are appropriate.
Additional comments: Minor comments
164. “traits of plants” or better “traits of plant species”. In any case from here and from the results it’s not clear the number of species on which you performed the measures. Please, specify.
Fig. 1/Results. Finally, I would add two plots describing the difference in alpha (richness) and beta diversity between the two ecosites. In addition, at the main difference in species composition should be shortly described at the beginning of the Results. You partly describe such aspect in the discussion but no mention in the result


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: SMALL-SCALE DRIVERS ON PLANT AND ANT DIVERSITY IN A GRASSLAND HABITAT THROUGH A MULTIFACETED APPROACH
Review round: 2
Reviewer: 1

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
Additional comments: I would like to thank the Authors for their careful reply to the comments and changes made in the manuscript. However, I can not agree with the statement that ‘measuring other environmental variables in the field a posteriori is not possible, since the communities were sampled in 2019 and ecological conditions might be changed’. It is hard to believe that soil pH, insolation, and water conditions will change within 2 years period. A simple, yet meaningful quantification of environmental variables (e.g. stable water conditions, insolation) can be obtained from digital elevation model, without any additional field works. Nice examples You can find in articles of Moeslund and al. (2013) and Kopecký et al. (2021)

A Moeslund, J. E., Arge, L., Bøcher, P. K., Dalgaard, T., & Svenning, J. C. (2013). Topography as a driver of local terrestrial vascular plant diversity patterns. Nordic Journal of Botany, 31(2), 129-144.
Kopecký, M., Macek, M., & Wild, J. (2021). Topographic Wetness Index calculation guidelines based on measured soil moisture and plant species composition. Science of The Total Environment, 757, 143785.


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: SMALL-SCALE DRIVERS ON PLANT AND ANT DIVERSITY IN A GRASSLAND HABITAT THROUGH A MULTIFACETED APPROACH
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comments
Experimental design: No comments
Validity of the findings: no comments
Additional comments: I found this version of manuscript really improved according to the reviewers' 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: LINKING INDIVIDUAL AND POPULATION PATTERNS OF ROCKY-SHORE MUSSELS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is an interesting paper which addressed an existing knowledge gap of within site variability. As the authors disclaim well in the concluding section however this study fails to address the environmental drivers which are responsible for this. These data and information are worth of publication nonetheless and provide a solid foundation for further necessary work.

I have some minor suggestions on the text, that is overall well presented

L27 which scales
L39 what is relatively constant crowding

L64 some definition or at least reference for engineers is needed otherwise this can be omitted

L79 add a reference
Experimental design: I believe all of the data collection was appropriate for the research hypotheses
Validity of the findings: Well written and discussed
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: LINKING INDIVIDUAL AND POPULATION PATTERNS OF ROCKY-SHORE MUSSELS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This article (Linking individual and population patterns of rocky-shore mussels) describes an attempt to link individual and population-level processes in rocky-shore mussels, based on various parameters measured over a year in one site. The data and the general goal of linking two levels of organization are interesting. The study, however, is essentially descriptive, and it lacks clear hypotheses or research questions. The Introduction, for example, is mostly a recapitulation of the reasons why rocky-shore studies are powerful. I am missing the knowledge gap, and some statement about the contribution that this study is providing. Below you will find comments and suggestions that can hopefully help you make these missing elements more visible.
Experimental design: The design seems appropriate to examine variation of individual and population level responses across time. As I clarify in my comments below, there are several points that need clarification.
Validity of the findings: The findings, as described in the Results, are valid. The Discussion does a good job at interpreting the results in the context of earlier research in the subject.
Additional comments: L33-34: the distinction between 2 rocky shores is a bit bleary. It seems they are 2 areas/plots within a site. I suggest referring to them as plots. Also, instead of referring to ‘the site’, for which we have no information, you can say ‘a site’ or ‘one site’.
Abstract: the abstract explains poorly the specific goals of the study and the results. You mention things like contrasting patterns, but at the end of the paragraph, it is unclear how these patterns help explain the link between the individual and the population levels. I suggest reworking the Abstract with a clearer emphasis on the research question.
L53-59: It is important to specify precisely the spatial scales you are studying here. You first talk about ‘scales ranging from 1 cm to 1 m’. Then you mention ‘hundreds of meters’ in the same context.
L62-67: This sentence can be simplified. Try avoid repeating the concept of engineer, and clarify what you mean by ‘conditions the development’.
L67-70: Unclear sentence. What is it that ‘varies’? The mussels? Their aggregation structure? And what do you mean by ‘structure’ as used in the previous sentence?
L83-107: These two final paragraphs of the Introduction are key for setting the scene of the study. However, I am missing important elements, which makes it difficult to understand why the work was done. As is, you give the impression that the study was a description of patterns at two scales. To strengthen the paper, I think it is key to specify the knowledge gap. Then, if possible, I would love to see some testable hypotheses. We know already that the rocky shore is a useful system to understand mechanisms behind species responses to biotic and abiotic factors. I think the manuscript could go beyond this, for example, by stating predictions about the strength of association between different individual and population level responses.
L118: Were the tide heights really measured? Or did you work with predictions? Please provide a source for these data.
L120: We need more details about how you determined bathymetric position. Did you use a surveyor equipment to measure this accurately for both rocky shores? Did the height differ between the two shores? Also, how did you link the tide data and the measured shore level? Did you validate it in situ?
L124-126: Its unclear what the different bathymetric positions are. Did you use 3 quadrats to represent the entire vertical gradient? Or three quadrats per shore level?
L127-128: Replace ‘picture of’ by ‘picture’. And what do you mean by ‘manual delimitation’?
L144: The larger animals were 0.5 cm? Seems really small.
L151: Did you really dry them at 160 degC? Rather 60 degC?
L166: Indicate the number of individuals used.
L172: Unsure what you mean by ‘fully covered area’. Why should recruitments on bare rock be excluded?
L200: Replace ‘An MA’ by ‘A MA’.
L205-214: The description of the ELEFAN model is clear; however, it is unclear how you produced the data used to parameterize the model. You had 17 animals of different size per shore. What was the raw data extracted from the animals? Did each animal provide 1 datum per growth ring interval, i.e., lengths vs growth rate? Please clarify.
L231-234: I got confused because you previously say that the data considered the variability in mussel cover across quadrats, but then you state that the estimates correspond to a full covered quadrat. Please clarify.`
L240-238: The terms ‘visually covered’ and ‘covered’ are equivalent. Please propose better descriptors for the two terms. I looked in the reference you provide, but the formulation of ‘crowding’ is different than yours. Please use a suitable reference.
L246-250: Extremely long sentence.
L255-256: The sentence ‘In addition, we tested for differences between shores; temporal variation; and the effect of adult density on the spatial distribution of recruit density’ seems unnecessary, given that you describe those effects bellow.
L266: I’m confused why you use a package for linear mixed-effects models to fit GLMs.
Also, why don’t you cite the references for only some of the packages used?
L285: ‘Immersion percentage’ is a bit misleading. Please specify that you are talking about time.
L209: The word ‘exposition’ does not add to the sentence.
L299-301: Please specify what you mean by significantly lower. Lower than which months? Perhaps simply describing them as the period of lowest values would suffice.
L313-315: The effect of the interaction between adult density and shore on the recruitment density is also interesting to examine. It seems that the slopes are crossing, suggesting that the effect of adult density depends on shore.
L313: Instead of saying ‘regardless of which shore was considered’, I suggest ‘at both shores considered’.
L319: Can you specify the date of this second increase?
L325-327: As mentioned in the M&M, it is not clear if you used the sclerochronology data to derive the von Bertalanffy growth curves. Here it sounds as if the curves emerge from a different dataset, while in Fig. 6 caption it sounds as if they were originated from the schlerochronology data.
L337: Standardized to what? Area?
L346: For every reference of bathymetry, you need to be careful about the direction. Bathymetry implies a depth, not a height. Is this how you treat them here? Is that how you treat it in Figure 3? On the intertidal zone, we usually speak about height above the mean lower water mark. Can you clarify?
L348: Why do you refer to ‘median length of individuals’? I imagine that each point in Fig. 8E corresponds to an individual. Or are those really medians? You also provide the maximal length, so perhaps this is an estimate for several animals. In that case you need to clarify number of individuals used.
L391-392: Do you mean asynchrony between recruitment and spawning dynamics? I suggest you specify it.
L417: Here you refer to air or sea water temperature?
L428-431: You use the term ‘variability’ quite loosely. What do you mean by variability of microclimates, for example? Or, why would more variable performance allow a second spawning? Please revise.
Discussion: I think the Discussion was much clearer than the Introduction. I suggest you bring some of the elements from the Discussion into the Introduction. This could provide the base for testable hypotheses.
Figures: Try using the same color code to distinguish shores across figures.
Fig. 3: One of the points is surrounded by a blue halo. Please indicate the reason for this.


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: LINKING INDIVIDUAL AND POPULATION PATTERNS OF ROCKY-SHORE MUSSELS
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment, see below
Experimental design: no comment, see below
Validity of the findings: I do not see a statement of data availability in the manuscript. Presumably the original data will be archived in an appropriate publicly-accessible repository. The original data are not available for review with the manuscript.
Additional comments: I have reviewed the manuscript and find it generally well written and describing a substantial data set. I have some points of clarification that I would like to see addressed, and some corrections made to the text and figures. Below I list three main concerns/suggestions, and then a series of points addressed by line number.

1.	I’ll start with a possibly embarrassing question: What species of mussel was being sampled in this study? Based on the location I would guess Mytilus edulis or Mytilus galloprovincialis, but based on searching for the terms “Mytilus” or “edulis” in the document, I can’t actually find an explicit listing of what species was present at the site.

2.	For the haphazard quadrat placement (lines 122-128), please provide more description of how the quadrat placement proceeded. A total of 3 quadrats per site per month was being used to characterize the entire shore height gradient at these sites, and presumably there is some variation in mussel bed patchiness at the small scale (centimeters to meters) that affects the various measurements being made. Was there any pre-filtering of potential quadrat placement sites, based on characteristics such as requiring some minimal number of mussels being present (i.e. excluding shore heights above or below the extent of the mussel bed at each site that would be bare of mussels, or excluding patches within the mussel zone that might have no mussels due to other characteristics such as being in a deep crevice)? Did the sampled shore height distribution differ at a site across time?

3.	I would appreciate the addition of a supplementary table with the coefficient estimates provided for each of the final LM, GLM, and LMM models. The manuscript currently includes tables of p-values, but not the actual coefficient estimates that are used to represent the various slopes shown in the figures.

Line 57: The Denny et al. 2011 reference would also be appropriate here where you discuss the within-site vs. across-latitude temperature ranges.

Lines 79-82: The discussion here of how environmental conditions might affect differential growth leaves out the potentially very important role that food plays in stress tolerance and growth. This paper makes the argument that shore height (bathymetric gradient) will play a role in mussel success, and that immersion time might be nearly doubled from the lower to upper reaches of the mussel zone (34% - 61%), which must affect time available for feeding, and thus access to food. The authors should discuss the potential interactive effect of food supply and other environmental stressors. A relevant reference on the importance of food ration for tolerating environmental stress would be Fitzgerald-Dehoog, Lindsay, Jeremy Browning, and Bengt J. Allen. "Food and heat stress in the California mussel: evidence for an energetic trade-off between survival and growth." The Biological Bulletin 223.2 (2012): 205-216.

Line 287: Either here, or in the Methods, please list what the local reference datum is for tide heights (is it mean low water?, mean sea level? etc.) and also what the full tidal range is (i.e. 0-6m). This will give the reader some frame of reference for what these shore height measures mean.

Lines 286-288: Based on my reading of the methods, I’m guessing that these estimates of immersion percentage are based only on still water levels, rather than incorporating wave run-up and swash. Please state explicitly whether any allowance for wave action is incorporated in these estimates, or if they are solely based on still water level. Based on the site orientation, I would assume that the East site is more directly exposed to ocean swells and might experience more splash and wave run-up.

Line 294: The line here says that East shore samples were almost perpendicular to the east-west axis. Based on Figure S1, should this line instead be almost perpendicular to the north-south axis?

Line 355: The text states that crowding index is negatively related to median length, but looking at Figure 8E it seems that the plane trends positive with increasing median length. This is an example of a case where having an additional table of the regression coefficient estimates would be helpful in checking what the text says.

Line 463: I would recommend replacing ‘fixed’ with ‘sessile’ here.

Line 489: The word “exondation” here could be translated to “emersion” to indicate the period of time when the mussels are expose to the air and not submerged by water.

Figure 2: The diagram here and the discussion Lines 436-479 seem to represent these beds as single-layer mussel beds, rather than multi-layer beds where top-layer individuals are not directly anchored to the rock substratum, but instead grow anchored on top of other adult mussels. Is that an accurate interpretation of the discussion and description of the mussel beds at your field sites? Are the mussel beds at this site limited to a single layer? Building multi-layer beds allows for an expansion of aggregation size without needing an increase in available substratum space, but the discussion seems to exclude this as an option for the populations at these sites. Mislan and Wethey 2015 present the case where a multilayer bed provides benefits to some mussels for avoiding heat stress. Mislan, K. A. S., and David S. Wethey. "A biophysical basis for patchy mortality during heat waves." Ecology 96.4 (2015): 902-907.

Figure 3: The axis title on the right side should say “altitude” rather than ‘altitud’

Figure 5: The units shown on both axes should be m^-2, not m^2

Figure 8A: biomass axis title should be m^-2, not m^2

Table S1: Correct spelling of CONDITION INDEX


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: LINKING INDIVIDUAL AND POPULATION PATTERNS OF ROCKY-SHORE MUSSELS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Review PeerJ-60692_v1

The authors of this manuscript have addressed all of my previous comments. I only have a few minor comments listed below for your consideration.

L29: 'exhibited stability pattern' is unclear. I also think that the word 'compensation' in line 30 is ambiguous, as it implies an active response. I don't think, for example, that mortality, which is mentioned in the following sentence, is a compensatory response.
L123: I don't agree that the quadrats were triplicates because they were placed on different shore levels.
L144: I believe is 'water height measurements' instead of 'measures'. Same comment applies elsewhere.
L199-200: Modify to 'where the sine wave oscillation begins'.
L296: Unclear what 'CI' stands for.
L298-300: Why did you only apply the post-hoc test when the interaction term was significant? Where you not interested on single term pairwise comparisons?
L454-455: You suggest that your data showed the presence of atresia, but that is not something you measured.
L474: As mentioned above, the term 'compensation' implies an active response. I suggest something like trade-off.


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: LINKING INDIVIDUAL AND POPULATION PATTERNS OF ROCKY-SHORE MUSSELS
Review round: 2
Reviewer: 2

Output:
Basic reporting: See below
Experimental design: See below
Validity of the findings: See below
Additional comments: I have reviewed the revised version of this manuscript, and I am satisfied that the authors have addressed my original concerns. The additional statistical results and parameter estimates given in the new supplemental tables are helpful. I have only a few minor comments on wording in the new version.
Line 116-117: This could be phrased more clearly as “Mussel patches consisted of only one layer of individuals in the study area.”
Line 126: Replace “coverture” with “cover”


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: THREE NEW SPECIES OF BYRSOPTERYX FLINT MICROCADDISFLIES FROM PERU (INSECTA: TRICHOPTERA) INCLUDING DNA-BASED LARVAL ASSOCIATIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: English is clear and unambiguous, easily understood throughout the paper.

Sufficient background and context is provided, literature references are appropriate.

Article structure is appropriate for this field, all figures and tables appropriately included. I would actually highly recommend one additional figure: a photo of the forewing showing the semi-dome modification described by the authors, in addition to the already included illustration. This morphological feature has not been observed in any other species in this genus, nor, to my knowledge, other genera within the family. A photo of the feature would be immensely helpful for other researchers and, based on the photos already included of genitalia and larvae, the authors should be capable of producing a photo of a cleared wing mount.

Results and conclusions are appropriate.
Experimental design: Methods well described, all aspects thoroughly detailed.

Research fills an identified knowledge gap, describing additional biodiversity for a hyper-diverse yet under-studied faunal group in a biogeographic region in need of further study. Observations are very complete, based on both morphological and molecular data, and including male, female, and larval diagnoses.
Validity of the findings: All underlying data have been provided and are being stored in the appropriate repositories.

Conclusions are appropriate in scale.
Additional comments: A very thorough description of three new species, a well-structured and well-written paper. My only recommendation would be the additional figure that I mentioned above, under 'Basic Reporting'.


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: THREE NEW SPECIES OF BYRSOPTERYX FLINT MICROCADDISFLIES FROM PERU (INSECTA: TRICHOPTERA) INCLUDING DNA-BASED LARVAL ASSOCIATIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Ref.:  Ms. No. 66094v1
Title: Three new species of Byrsopteryx Flint microcaddisflies from Peru (Insecta: Trichoptera) including DNA-based larval associations

The manuscript constitutes an important contribution to the caddisfly taxonomy, especially on Peruvian fauna. Following the proposed review criteria, I should highlight some points:
1-	The text is clear, and the English is completely understandable.
2-	The theoretical background is sufficient, and the references are updated.
3-	The structure and figures of manuscript are good.
4-	The objectives are clear, well defined, and meaningful; the methods are appropriated and can be considered innovative to taxonomy of the order; and the results are very relevant, including the molecular association of adults-immature stages.
5-	Corrections and suggestions are in the manuscript file.
The manuscript is well written, provides relevant information about the Peruvian caddisfly fauna and, consequently, makes an important contribution to the Neotropical caddisfly taxonomy. In this way, I strongly support the publication of manuscript in the PeerJ.

With best regards,
Experimental design: .
Validity of the findings: .
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: THREE NEW SPECIES OF BYRSOPTERYX FLINT MICROCADDISFLIES FROM PERU (INSECTA: TRICHOPTERA) INCLUDING DNA-BASED LARVAL ASSOCIATIONS
Review round: 1
Reviewer: 3

Output:
Basic reporting: (1) If this is the case, make it clear in the text that the larvae of B. mamaocllo sp. nov. and B. mancocapac sp. nov. do not differ significantly from other described (and illustrated) larvae of the genus. Otherwise, I believe it is necessary to include the illustration of the larvae in the manuscript, as not all the structures described can be seen in the photographs.
(2) To make the manuscript easier to read, I strongly suggest naming and indicating the main structures of the male genitalia in the figure of at least one of the new species.
Experimental design: (1) The method used to associate larvae and male adults was described in detail in the manuscript, but nothing was said about how the association of females was carried out. It is necessary to emphasize that all species described in this work occur in the same localities, therefore, it is not advisable to base the male-female association by co-occurrence.
(2) Describe in the material and methods for which structures of the male genitalia a terminology different from that of Harris & Holzenthal (1994) was adopted.
Validity of the findings: no comment
Additional comments: The work presents a clear and objective writing with small suggestions for changes. Major comments refer to elements that need further clarification to ensure that other readers can easily understand the work. The illustrations are in excellent quality. I congratulate the authors for their efforts to use different sources of information to associate larval and adult stages.