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: TRANSCRIPTOME AND STRUCTURE ANALYSIS IN ROOT OF CASUARINA EQUISETIFOLIA UNDER NACL TREATMENT
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript in english, which  nedds Some improvement. Some sentences are unclear.
TITLE :     Title should be changed. There is no elucidation of mechanism (no functional approach); It is just e description of genes regulated. last there is no real link done between the histology and the transcripto.
ABSTRACT: Conclusion should be modified

Introduction : Litterature is not recent enough. Intrduction should be reconsider by including more recent bibliography. There are lots of recent review and data on salt stress responses in plant.
Last the introduction focus on a list of genes ...but does not cite other transcriptomics studies neither the gene families that were modulated during salt stress. It should be added to compare if Casuarina equisetifolia has a similar response or not regarding salt stress.
See : Park etal 2016 Mol Cell Biol
Yu et al. 2020 TIPS
Farhat et al., 2019
van Zelm et al ARPB 2020
Morton et al 2019 Plant J
Liu et al 2019 BMC PB on populus (an other tree)

Structure of the manuscript is standard.

Figures are not good. Often too small and legends are not precised enough. See detailes comments.
Experimental design: Research question is unclear. Salt stress mecanisms in casurina equisetifolia are not precisely elucidated and not compared to not tolerant species. Thus difficult to see what makes this species more tolerant.
Considering technics, there are some weakness; The study is done with only 2 biological replicates (transcriptomics) and this is not the actual standard which requires 3 biological replicates.
Statistics are somhow inapropriate and should be reconsidered regarding to the low number of samples (non parametric tests).
I am also surprised of the sample clustering; Ah close to 6h = OK; 24h clustering with 168h (7days)...I am quite surprised. Thsi could be due to the low number of replicates and to a weakness of statistic methods; Please you may check.
Validity of the findings: The subject is oruiginal but should be more exploited in the discussion. What makes casuarina equisetifolia more tolerant? Comparison with other study. Other works done on Casuarinacea and salt stress should be considered (Ngom et al, 2016 FIPS; Djighaly et al, 2018 annals of forest science). There are data about casuarina in salted condition and authors should see if their observation are similar or not. There is a strong part focusing of transcription factors and the paper of Diédhiou et al. BMC Plant Biology 2014 is not cited, although it is a study of FT in actinorhizal species (Casurina and alnus).
Papers of gaca et al Int J Mol Sci. 2019 should also be considered, soame for Scotti-Campos P, J Plant Physiol. 2016
All these paper deals with salinity and casuarina;and my give some interesting comparison sources.
In brief, results should be more analysed, and result part of the paper reorganised.
Discussion and conclusion should include more recent litterature and try to do a comparison of transcriptomic data and histology results; Did you find gene inviolved in cell remodelling ?
Additional comments: The research is of novelty; However the main default is that there is only 32 biological replicates for transcriptomics which does not respect the actual standard (3). Then staistical anlysis is weak. Lots of litterature on saline stress in plants and in casurinaceae is missing.
Detailes comments are provided as a separated file.
Study nedds to be reconsidered to fit the standard for publication;
Review of the manuscript 2021:02:58311:0:1:NEW 4 Mar 2021
Comprehensive elucidation of the mechanism of salinity resistance by cell structure and transcriptome analysis in root in Casuarina equisetifolia
Tittle
Line 1 : Title should be modified to reflect the study.
Introduction
Line 52 to 77 This part lack of recent references on salt stress in plant and on transcriptomic studies done on this problematic. What are mechanisms of tolerance? Which sets of genes are implicated?
Then it will be easier to compare (in the discussion part) and to explore what makes Casuarina equisetifolia more tolerant. This is the main question of the paper and it does not show up.

Papers to be considered (among other):
Park et al 2016 Mol Cell Biol
Yu et al. 2020 TIPS
Farhat et al., 2019
van Zelm et al ARPB 2020
Morton et al 2019 Plant J
Liu et al 2019 BMC PB on Populus (another tree species)

Line 85 : dtat on Salt and Casuarina equisetifolia are not presented. There are papers
(Ngom et al, 2016 FIPS; Djighaly et al, 2018 annals of forest science). There are data about casuarina in salted condition and authors should see if their observation are similar or not.
Line 95 : Why the fact to have genome gives opportunity? You should develop.
Line 98: Root = first perception  give a reference
Line 100 : explain why. I do not exactly agree with that. Roots are sometimes difficult to work with ; RNA extraction can be tricky with polyphenol and polysaccharides, presence of substrat … At the tissue level, structure is not simple …
Line 104 : cell structure is absolutely not connected with your transcriptomic data. It’s a pity. You should try to see if some DEG are linked to membrane integrity, or to a stage of cells structure degradation.

Line 109 : OK: Can you explain how and which one you will use? This part is absolutely not discussed.

Material and method
Line 120 : why did you choose this time course (previous results ? Please explain. Line 121-122: unclear. Please rewrite.
L1329-132: be sure that these statistics test are adapted to your experiments, generally with a low number of replicates (3), you should use non-parametric tests. Get help from a statistician.

Line 133 : title not adapted. You do histology at two level : tissue level and ultrastructure…but not only electron microscopy
Line 145: Define ‘new root’? How do you select them? Which time course. The sample names should be here. How many replicates …
Line 151 : 20mg seems a bit overestimated for total RNA.. Please verify.
Line 152 precise which Illumina sequencing; Size of the reads?

As a matter of clarification, I would group : RNA extraction for illumina with the mRNA seq experiments parts.
Moreover, I would group RNA extraction for QPCR with QPCR.
Line 158 : & 159 : 2 replicates only? This not the standard for publication of transcriptomic (generally 3 replicates are needed). This also can be a problem with the results of clustering. You clustered 1H and 6h and 24H was grouped with 168h. I am a bit surprised that 24h of salt stress is close to 168h which makes 7days .?...You should precise. Change also the sample names, as CK is troubling (CK means generally cytokine). I would prefer control.
Line 181-185 : unclear. Please reformulate and clarify.
Line 186: The method used is old fashioned for ortholog finding. You should use other tools (orthofinder …). If you use BLASTN you cannot talk or orthologs. By the way, the reference cited is not adapted; you should find another one describing the methodology you used.

Results
Morphological changes in roots
Line 202 : did you compare this observation with the one of Ngom et al 2016 or Djighaly et al 2018?
Line 202 – 205 : not clear with the figure.
Line 206 : Pictures are too small to see the description you do.
Line 214: blocked the deleterious ion transport = you cannot say that. With these observation, you have no proof of that.
Fig 1 is cited …but Differences are really not clear for the root. Please explain more clearly

Changes in ion content
You should describe a little more the table and give values.

	Ultrastructural changes in roots
Put this part before the change in ion content.
Line 236 : Did you count?

	Response of differentially expressed genes to salt stress in C equisetifolia
L292 : dynamic changes : how do you evaluate that ?

Figure 4A :is cited .. but I have a problem with it. See below. Considering Fig 4A : how can you cluster H1 and H6 and the  H24 and H168: patterns are really different. Please explain what is TPM. I cannot understand Fig 5. I don’t understand the fig (see below).

This results part should be more organised; in its present form it is confusing and we cannot really see what is the finding of the study regarding the initial question.
Fig S2 is really interesting and should be put as principal figure.

There is a strong part focusing of transcription factors and the paper of Diédhiou et al. BMC Plant Biology 2014 is not cited, although it is a study of FT in actinorhizal species (Casuarina and alnus).
Papers of Graca et al Int J Mol Sci. 2019 should also be considered, same for Scotti-Campos P, J Plant Physiol. 2016

Discussion
Considering all these remarks, paper should be completed and rewritten. Of course discussion should be updated regarding the new results form.
Line 332 :  ‘High level of salinity’: can you precise what is a high level? Maybe you can compare to sea water ?
Line 337-340 : sentence is unclear.
Line 341 : Not clear.. Please give more information.
Line 342 : Histological = no visible change…but what about MET?
Line 348 : It could be interesting to compare your resultas to other species tolerant and not tolerant. My concern is : is there a transcriptomic profile linked to salt tolerance ? What makes the Casuarina equisetifolia more tolerant compared to other species?
Part on PCD should be placed after the ROS part.
You should comment on what will be the next step of the study. What will you do with all these DEG? Will you develop functional studies to validate your hypotheses?

FIGURE 1 : legend are not descriptive enough. Normally the legend should be  very clear and precise and autonomous regarding the text. Then root pictures are big and I don’t understand why there are two range of root picture; This should be explained. Fig 1B : pictures are too small and we cannot distinguish what is described in the results part. Scale bars should be specified for roots and more visible for 1 B;

TABLE 1 :Legend is not specified enough. Statistics should be mention. How many replicates …

FIGURE 2 : Legend not specified enough. Picture are too small. Put some of them as Sup data and focus on the most important and then you can put them in bigger size.

FIGURE 3 : Incomplete legend.

FIGURE 4 Incomplete legend. How did you obtain Fig 4A: from what I understand, this is just raw level of mapping? It is not based on Fold change. Please explain. Fig 4B: too small. Fig 4C: not clear and should be in a separated figure.

FIGURE 5: legend are not precise; What is this figure; Is it a comparison of DEG and QPCR? Please explain. And what are the to range of histogram (TPM and relative expression). It is really confusing and unclear.


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: DETECTION OF SYRUP ADULTERANTS IN MANUKA AND JARRAH HONEY USING HPTLC-MULTIVARIATE DATA ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript (62039) reports on the application of HPTLC to honey adulterants.
Recently, HPTLC has been applied to chemical profiling of natural products including honey. The authors combined various chemometrics methods to HPTLC for the purpose.
Despite the efforts, there is lack of novelty in the manuscript. There are already quite number of works of HPTLC to honey samples, both for sugars and minor floral metabolites. Especially, to prove the feasibility of the methods, more number of honey samples are required.
The application of HPTLC did not show any clear advantages over other methods. For examples, compared with GC analysis, the advantages are not clear, though the authors said GC is expensive (actually, the cost of instruments are not that different).
Experimental design: The target of metabolites are in this study, sugars and organic phase metabolites. The analysis of the honey samples adulterated with sugars by HPTLC is not that new. The analysis of non polar metabolites are already well known. Even in the study, there is not detailed work for non-polar part. Also, in non-polar metabolites of honey, there are many terpenoids, non-phenolic metabolites, but in the manuscript there is no data interpretation about there terpenoidal metabolites.
Validity of the findings: Data obtained in the study is validated but there must be more honey samples for validating feasibility.
Additional comments: It is interesting to apply HPTLC to adulterated honey samples. However, targeting on sugar detection is already well known. There is not much novelty in the method.


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: DETECTION OF SYRUP ADULTERANTS IN MANUKA AND JARRAH HONEY USING HPTLC-MULTIVARIATE DATA ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The text is not well structured,it also contains numerous small paragrahps that have to be merged. The background and justification of the study is not enough. For example, if there are several methods for honey autentication, what are the real avantages of implementing HPTLC? Moreover, even if there is a raw data file, this have to be properly presented (please see specific comments). Finally, results are not described enough and the discussion is more focused on explining statistical analysis procedures (which are not developed by the authors) rather than focus on discusing their own results.
Experimental design: The research question is well stablished, however, not well expressed (please see specific comments). There are important missing information on methods which can change results obtained when trying to replicate the study.
Validity of the findings: There was just one replicate for autentic honeys and the syrups, and results of each multivariate data anlysis is not properly interpretated.
Additional comments: The topic presented by the authors is original and used a reemerging analytical technique in combination with multivariate data analysis. Moreover, the samples analyzed (honeys) are quite interesting. Nonetheless, there are several format and content issues to be corrected. Please, get much better quality figures.


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

Input:
Title: MICROBIAL GROWTH IN BIOBEDS FOR TREATMENT OF RESIDUAL PESTICIDE IN BANANA PLANTATIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This an interesting study about the possible application of bioded made with local material to treat wastewater polluted with the fungicide ethylenebisdithiocarbamate. However, this work has several points that must be deeply clarified and improved, for example, the aim proposed is not discussed appropriately in relation to the obtained results, there are a lot of figures but the discussion is very poor as well as the association to other studies made by other authors. If one of the aims was to study the microorganisms distribution in the biobeds the conclusions should refer to that.
Your introduction needs more detail. I suggest that you improve the description at lines 63- 75 to provide more information and justification for your study (specifically, you should expand upon the knowledge gap being filled).
The raw data were shared and the structure of the article conforms to an acceptable format of standard sections.

More details about my revision of the different section are below:
Introduction
Lines 63 to 75: There are works about microbial population and enzymatic assays on biobeds and these articles are not mention neither discussed. The studies show different techniques to determinate for example Bacterial abundance (by qPCR) or bacterial and fungi population. Same of them are:
-Fate and effect of imidacloprid on vermicompost-amended soils under dissimilar conditions: Risk for soil functions, structure, and bacterial abundance. Jean Manuel Castillo Diaz, Fabrice Martin-Laurent, Jérèmie Beguet, Rogelio Nogales, Esperanza Romero. Science of the Total Environment 579 (2017) 1111–1119
-Aging of biomixtures: Effects on carbofuran removal and microbial community structure. Víctor Castro-Gutierrez, Mario Masís-Mora, María Cristina Diez, Gonzalo R. Tortella, Carlos E. Rodríguez-Rodríguez. Chemosphere 168 (2017) 418-425
-Indigenous biobed to limit point source pollution of imidacloprid in tropical countries. Totan Adak, Bibhab Mahapatra, Harekrushna Swain, Naveenkumar B. Patil, Guru P. Pandi G, G. Basana Gowda, M. Annamalai, Somnath S. Pokhare, Sankari Meena K, P.
C. Rath, Mayabini Jena. Journal of Environmental Management 272 (2020) 111084.

In general, the way that the results are presented is not attractive. The best form could be, for example using a unique title: Physicochemical parameters evolution. This style would help to have a discussion more integrated.
Lines 200-203: the description of results should be more appropriate to the aims of the work. For example Figure 2 shows that the pH follows the same tendency in both substrates (soil and biomixture) with a slight increase with the incubation time. Also, the behaviour is similar for the three profundities.
This comment is for all parameters described. ¿Why the analysis with the profundity is avoided in almost all parameters?.
In Figure 5 is not necessary (and not correspond) to show the values corresponding to soil and biomixture no polluted. If the values are very low the authors should use an axe Y secondary on the right side.
I recommend see other similar studies to improve the presentation of the results, for example (see the type of figures used):

-Indigenous biobed to limit point source pollution of imidacloprid in tropical countries
Totan Adak, Bibhab Mahapatra, Harekrushna Swain, Naveenkumar B. Patil, Guru P. Pandi G , G. Basana Gowda , M. Annamalai , Somnath S. Pokhare , Sankari Meena K , P.
C. Rath, Mayabini Jena. Journal of Environmental Management 272 (2020) 111084.

Figure 7 and 9: For this type of figure is better to use the Y axe in the logarithm scale (see the last paper recommended).
Figure 8 is not necessary; the analysis can be only in the text with the corresponding r2. Possibly exist other correlation tests that can be applied, if other environmental factors can also simultaneously influence the abundance of microbial populations may be the more appropriate apply a test like PCA. See other studies, for example:
-Agricultural effluent treatment in biobed systems using novel substrates from southeastern Mexico: the relationship with physicochemical parameters of biomixtures. Virgilio René Góngora-Echeverría, Fabrice Martin-Laurent, Carlos Quintal-Franco, German Giácoman-Vallejos, Carmen Ponce-Caballero. Environ Sci Pollut Res, DOI 10.1007/s11356-017-8643-z
Experimental design: The work is within Aims and Scope of the Journal.

Section methods. The authors need to clarify the following:
Were the materials required for biomixture treated?, i.e, these were cut into pieces of determinate size or were used directly.
Line 100: why a concentration of 1000 mg/L was used?. Justify.
Lines 104-105: to add the different depths of biobed-column (surface, medium, and bottom) in cm.
Discussion
Line 246-248:  It would be interesting found a relationship between physicochemical parameters, microbial population and compound degradation under a point of view statistic. There is a lot of information but must be rigorously analysed.
Lines 258-264: the paragraph describes other works but no explore the relationship with the results obtained in this work. Rewrite.
Line 266-271 and 273-279: Again, rewrite taking into account the owner results.
Line 281: Organic matter: something to say about the results of organic matter vs. profundities?
Lines 311 and 312: The sentence is not clear. Improve the idea: At 85 days of pesticide degradation, the deepest level’s concentration decreased to an extent acceptable for soil organisms growth, close to 1 mg L-1
The authors can give some explanation about why the concentration of ETU increases between 14 and 42 days in the medium and bottom of the biobed.
Lines 320 and 321: How is this conclusion obtained? The information can be seen in the figures?. Please, explain better the idea of the following sentence:
It was found that the number of cells that could be necessary for EBDC degradation should be greater than 100 x 105 cells mL-1 in bacteria and close to 20 x 103 cells mL-1 in fungi.
Validity of the findings: -The conclusions should mention something about the effect of profundity.
-The concentration (3.7 mg L-1) of the pesticide at 85 days: is an average value between the different profundities?.
Line 382 to 384: I do not think that colony and cell counting techniques are simple. I think that indirect technique like analyses of the enzymes (for example FDA) can be more simple.
Additional comments: Other comments:
Line 134: replace NH2OH by NH4OH
Lines 158 and 164: should say “soil or biomixture” since the assay was done for both materials.
Lines 197 and 198: this sentence and the Table 1 must be in Material and methods section after the description of the material.
For CEC the units used generally is meq/g. Why the reported values have a subindex: cmolc kg-1?
In Figure 5 the ET concentration increases at the medium and the bottom. Do the authors have any explanation?


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: MICROBIAL GROWTH IN BIOBEDS FOR TREATMENT OF RESIDUAL PESTICIDE IN BANANA PLANTATIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript describes a laboratory-scale biobed system for the treatment of pesticide EBDC from a banana plantation. While the aims and subject (crop and pesticide) of the study are of interest, the manuscript suffers from issues with readability, and there are important concerns related to the methodology employed and subsequent conclusions drawn.

The manuscript would benefit from review and contribution from a proficient English writer as there are several issues with grammar/syntax, flow and readability. For example, line 19-20: “frequently unused pesticide mixture (solution) is not disposed of properly” should be “unused pesticide mixture (solution) is frequently improperly disposed of…” and lines 46-67 include a dependent “because” statement that is not resolved. These are two of many examples.

The introduction is sparse with current literature describing biobed microbial community dynamics and treatment efficacy. Furthermore, lines 72-75 contain references and information that are not relevant to the study at hand.

The use of subheadings in the discussion (as used in Results) should be avoided and discussion of these topics integrated into a broader and flowing narrative consideration of the results.

While several physicochemical explanatory parameters of the biobed were investigated, the presentation of all this data is not entirely necessary and would be more suitable as supplementary material in an improved manuscript that included expanded analysis (see further comments below). In the figure legends of line graphs (Figs 1-7), replicate numbers and error bars are not described/explained.
Experimental design: While the research topic is broadly relevant as a novel biobed treatment application (i.e., banana crop and EDBC), the research questions is vaguely stated and is not appropriately interrogated with the methodology used. Understanding physicochemical parameters of a functioning biobed are certainly relevant for understanding overall system dynamics and with respect to monitoring, but this research stops short of characterizing the health, diversity, nature/characteristics and stability of the biobed microbial community and linkage between these communities and the treatment efficacy of EDBC in any way. Cell counts (direct or CFU) of microbial cells are not representative of the microbial population present or micorbial activity, merely those ones capable of growth on that particular medium. Indeed, it is demonstrated within the paper itself (Figure 8), that there is little to no correlation between any cell counts and pesticide concentration.

There are also concerns with the detail provided for the methodology section related to the setup of the biobed systems – (i.e., replication, experimental design, how samples were collected, irrigation). Furthermore, the biobed systems as described appear to be considerably small in size (50 x 9.5 cm) and received one pulse of pesticide rinsate inflow/input and thus applicability to full-scale, on-site systems is of questionable utility.

Lastly, recent/current investigation of biobed treatment systems employ microbial community profiling techniques (i.e., sequencing) to specifically characterize “microbial dynamics” of biobed treatment systems, which any cursory literature search would reveal. As conducted and presented, this paper does not deliver contemporaneous results that advance understanding of the field.
Validity of the findings: Within the presentation and discussion of results, the rationale for the experimental design is not well explained or connected and thus the impact the data has is not clear. As previously mentioned, the sole examination of bacterial counts and the use of small bioreactor systems severely hampers the ability to draw any conclusions as to the nature of “microbial dynamics” in the treatment of EDBC using banana compost.

The conclusive statements related to a specific number of microbial counts being required for EDBC degradation is not supported given the lack of proper controls and demonstration that the microbes contribute substantially to the degradation (other factors such as sorption, dilution, half-life degradation are not considered or examined), and by the authors own statistical analysis. Replication is not made clear throughout (i.e., in figure captions) and there is limited impactful discussion of their experimental design (i.e., the four treatments analyzed). The authors also discuss microbial species within biobeds that have been identified in other research and draw unsupported and inappropriate connection to the present study, as no similar analysis was conducted.

While the authors are certainly correct to state that is necessary to develop cost-effective means of biobed assessment, particularly in remote areas/situations that rely on local borrow-sources, however the conclusions that the authors understand the “minimum population of cells required for treatment” is not supported by current experiment. Further experimentation using appropriate controls, replication, and demonstration of biobed efficacy over biobed management lifecycle and/or continuous pesticide treatment is required in order to support the claims of understanding “microbial dynamics”.
Additional comments: While the overall objective of the study is sound and of utility to the broader field of investigation, the methodology used to investigate this crop/pesticide treatment, and the overall design of the experiment do not support the ultimate conclusions made. The authors must consider i) the role that other means of pesticide degradation (sorption, half-life, etc.) might play in the observed concentration reduction ii) the scale of their experimental system and the applicability it may have to large-scale, on-site systems iii) adjusting their experimental design such that several of the “future research” questions may indeed be answered (i.e., replacing materials, increased irrigation, continuous or pulse-pesticide application, sequencing of communities, and use of control biobeds/matrices).


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: MICROBIAL GROWTH IN BIOBEDS FOR TREATMENT OF RESIDUAL PESTICIDE IN BANANA PLANTATIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: pass
Experimental design: pass
Validity of the findings: pass
Additional comments: I consider that the manuscript was deeply improved, the discussion now is more clear and it is well connected with the aims and the obtained results. I think that the results are valuable for monitoring the biobeds in the banana production because suggest bacterial and fungal populations (or cell count) to optimal working of the biomixture or for its replace. I agree with the authors when they say that the techniques used in this work were convenient due to their low cost and simplicity (no require expensive equipment or reagents). This is key factor to apply this technology in producing areas.
I have minor comments that annotated in PDF files (57885-v1).


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: FUNGI IN SOIL AND UNDERSTORY HAVE COUPLED DISTRIBUTION PATTERNS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Please see the review report.
Experimental design: Please see the review report.
Validity of the findings: Please see the review report.
Additional comments: In this study, Boraks and Amend conducted a survey of fungi in soil and phyllosphere in a tropical forest. They found that (a) soil and phyllosphere had different fungal community composition; (b) generalist fungi (present in both soil and phyllosphere) did not have larger range sizes; (c) soil specialists were locally abundant but phyllosphere generalists were abundant over a larger geographic range.

This manuscript is well written. In addition, it contributes to a growing body of literature on microbial dispersal. I also found that the figures are aesthetically pleasent. However, their method of range size calculation needs to be justified and some statements need to be clearly clarified. Below, I provide some comments that outline my major concerns and provide recommendations to hopefully improve the manuscript.

Major comments:

The authors used semi-variogram to calculate range size. However, this method is usually used to calculate spatial autocorrelation range rather than range size. In ecology, area of occupancy (AOO) and extent of occurrence (EOO) are more widely used to calculate range size (Choudoir et al., 2018). Citations are needed to justify the reasons why they used semi-variogram to calculate range size.

Choudoir, M.J., Barberán, A., Menninger, H.L., Dunn, R.R. & Fierer, N. (2018). Variation in range size and dispersal capabilities of microbial taxa. Ecology, 99, 322–334.

L131-132: A major limitation of this study is that plant species identity information was not included in the analyses. This is particularly relevant for phyllosphere-associated fungi. For example, in Figure 1 a certain proportion of variation in community composition of phyllosphere-associated fungi can be explained by plant species identity. Could the authors add this information into their analyses? If not, please address it in the discussion.

L189-19, Figure 2 and Figure 5: Why was the occupancy calculated at the within-transect level? Wouldn’t it ignore broad-scale dispersal patterns, for example, inter-transect and inter-island dispersal? This needs to be justified. I question whether their results and conclusions still hold when looking at larger spatial scale.

Figure 4: Please report the cross-validation results of kriging. That is, the accuracy of spatial kriging prediction.

Minor comments:

L224-226: Please report the R2 from PERMANOVA.

Figure3: Ground and understory? Maybe soil and phyllosphere? Also, I don’t see there are any shaded areas.

In some places “p-value” was used, while in other places “P” was used. Please make it consistent throughout the 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: FUNGI IN SOIL AND UNDERSTORY HAVE COUPLED DISTRIBUTION PATTERNS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Introduction
- line 90 - not sure if 'spore' should be 'spores' or 'spore dispersal', or was desired as is.
- line 95 - I think it would be helpful to briefly define Xenospores here (e.g., 'Xenospores (i.e., spores that are dispersed vs. those that stay where they are produced).').

Methods
- line 147 - My understanding is that the MiSeq and NextSeq are unique Illumina instruments/platforms (see https://www.illumina.com/systems/sequencing-platforms.html). Can you please clarify which platform was used? Also, just FYI, I've noticed that for Illumina instruments with two-color chemistry, such as the NextSeq and NovaSeq, introduce high-quality, poly-G tails of various lengths to R1 reads, which are often not detected but can affect downstream analyses. So, depending on which platform was used, this can be relevant.

Results
- Figure 1A - I suggest to add outlines to sample points/triangles, as well as an alpha value to add transparency so that the number of points and their overlap is more clear. I also suggest to include the results from PERMANOVA here if relevant.
Experimental design: Methods:
- line 157 - More and more I see debate over the use of deseq2's VST vs. other sample normalization methods. I'm fine with it's use here (i.e., except where noted below re: Fig. 5), but just want to highlight this and suggest you continue to explore novel methods for normalization as they develop.
- line 165 - Similar to above, more often today I see debate over the use of Bray-Curtis distance outside of analyses of beta-diversity where the goal is to emphasize differences based on 'rare' taxa. Issues surrounding the sparse- and compositional-nature of these type of data point to using pseudo-counts/matrix completion and/or compositionally-aware diversity metrics to avoid this emphasis, which apparently stems in part from BC's inability to handle the sparsity issue. Again, your results are likely robust to this and I'm fine with the use of BC here, but I recommend to explore a new metric called robust Aitchison that can be implemented in the deicode package in qiime2. This metric is compositionally aware and uses matrix completion to deal with the sparsity issue. There is also a phylogenetically-aware version, which is my second comment that you may consider using a phylogenetically-aware distance metric, such as unifrac. I understand the difficulty in constructing fungal phylogenies with short-read ITS data, but there may be a way to use fragment insertion into a larger backbone context, to get a tree that can be used.
- line 165 - Did you explore permdisp in parallel to permanova? It is typically not appropriate to interpret the results of permanova if the results of permdisp are significant i.e., indicate differences in group variances.
- line 195 - The other analyses using VST normalized read counts above appear to be based on presence/absence alone and thus are appropriate. However, as I describe further below for the analyses in Figure 5, any analysis using relative read counts should consider that the read count data are compositional (e.g., you can not tell, from sample-to-sample, if taxon A's abundance is going up vs. taxon B's abundance going down). One solution is to use reference frames (see https://www.nature.com/articles/s41467-019-10656-5). I highly recommend you consider this to accurately estimate these range sizes.
- line 211 - The same concern I have for your range size estimates holds here - please exert caution in using raw/VST-normalized read counts for comparing relative abundances among groups.

Results:
- Figure 5 - I suggest to examine the log-ratio of local abundances of generalists to specialists, for technical reasons. The VST normalization used previously is debated as I noted, but should be fine to use for the diversity analyses above. However when comparing relative abundances of groups, it's been shown that the compositional-nature of these data causes problems, that VST is not robust, and that reference frames (e.g., ALR, ILR, CLR transforms) or similar should be used (see https://www.nature.com/articles/s41467-019-10656-5).
- Figure 4 - I suggest to map instead the log-ratio of leaf to soil for each taxon, per my concerns re: comparing read counts among groups above.
Validity of the findings: Results:
- My only concern surrounds the analysis of VST-normalized read counts in quantitative analyses comparing groups (i.e., I have no concerns when they are used for typical diversity analyses or presence-absence/qualitative analyses). It has been shown that the compositional nature of next generation sequence data causes problems in interpreting 'changes in relative abundance' from sample to sample (see https://www.nature.com/articles/s41467-019-10656-5). Therefore I recommend to use reference frames, or log-ratios, if possible. I have provided specific suggestions where I was able to above.
Additional comments: Thank you for preparing such a wonderful article! It was a pleasure to read and the scope and findings are very interesting. I look forward to continuing to follow your 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: FUNGI IN SOIL AND UNDERSTORY HAVE COUPLED DISTRIBUTION PATTERNS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Overall, the paper could use some stream lining to make the authors' message clearer to the reader. I have outlined some issues below, as well as in the "General comments to the author".

The authors seem to switch their vocabulary readily making it hard to follow. For example, in the introduction the authors use habitat breadth, habit specificity, and habitat occupancy, but do not define what they consider habitat to be. Is it solely soil versus phyllosphere? This seems more like substrate rather than habitat. Use of habitat here has issues as habitat is the environment of an organism and is an amalgamation of different factors (plant species, soil conditions, climate, co-occurring organisms). I think it would benefit the paper if you outlined major differences in the environment of the phyllosphere (UV, low nutrients…; Vorholt 2012 outlines this well) versus bulk soil (highly heterogeneous environment, high competition…). What do fungi need to be able to survive in one, the other, or both? Why would you expect some fungi to be "specialized" to soil or the phyllosphere?

In considering habitat breath and range size, the authors do not define what they mean by these terms. High throughput sequencing data limits one’s ability to detect habitat breadth and range size as it amplifies all target DNA in a sample which includes DNA from living and dead cells, as well as hyphae and spores. It is impossible to know what life stage a fungus is at and what substrate it is using based on sequencing data alone. It seems that here the habitat breadth and range size only include where the fungus has been detected. As a suggestion, you could use FunGuild to determine broad lifestyles of fungi providing possibly more insight into what they are doing.
Experimental design: Please be more explicit in your methodology for HTS as the quality of HTS data is dependent on the how sample libraries were made and for reproducibility of the results.
a. For example, you do not state whether library preparation was a one-step or two-step process and whether PCRs were done in triplicate to account for stochasticity in PCR amplification (see comment below).
b. Negative controls during extraction and PCR are not mentioned until they are referenced for contaminant removal (lines 154 to 156). Did you include positive controls in your sample preparation and workflow? Positive controls or mock communities allow for assessment of PCR and sequencing bias, additionally they allow assessment of the validity of OTU delimitation (see Bakker et al. 2018; Taylor et al. 2016; Nguyen et al. 2016). If mock communities were not used, then that should be explicitly stated as there is no way to verify that read abundance matches biological abundance, so analyses in which read abundance is used (even if normalized) are questionable. This is due to different copy numbers of rRNA genes in different fungal lineages (see Bakker 2018).
c. An additional issue is that no filtering of OTUs was done and singletons were kept, although this was not mentioned until the results section. It is generally recommended that OTUs with low read abundance and particularly singletons should be filtered out due to the change of errors when sequencing to that depth (see Edgar, 2013; Tedersoo et al. 2010; Dickie 2010; Nguyen et al. 2016). I would recommend verifying results without singletons or low abundance OTUs and including these results alongside the ones reported in the manuscript.
d. You don’t mention read normalization until Line 216 of the results. Please explicitly state how you normalized reads in your methods. Here I am assuming by normalizing the reads you mean rarefaction. Did you use size-based rarefaction or coverage-based rarefaction (Chao & Jost 2012)?

Please see more specific line-by-line comments in the "General comments to the author section" and broader comments in the "Validity of the findings".
Validity of the findings: I have concerns regarding assumptions that are made in the paper. For one, the authors state “Fungi are cosmopolitan…” (line 40) and “…so many are habitat generalists” (line 65-66). While yes fungi are found everywhere, specific species and groups of fungi show strong distribution patters that align with not only their symbiotic status (plant-associated, animal-associated, lichen-associated, free-living…), but also the abiotic and biotic environment (see Tedersoo et al. 2012; U’Ren et al. 2012; Bahram et al. 2013; Huang et al. 2018; U’Ren et al. 2019). As part of this, I think the models that are used lack important aspects of the environment that might add more depth to their results at both larger and smaller spatial scales. This includes host species (see below for specific question regarding this), geographic location (two distinct islands were sampled), forest type, and climatic variables (precipitation and temperature; this may correlate with geographical location if the main differences are between the islands). Lastly, limitations to the methodology are not clearly stated in the discussion section. Sampling was conducting by swabbing leaf surfaces and soil followed by metabarcoding. There is no way to distinguish what a particular OTU was doing (actively growing, dead, or a dormant spore) when it was sampled, so there is a huge caveat on the distribution patterns. Do these patterns represent the true distribution of fungi or the dispersal ability of fungi?

Please see more specific line-by-line comments in the "General comments to the author section".
Additional comments: In this paper, the authors calculate the range size of specialist and generalist fungi from both the bulk soil and phyllosphere using data from metabarcoding of the ITS1 region. In all, I found this paper interesting and novel in its goal. As the authors state, the calculation of range size of fungal species is an underexplored area, and the authors use a variety of methods to answer their main questions. The inclusion of two methods for analyzing distribution patterns (the area-occupancy analysis and semivariograms) was compelling, and the authors do a good job outline the differences between the two methods. I think this paper would be of interest to both mycologists and ecologists.

Line edits:

Line 63: change “specie’s” to species’

Line 65: change “withing” to within

Line 65-69: I disagree with the authors’ assessment that many fungi are habitat generalists, and that fungal species are cosmopolitan. It harkens back the Bass Becking hypothesis “Everything is everywhere, but the environment selects”. The authors provide no evidence to support these statements other than the occurrence of one species Aspergillis niger in the GlobalFungi database. This example has some inherent issues and I don’t think it can be considered a cosmopolitan species as it is a species complex and requires use of multiple loci to delimit species (da Silva et al. 2020). The GlobalFungi database uses records from HTS and includes only ITS sequences (Větrovský et al. 2020). HTS detects all fungal structures present in a sample, including spores. Aspergillus spp. are known to produce prolific numbers of aerial dispersed spores. It is impossible to know the life stage and function of a fungus isolated via HTS techniques.

Line 103-113: The final paragraph of the introduction does outline the goal of the study but is difficult to follow. It seems that the statistical analyses and results are outlined with subheadings that are meant to divide the analyses by the question they are meant to answer. Is that right? If so, the final paragraph is not written in a manner that it is easy to match the analysis and results to the question it is addressing.

Line 124: Are all the six transects in the exact same forest type, same soil type, same climate? They are across two different islands, but how similar are the habitats between islands?

Line 126-129: Do you have a reference for this sampling methodology that is swabbing leaf surfaces and soil? I have never heard of taking swabs of soil. The Qiagen DNeasy PowerSoil DNA isolation kit usually requires a sample of up to 0.20-0.25 g of soil. Where you able to isolate that much soil with a swab?

Line 131-132: Please change this sentence to “Plant host identity influences phyllosphere mycobiome..” and put proper citations. Also, here do you mean you swabbed multiple surfaces with the same swab? Or were multiple swabs taken, one for each host? If the latter, then were these extracted separately or together?

Line 142-143: Where PCRs conducted in triplicate? This is important to remove some of the natural stochasticity present in PCR amplification (see examples in Daru et al. 2019; Glassman et al. 2017; Bakker 2018). Was this a two-step PCR in which barcodes and Illumina adapters were put on in the second outer PCR?

Line 151: Did you try to make contiguous sequences out of the forward and reverse reads prior to discarding the reverse reads? Reverse reads are generally going to be lower quality than forward reads, but do provide value as they verify the lower quality end of the forward reads.

Line 151: How many chimeras were removed (total number and % of reads)?

Line 152: Why did you cluster at 97% sequence similarity?

Line 156: What percentage of OTUs could not be assigned to a fungal phylum?

Line 166: Change “Permutational” to permutational

Line 167: What were your explanatory variables in the PERMANOVA?

Line 171 to 173: Please move this sentence (“Fungal communities…to the soil below”) to the introduction.

Line 178: Why did you use a student T-test here? You conducted the test at two different spatial scales. Why not use an ANOVA? That would reduce you change of a type I error.

Line 188: What is S2?

Line 196-198: Please move this sentence (“Range size…transect.”) to the introduction.

Line 242-243: Please move sentence (“OTUs were first categorized…for each OTU.”) to the methods.

Line 249-250: Please move sentence (“Spatial autocorrelation…a Mantel test.”) to the methods.

Line 252-260: Please move section (“Range sizes were modeled using…were removed from downstream analysis.”) to the methods. Please not that I was unable to find Table S3. Do you mean Table S1?

Line 266-267: Please move sentence (“Including the culled…range-size comparisons.”) to the methods.

Line 272-273: Please move sentence (“To visually illustrate…but of the same transect.”) to the methods.

Line 289: Change “fugal” to fungal.

Line 325: Remove “1)” and “2)” (line 328). These are in separate sentences which makes the numbers confusing.

Line 327: Change “Fig.s” to Fig. Change “axis” to axes.

Line 369: “This result this study” should be changed.

Line 382: Here in your conclusion, you mention physical and temporal characteristics, but you have not outline how the phyllosphere and soil environments differ. Also, I am unsure of the temporal characteristic except in Fig. 1a in which you state that axis 2 corresponds to differences in the sampling times and islands, but sampling time was not including in any other analyses, correct? I do think it would improve the depth of your results if you included more physical and even the temporal aspect in your analyses as outlined above.

Figure 1a: In the legend, you mention that axis 2 corresponds to differences in sample date and island. Was this included in your PERMANOVA?

Figure 1b: Not referenced in your manuscript. Also, this is not particularly informative, and you may consider either modifying it or removing it.

Figure 3: Please change the titles of the graphs to match the habitat types you are using (soil and phyllosphere). Changing them here to ground and understory is confusing. If these represent other groups than soil and phyllosphere then it should be explicitly state. Also in this figure you mention that the outliers can be found in Supplemental Table S1, but in the manuscript you state that it is found in Table S3.

Figure 4: Very interesting figure. Could you place the names of the OTUs in the legend or in the manuscript, so they can be cross referenced in Table S1?

Figure 5: What is the y-axis? I understand that it is normalized OTU reads, but the axis range (0 to 10) doesn’t seem to match that.

Supplementary figure S1: Figure is cut off. Also, it would be helpful if the transects were labeled to match Table 1.

Supplementary Table S1: Why are all the OTUs called OTU97_***?? In Table S1, the outliers removed for Figure 3 should be clearly marked.

In the supplemental material, there is a flowchart, but I couldn’t find a reference to it in the manuscript. I think it is useful for understanding the workflow though.

Do you have plans to deposit the reads into the Sequence Read Archive?


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: FUNGI IN SOIL AND UNDERSTORY HAVE COUPLED DISTRIBUTION PATTERNS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I thank the authors for carefully addressing my concerns. I don't have further comments and suggestions.


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: FUNGI IN SOIL AND UNDERSTORY HAVE COUPLED DISTRIBUTION PATTERNS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Review for Milazzo et al. 2020 revision. High-throughput metabarcoding characterizes fungal endophyte diversity in the phyllosphere of a barley crop. Phytobiomes Journal.

In this paper, the authors calculate the range size of specialist and generalist fungi from both the bulk soil and phyllosphere using data from metabarcoding of the ITS1 region. In all, I found this paper interesting and novel in its goal. As the authors state, the calculation of range size of fungal species is an underexplored area, and the authors use a variety of methods to answer their main questions. The authors’ revisions greatly improved the paper and clarified their methods.

The authors have clarified some of the vocabulary they are using with the exception of niche and niche breadth. Niche most commonly means the environmental conditions and resources required for an organism to persist (as Elton and Hutchinson defined it), but can also mean just the environment. In this paper, it seems that niche means only the physical limits tolerated by species of fungi. Niche breadth is an important aspect of their conclusions, so how the authors are defining it here should be explained explicitly.

Line 270: Change “were” to was

Fig. 1: For 1a, you mention that separation is along axis 2. Are the points from each island well mixed or separated? Could you mention this or possibly change the color or shape of the points based on which island the sample was from?

Fig. 5: You do not have labels for panels (a and b) but refer to panel b in line 399.

Supplemental Figure S1: Figure is labeled S2 in file.
Experimental design: Methods are well clarified.
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: WHO’S WHO IN MAGELONA: PHYLOGENETIC HYPOTHESES UNDER MAGELONIDAE CUNNINGHAM & RAMAGE, 1888 (ANNELIDA: POLYCHAETA)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The submitted manuscript is very well written, and structured conforming to all professional standards. The text includes sufficient information both in the Introduction and Material and Methods referencing all relevant prior literature. In this last section, the “Methodological considerations” deserves special mention and gives the work added value. The Results are relevant to the hypothesis and the Discussion includes everything previously published on this field. Literature used is also relevant and well referenced. Figures are all necessary to the content of the article, of very high quality, and appropriately described and labelled. Raw data supplied in a supplemental file are robust.
Experimental design: The submission describes original primary research, clearly defining the research question, which is undoubtedly relevant and meaningful. The investigation has been conducted rigorously and to a high technical standard. Methods are described and widely justified with a lot of information. The research has been conducted in conformity with the prevailing ethical standards in the field.
Validity of the findings: The data on which the conclusions are based are robust. The Conclusions are appropriately stated, connected to the original question investigated, and limited to those supported by the results.
Additional comments: I think the manuscript of Mortimer and colleagues represents an outstanding work, which I believe should be published as is. Although I am not a specialist in Magelonidae and my experience in phylogenetic analysis is limited, as it undoubtedly is in the case of the authors, my long experience in the study of the morphology and taxonomy of polychaetes allows me to evaluate it as a very relevant 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: WHO’S WHO IN MAGELONA: PHYLOGENETIC HYPOTHESES UNDER MAGELONIDAE CUNNINGHAM & RAMAGE, 1888 (ANNELIDA: POLYCHAETA)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well written and each section has been carefully constructed. The references are thorough and sufficient historical information is provided. The illustrations are well done and in particular those that depict the critical morphology are excellent.
Experimental design: This study, unlike most phylogenetic papers deals with morphology and not molecular results. The reasons for this are presented up front and although unnecessarily long and drawn out do capture most of current problems and vagaries with current phylogenetic studies. To be honest, it is refreshing to see morphological characters used directly in an analysis instead of mapped on to a tree produced from molecular data. The large number of characters and states is impressive and is what is be expected from biologists who are thoroughly familiar with the morphology and biology of their organisms.

Methods are described in great detail. However, the philosophical basis of abduction and the approach seems overly long for a paper like this and most of the methods and arguments have been previously dealt with by Fitzhugh in numerous papers. I recommend that the large up front section be condensed somewhat or moved to an appendix.
Validity of the findings: The results as presented are well explained and quite clear. I am not so worried about the fact that Magelonidae is a stand-alone taxon. I think every user will understand that the genus Magelona and family Magelonidae have the same meaning and that both terms will be available for use by ecologists and systematists as they prefer. This situation is no different from Apistobranchidae--Apistobranchus or Longosomatidae--Heterospio albeit the latter has a problem with nomenclature and priority. Perhaps the authors could provide a few additional examples such as these.

I would be interested in knowing more about how the various clades relate to currently perceived species groups such as the so-called M. mirabilis group (no frontal horns, etc). I was a little surprised to see that species having the large curved hooks were in different parts of the tree. In those instances where certain characters are somewhat unique, some further discussion would be of interest regarding their placement.
Additional comments: As noted in the other comments, it is refreshing to see a phylogenetic analysis that uses the actual morphology of the animals instead of mapping these characters onto a tree based on molecular results.

I recommend but do not require that the large methods section on Scientific Inquiry be moved to an Appendix. The methods are already long given all the space provided for text and figures documenting the characters being analyzed.


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: WHO’S WHO IN MAGELONA: PHYLOGENETIC HYPOTHESES UNDER MAGELONIDAE CUNNINGHAM & RAMAGE, 1888 (ANNELIDA: POLYCHAETA)
Review round: 1
Reviewer: 3

Output:
Basic reporting: Overall, this is an excellent article. It is very well written and structured according to the standard formats, with a perfect scientific English and the most accurate terminology for the subjects covered. It is very well illustrated, with figures relevant to the topics discussed, and all appropriate raw data are provided. The relevant hypotheses and results are all discussed, making an appropriate unit of publication. If something is missing, is a possible “What next?” final section, pointing possible steps to be followed in the future from the point where the present article ends. This is partially achieved in the section “Morphological characters in future specimen descriptions”, but these statements do not seem to be sufficiently highlighted in the work or abstract, and end somehow lost due to the following sections of the text . In its whole, the work is a pleasant and easy reading for those interested in the subject, and a step forward in the field.

Parts of the article, with a special emphasis in the “Methodological considerations”, can be used as a reference text, being profusely supported with numerous and relevant citations.

Examples are provided to illustrate some criticism discussed in the text, but this is always done in accordance to the professional standards of courtesy and respect expected inter pars, and in the spirits that Science should be open to discussion and avoid dogmatic standings, specially when criticism or the different points of view are perfectly argued and performed in a positively.

The article is abundantly backed with literature references, but I miss the references concerning the original authorship of some taxa cited in the text (see General Comments for the Author, below).

The purpose of the article is clear and well backgrounded, and while it could be argued that the Material and Methods section is probably too long and that it covers issues previously published elsewhere, it is clearly necessary to keep it that way, as the Authors are avoiding some of the actual fashions and practices in phylogenetic studies, and need to explain not only how they did their study, but very specially why they did it that way, avoiding those practices. This section covers aspects of the Philosophy of Science that in spite of being fundamental, seem to have been increasingly overlooked in the way that some tools, analyses and methodologies are applied in the field, and how incorrect and unfounded conclusions are being retrieved from the erroneous application of those same tools, analyses and methodologies to the object of study.

The information included in the section “Methodological Considerations” might have been published elsewhere, but I doubt that many of the potential readers of the present article have actually read it. This is a perfect way to resume all that information in one single article, moreover in the field of the systematics of annelids, and to provide the potential readers with the necessary background to follow the present article and to discuss it. I imagine that the present article might cause some discussion, which is also the driving force that makes Science and our knowledge to progress, so before start arguing, everybody interested should be familiar with the Authors’ arguments.
Experimental design: The research question, the establishment of phylogenetic hypotheses under Magelonidae (Annelida), was perfectly established and defined, and the intention to fulfil this important gap in the knowledge of the group was correctly addressed. It followed a correct experimental design, using the methodology proposed and explained in detail, and established according to the initial question and the required needs to approach its study.

The investigation was performed rigorously and following the highest scientific, technical and ethical standards. Moreover, the authors show a perfect knowledge of the group under investigation and of the methods employed. This not only allows them to use all information available, many of it obtained first-hand and including unpublished data, but also to recognize the flaws and gaps in the understanding of the group, to better establish the necessary methodological approach needed to achieve this important contribution.

While reading, it becomes clear that the present article is the result of a long process of maturation and careful deliberation, integrating the results of a previous approach done almost 20 years ago, in an unpublished PhD thesis. Moreover, a previous draft of the article is mentioned in the text, which supports the idea that the present article is the result of multiple critical readings preceding the present version, and explains in part its strength and cohesion. This becomes perfectly clear in some parts of the text, which are almost flawless. As stated previously, a perfectly matured and deliberated article.

All methods and raw data are explained in detail and provided to the readers, so the results and the consequent conclusions can be not only replicated but also used and integrated in future approaches to the same research question.
Validity of the findings: The present article fulfils and important gap in the knowledge of Magelonidae (Annelida), but its scope goes further beyond the interest of specialists in this particular taxonomic group, or in annelids in general. The Authors present an approach to phylogenetic studies which, is spite of being perfectly established and supported for decades, has been somehow disregarded by a scientific community increasingly in the pursuit of quick and poorly debated publications.

The approach used in the article is far from being new, but it will be seen as such by many unfamiliar with the methodology and the philosophy supporting it, and confidently it will create a much-needed debate in some spheres of Systematics, hopefully in a positive and open-minded way. Some arguments are complex and probably uncommon for many of the current taxonomists, as they were for me, but that does not mean that they should not be considered, fully discussed and integrated in the performed studies, always with base on solid and scientific arguments. I see this possibility of opening a much-desirable debate as one of the big strengths of the present article.

The conclusions achieved are sound and appropriately stated, clearly connected with the original question investigated and the methodology used, and strictly limited to those supported by the results. As commented in a different section of this review, I would have appreciated a final and slightly more speculative section, a “What next?” section debating possible consequences, implications and applications of the obtained results, and pointing possible issues to be investigated in the future, resulting from the conclusions of present article. as stated previously, this is partially achieved in the section “Morphological characters in future specimen descriptions”, but these statements do not seem to be sufficiently highlighted. However, the lack of such hypothetical section does not compromise the interest of the present work.

As a possible flaw, in the Discussion section of the article I miss the inclusion of the genus Maea Johnston, 1865 in the debate about the possible validity of the genera previously assigned to the family Magelonidae, similarly to what is done with the genera Meredithia Hernández-Alcántara & Solís-Weiss, 2000, and Octomagelona Aguirrezabalaga, Ceberio & Fiege, 2001. The type species of the genus, Maea mirabilis Johnston, 1865, appears in both consensus trees in a clade clearly different from the one including the type species of Magelona, M. papillicornis F. Müller 1858. The consequences of such placement should be properly discussed in the text, as it is done with the other two genera synonymized with Magelona.

Finally, another strength of the article that I particularly appreciate as a taxonomist, is that after its reading it becomes clearly evident that the current Systematics needs to go one step further from the basic mechanics of describing taxa or delineating fast phylogenies, to address the epistemological sense of what is being done and, very specially, why it is being done. Only by doing this, Systematics can be considered as a fully established scientific discipline.
Additional comments: First, I would like to thank the Authors and the Editor for the given possibility of reading and reviewing the present work. I have read the manuscript with great interest and pleasure, and I think it is a very good work, very well-structured and thoroughly revised before its submission, which must be acknowledged. I did some comments and corrections directly in the provided pdf, as well as a couple of corrections in table S1, which will be provided to the Authors as attachments to this review. Please, check the attached pdf, and notice that the last three pages correspond to table S1, added to the pdf of the manuscript originally provided.

I would like to highlight some comments and corrections done in the provided pdf, as well as to discuss some other questions that I think the Authors need to consider and/or to address in order to improve the present version of the article. There isn’t a real order of importance of these questions, so they will be provided following the order in which they were noticed while reading the manuscript.

1) In lines 56–57 it is stated:

“Magelonids were given the rank of family by Cunningham & Ramage (1888) but their unusual morphology has often led to difficulties in relating them to other annelid groups. Johnston (1865), puzzled by their peculiar external form, placed them at the end of his catalogue, under the family Maeadae.”

This sentence is not completely correct, as the first author to recognize the uniqueness of magelonids in order to give them the rank of family was Johnston (1865), based on his new genus and new species Maea mirabilis, as Maeadae, a family name that would have priority. McIntosh (1877) included magelonids in the Spionidae, after synonymizing Maea Johnston, 1865 with Magelona Müller, 1858, but Cunningham & Ramage (1888) considered the magelonids to form a distinctive family again, this time based on Magelona, and once more relying on its unique morphology: "We have formed a special family for it, as it cannot be admitted into the Spionidae, with which it is most nearly allied, or into any other family."As stated above, the family name Maeadae would have priority over Magelonidae, but this latter name is to be used, according to article 40.2 of the ICZN. There is also a recommendation (Recommendation 40A), to cite the family-group name maintained under the provisions of Article 40.2.1, with its original author and date, followed by the date of its priority as determined by Article 40.2.1 enclosed in brackets. In case the Authors would like to follow this recommendation, or at least to refer it, the name of the family should be referred as Magelonidae Cunningham & Ramage , 1888 (1865).

2) Note that McIntosh’s 1877 paper "On the structure of Magelona" published in the pages of the Annals and Magazine of Natural History, Series 4, 20(116):147-152 is exactly the same work published previously the same year in the Proceedings of the Royal Society of London, 25(178): 559-564. It would be desirable to cite this latter paper, instead. As an alternative, the Authors can cite both papers, in the same way they are citing the German 1879 paper, and its 1911 English translation in the current article.

3) The references with the original descriptions of some taxa cited in the text are missing in the bibliography. Considering that each one of those names reflects a different hypothesis, please, include their full bibliographic reference. I have gone through the text and tables, and compiled the following list of missing references, but please, revise it:

Aguado & San Martín, 2004
Carrington, 1865
Clarke, Paterson, Florence & Gibbons, 2010
Ehlers, 1874
Ehlers, 1908
Eliason, 1962
Fauvel, 1928
Gallardo, 1968
Glémarec, 1967 [note: 1967, not 1966]
Gravier, 1905
Harmelin, 1964
Hartman, 1944
Hartman, 1960
Hartman & Reish, 1950
Hartmann-Schröder, 1962
Maciolek, 1985
Magalhães, Bailey-Brock & Watling, 2018
Malmgren, 1867
Mohammad, 1970
Monro, 1933
Mortimer & Mackie, 2006
Mortimer, Gil & Fiege, 2011
Müller, 1776 [note: O.F. Müller]
Müller, 1858 [note: F. Müller]
Sars, 1851
Sars, 1853
Shakouri, Mortimer & Dehani, 2017
Wesenberg-Lund, 1949
Zhou & Mortimer, 2013

4) Please, revise the in-text citations, as in some cases they do not seem to follow the journal’s reference format. For instance, for three or fewer authors, to list all author names, for four or more, to abbreviate with ‘first author’ et al. Moreover, multiple references to the same item should be separated with a semicolon (;) and ordered chronologically. According to the same reference format, for two or three authors, the last name should be preceded by “&”, instead of “and”. Please, revise also the whole reference section and edit it according to the journal’s reference format, and include the DOIs of the works cited, if available.

5) Outgroups – The work does not provide much information concerning the included outgroups, such as where the specimens were collected or the vouchers deposited, with the corresponding collection numbers. Please, revise this absence and include the corresponding and relevant information in Table 2.

6) In lines 988–989 it is stated:

“Where specimens were not available for observation, the remaining taxa were chosen based on the level of information and detail in the original descriptions.”

For completeness, and to clearly state which taxa were not considered, please, provide the full list of the taxa excluded from the analyses (as well as the full bibliographic reference containing the original description). This list seems to include the following valid taxa, but please revise and complete it, if necessary:

Magelona agoensis Kitamori, 1967
Magelona americana Hartman, 1965
Magelona capax Hartman, 1965
Magelona capensis Day, 1961
Magelona japonia Okuda, 1937
Magelona japonica koreana Okuda, 1937
Magelona kamala Nateewathana & Hylleberg, 1991
Magelona longicornis Johnston, 1901
Magelona methae Nateewathana & Hylleberg, 1991
Magelona mickiminni Nateewathana & Hylleberg, 1991
Magelona noppi Nateewathana & Hylleberg, 1991
Magelona pectinata Nateewathana & Hylleberg, 1991
Magelona petersenae Nateewathana & Hylleberg, 1991
Magelona pettiboneae lanceolata Jones, 1963
Magelona rosea Moore, 1907
Magelona sachalinenis Buzhinskaja, 1985

7) In lines 992–995 it is stated:

“All ingroup taxa included in the analyses are listed in Table 2, including which specimens were observed and the institution from which they were borrowed. Table 2 also indicates whether observations were based solely on published material.”

Some observations were based on type material, while others solely on published material. In what concerns this last alternative, only in one case it is stated that the observations were based on the redescription of the species, while other redescriptions also available apparently were not used (e.g., the redescription of Magelona pitelkai by Jones (1978)). Was there a reason for not using other redescriptions?

8) In table 3, the characters 26 and 27 are described respectively as “Chaetigers 1‒7 neuropodial lamellae position relative to notochaetae” [character 26] and “Chaetigers 1‒7 neuropodial subchaetal lamellae position relative to notochaetae” [character 27]. It seems to exist a lapsus in both character descriptions, and that they should state “relative to neurochaetae”, instead. Please, confirm if this is correct. In the Results section, revise also the discussion concerning clades a, b and r (lines 1589, 1596 and 1658, respectively).

9) This is probably irrelevant, but in several other publications covering the same taxonomic group, the expression “vis-à-vis” is written using Italics. Please, confirm that Italics is disregarded in the present article on purpose.

10) The distribution of the granular bodies present in the surfaces of the segments seem to be a promising character to be further investigated, while they also raise some questions concerning their possible nature and function, as in some occasions they seem to stain differently under the same stain. For instance, in some cases they stain as dark granular bodies with methyl green (e.g. Fig. 16A) while in other cases and with the same stain, they appear as white granular bodies (e.g. Figs. 16B, 16C). It would be interesting to apply different specific stains to the same specimens, in order to identify the molecular nature of those granular bodies and their possible function.

11) The figures are excellent, but in some cases the labels are difficult to see. Please, revise them and confirm that they are all clearly visible and contrasted in relation to the background. Use appropriate types of fonts and sizes and try using bold. Scales seem to be missing in some figures, please revise them and make sure they are always provided. If necessary, an approximative value can probably be stipulated for the scale. Please, try to standardize the type of scales used as much as possible. In some cases, the labels seem to have been partially cut (e.g., check the label “Scoop-shaped neuropodial lamellae” on figure 9H).

12) The figure captions needed to be standardized through the whole text. I tried to do it, according to the structure that seemed to be used most often by the Authors. Please, revise them and confirm that the same structure is uniformly used through the whole article, or change it accordingly to the Authors preferred structure, and use it consistently.

13) In line 1803 it is stated that in Brasil (2003) Magelona jonesi occurred in clade VIII.3, when it occurred in clade VIII.4.

14) In lines 1853–1855 it is stated, in relation to the genus Magelona F. Müller, 1858:

“The consequence is that ICZN (International Commission on Zoological Nomenclature, 1999)
Article 13.1.1, where a name must be ‘accompanied by a description or definition that states in  words characters that are purported to differentiate the taxon,’ cannot be reasonably applied.”

However, Article.13.1.1 applies only to names published after 1930, which is not the case of Magelona F. Müller, 1858. The articles that are relevant here, concerning the validity of the generic name, seem to be articles 11 and 12, and the name Magelona F. Müller, 1858 accomplishes both. Thus, the definition of the name Magelona could be the same as the used for Magelonidae, as both seem to represent the same phylogenetic hypothesis (i.e., Magelonidae as a monogeneric family, with Magelona as the only valid genus).

In the list of synonyms of the genus Magelona the genus Maea Johnston, 1865 is missing and should be added. Consider also the possibility of adding the name Rhynophylla Carrington, 1865 to the list.

15) Directly related with the previous point, why not to consider the definition of Magelona to be the same as Magelonidae? This way, it would not be an empty placeholder, as the family is clearly differentiated and defined. According to the results, and if I understood it correctly the same phylogenetic hypothesis defining Magelonidae, also defines Magelona.

16) There is an interesting issue that seems to have been overlooked. The Authors used the descriptions provided by Uebelacker & Jones (1984) of a series of taxa from the Gulf of Mexico which were described but not formally named by those authors.

Two of these species, Magelona sp. D and Magelona sp. H were later redescribed and formally named as new species by Hernández-Alcántara & Solís-Weiss (2001), as Meredithia spinifera Hernández-Alcántara & Solís-Weiss, 2001 [= Magelona sp. D] and Meredithia uebelackerae Hernández-Alcántara & Solís-Weiss, 2001 [= Magelona sp. H].

While the first species was described and named by Hernández-Alcántara & Solís-Weiss (2001) based on a mixture of material from the Gulf of California and the Gulf of Mexico (type locality = Gulf of California), with the material from the Gulf of Mexico being part of the material used by Uebelacker & Jones (1984) in their description, the second species was described based only in material from the Gulf of Mexico (type locality), all of which originally used by Uebelacker & Jones (1984) in their description. While each one of both duplets represents theoretically the same species hypotheses, they are not necessary grouped together in the two consensus trees provided in the work.

It can be argued that in the case of M. spinifera/M. sp. D, the differences obtained in the consensus tree of Brasil (2003) could be due to the mixture of specimens from two different populations isolated geographically in the description of M. spinifera. However, the differences obtained for the second duplet, M. uebelackerae/M. sp. H, can only be the result of the different ways that the same specimens were interpreted and their characters described by the different authors, and so, they can give an idea of the subjectivity introduced by different authors while studying the same subject, and how and where the species descriptions can be improved.

The analysis of the character matrix of the table S1 shows that there are more differences stated for the duplet M. uebelackerae/M. sp. H (using the same specimens in the two descriptions) than for the duplet M. spinifera/M. sp. D (using a different mixture of specimens in the two descriptions). The analysis of these differences and how they affect the way the duplets are grouped in the two consensus trees, can give an idea of how the interpretation of each one of those characters affects the results, and how and where descriptions should be improved in order to provide more accurate and reliable results. The analysis of the differences in the characters of the duplet M. uebelackerae/M. sp. H could be particularly interesting. Many of the observed differences seem to be related with the description of the morphology of the parapodia, the importance of which is precisely highlighted by the Authors in the present article

17) Finally, and as already observed in a previous section of the review, the genus Maea Johnston, 1865, with M. mirabilis Johnston, 1865 as type species, should also be integrated in the discussion concerning the validity of the different genera included in the family Magelonidae, performed after the subsection “Magelona F. Müller, 1858, emended” (line 1849).

The type species of the genus, Maea mirabilis Johnston, 1865, in placed in both consensus trees in a clade different from the one including the type species of the genus Magelona, M. papillicornis F. Müller 1858. The consequences of such placement should be properly discussed in the text as it is done with the other two genera synonymized with Magelona, especially with Meredithia, which synonymy with Magelona is confirmed.

Thank you!


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

Input:
Title: THE SYNDEMIC CONDITION OF PSYCHOSOCIAL PROBLEMS RELATED TO DEPRESSION AMONG SEXUALLY TRANSMITTED INFECTIONS PATIENTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: I think the author might consider two more factors: the type of STI and the severity of the STI.
Since they recruited participants with different STIs...

Does the severity of STI have an impact on depression tendency?
Is it possible that certain type of STI is particularly associated with depression? If so, what is the possible explanation?
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: THE SYNDEMIC CONDITION OF PSYCHOSOCIAL PROBLEMS RELATED TO DEPRESSION AMONG SEXUALLY TRANSMITTED INFECTIONS PATIENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Hello authors, I hope you're all safe and well. Overall the article is well written. Only few minor changes are required.

The references, mentioned at the end of their respective paragraphs/sentence, are written as for example (Hans and Kim, 2020). According to APA format 'and' is only written when the reference is mentioned in the sentence or para for example Hans and Kim (2020) mentioned....

Corrections that need to be made for those references have been provided (Details for some have been given form more clarity):

Line 71: (Han & Kim, 2020)
Line 72: (Sowislo & Orth, 2013)

Line 76: The reference in this line has been written as Gilbert and Allan in 1998 (Gilbert and Allan, 1998). The correct format has been provided which is:

first proposed by Gilbert and Allan (1998) and has been confirmed

The sentence from the article has been mentioned as well to provide more clarity of the correction.

Line 80: (Carrera & Wei, 2017)
Line 166: Cheung and Lau (1985)
Line 167: (Hu & Ai, 2016)
Line 180: (Hays & DiMatteo, 1987)

Line 184: In this line either the authors can mention the reference as Zhou and his colleagues (2012) or mention it as Zhou et al. (2012). Or rephrase it in a manner that they provide the reference at the end of the respective para i.e (Zhou et al., 2012). However according to the APA format they cannot mention both formats.

For example
This study uses a Chinese version of the ULS-8 Loneliness Scale. The translated version of the scale was created by Zhou and his colleagues (2012) for their own research on Chinese participants.

Line 190: (Gilbert & Allan, 1998)
Line 198: (Gilbert & Allan, 1998)
Line 205: Van Orden et al. (2008)
Line 308: play (it was misspelled as paly)
Line 310-311: (Orth & Robins, 2013)
Line 314: (Sowisho & Orth, 2013)
Line 324: (Gilbert & Allan, 1998)
Line 329: (Gilbert & Allan, 1998)
Line 335: 'developing depression' instead of 'development depression'
Line 345: ‘can’ instead of ‘and’
Line 348-349: (Griffiths, 2014; Griffiths, 2015)

Line 386: References

All the references mentioned under this line need to be corrected into APA format unless another format is being used, as required by the journal in which the article will be published. If the latter is the case then all the references within the article needs to be consistent with that format. Incase APA pattern is being used then refer to the APA manual 6th edition to make the necessary corrections

In the following feedback is provided for background and context:

Line 111-112: Although researches of the most common STIs have been provided, the study included participants with all forms of STIs. Mention the rationale for this using other researches.

Also in Table 1 titled;  Sociodemographic characteristics and their associations with depression among STIs patients in Shanghai, China

Under the heading  'Types of STI'

Various STIs have been mentioned in the category of others

Provide the process to explain how the STIs were chosen to come under this category with reference to previous researches.

Line 149: Provide a rationale for the use of DSM-IV criteria based test when DSM-5 exists

Since DSM-5 is now in use for the purpose of diagnoses and tests like Beck Depression Inventory (BDI-II), a screener, has also been updated in context of the symptoms provided by the DSM-5. A concrete rationale needs to be provided as to why this scale was used with reference to research.

Line 236-237: Six sociodemographic variables (age, gender, education, income, marital status, residence status) have significant effects on depression.

Provide research references for each of the demographics, mentioned.

Line 338: The coexistence of psychosocial problems is evidenced based on previous studies.

Mention references for those previous studies.

Line 338-339: The self-esteem antecedent model argues that self-esteem has an effect on social support

Provide reference for self-esteem antecedent model

Line 343: Provide reference for self-esteem consequence model
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: MASS MORTALITY OF PEARL OYSTER (PINCTADA FUCATA (GOULD)) IN JAPAN IN 2019 AND 2020 IS CAUSED BY AN UNIDENTIFIED INFECTIOUS AGENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This paper is devoted to identification of the causative agent of mass mortality in 0-year-old pearl oysters and anomalies in adults which occurred in Japan in the summer of 2019 and 2020. The incidence was named as “summer atrophy” to discriminate against the diseases reported previously such as Akoya oyster disease and marine birnavirus infection. Based on the filterability and sensitivity to chloroform of the pathogen, the causative agent was suggested to be a non-enveloped virus. Although this paper has potential contribution to the progress of our knowledge in marine science and aquaculture, some issues remain to be considered carefully as follows.

Major comments:
1)	It seems reasonable to conclude that the causative agent is a non-enveloped virus considering the results obtained in this paper. However, it may be more confirmative if the authors set up experiments with pathogenic samples after heat treatment to examine whether or not pathogenicity remains.
2)	It is curious about no apparent histopathological effects (lines 221 – 223). Figures 2 and 3 show severe changes in the structure of mantle accompanied by the formation of black spots and the loss of luster on the nacre. Is it relevant to have observed migrating cells in the connective tissues (probably in mantle)?

Others
Line 44: peaking -> attaining the peak.
Line 49: improve the sentence.
Line 64: improve the name of Fisheries Research Institute.
Line 73: and/or.
Line 91: running speed? Day and night period.
Line 106: explain 3+.
Line 182: examined -> observed.
Line 195: Why are the results not shown in Table?
Line 208: um -> (micro)m.
Line 267 – 269: reference is needed.
References: use the consistent format.
Table 4: ND data columns can be removed.
Figure 3: better to mark the places with deposition of brown pigment and loss of luster.


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: MASS MORTALITY OF PEARL OYSTER (PINCTADA FUCATA (GOULD)) IN JAPAN IN 2019 AND 2020 IS CAUSED BY AN UNIDENTIFIED INFECTIOUS AGENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. This paper has a substantial weak point, in which  "summer atrophy" of Akoya pearl oyster is not well defined and validity of diagnostic criteria used in this manuscript (nacre abnormality) is not clear. The characteristics of the disease is described in the introduction part, but no references are given. The authors should have described the disease more in detail with their own data or using references. I would like to recommend the authors give references for the description of the disease, mortality of affected oysters with the detailed description of methods to calculate the mortality. Although I guess that peer-reviewed references are not available as the disease is a newly emerging disease, any information sources including media release by local governments or special/temporal reports from local fisheries experts/organization should be given. I recommend the authors to compare the occurrence of nacre abnormality among much more Akoya oysters with atrophied muscle and healthy oysters and among areas/populations with/without mortality probably caused by the disease to give validity to the diagnostic criteria.

2.  Many incorrect or unclear expressions are found in English and scientific wordings. I strongly request the authors to have professional English editing for the revised manuscript.
I am sorry that I cannot give suggestions to improve the expressions, as my native language is not English.

3. Some other basic comments
1) Mortality of oysters at the collection sites should be given in Table 1 to the severity of the diseases in the sites, if available.
2) "average" is not a mathematical word. "mean" should be used in scientific papers.
3) Give the amount of diatoms given to oysters during the experiments.
4)  The authors stated that nacre abnormalities was used for diagnosis, as scarification of oysters was needed to know extent of atrophy(line 101-103). However, it is also difficult to examine the nacre abnormalities without killing oysters. It is unclear why the nacre abnormality was used for diagnosis.
5) It is unclear whether oysters were individually separated or the two groups were separated.
6) The results of Test 2 should be given in a table.
7) In Table 4, the lines of "ND" can be deleted.
Experimental design: The experiments are well designed.
Validity of the findings: The authors used student's t-test for comparison of shell score. However, the shell score is obviously non-parametric data, for which non-parametric statistical methods should be used instead of t-test.
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: MASS MORTALITY OF PEARL OYSTER (PINCTADA FUCATA (GOULD)) IN JAPAN IN 2019 AND 2020 IS CAUSED BY AN UNIDENTIFIED INFECTIOUS AGENT
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment.
Experimental design: Although the study provides convincing results on the nature of the etiological agent, further tests could have been carried out in order to infirm/confirm the nature of the agent. For example, PCR tests specific for birnaviruses/Akoya virus (if available), as well as using a virucidal treatment for the infectious hemolymph that would inactivate the agent not allowing infection in healthy oysters. Electron microscopy performed on infected oyster tissues would also provide useful information and strengthen the manuscript.
Validity of the findings: No comment.
Additional comments: I recommend the publication of this manuscript as it provides useful additional knowledge to the field of oyster diseases. Further work will hopefully lead to the identification of this potentially very serious etiological agent.


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: MASS MORTALITY OF PEARL OYSTER (PINCTADA FUCATA (GOULD)) IN JAPAN IN 2019 AND 2020 IS CAUSED BY AN UNIDENTIFIED INFECTIOUS AGENT
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The revised manuscript has been much improved based on the experiments of thermal treatment. As for the second major question of the reviewer, a certain metabolic change may have happened by summer atrophy disease, resulting in “increased numbers of migrating cells”. If so, not only histopathological experiments but also biochemical or molecular biological approach will be required. However, this paper contributes to the understanding of pearl summer atrophy disease of pearl oyster even at the present stage, because the pearl oyster aquaculture industries have not established yet any effective strategies against this enormous infectious disease.

Minor comments.
Table 4: death -> the dead?
Table 5: seriese -> series
Table 8: What is “suer atrophy” in the title?


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: MASS MORTALITY OF PEARL OYSTER (PINCTADA FUCATA (GOULD)) IN JAPAN IN 2019 AND 2020 IS CAUSED BY AN UNIDENTIFIED INFECTIOUS AGENT
Review round: 2
Reviewer: 2

Output:
Basic reporting: The usage of 'tense' is so unique that it may lead readers to misunderstandings.
They frequently use simple present tense to describe the observations and opinions of limited numbers of previous reports, which have not still been widely recognized as truth.
I think the authors should reconsider the usage of tense in this manuscript.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: GRAFT SURVIVAL OF PINUS ENGELMANNII CARR. IN RELATION TO TWO GRAFTING TECHNIQUES WITH DORMANT AND SPROUTING BUDS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The main issue is:

Most of the manuscript sections, as noted in the text, need to be significantly improved in English writing, with better order and organization for the sentences used. The reviewer made all necessary corrections and improvements and highlighted them in the various parts of the manuscript. All suggested corrections must be implemented, and the highlighted errors must be corrected.
Experimental design: No comment.
Validity of the findings: The main issue is:

The discussion section should be greatly improved. In other words, a more in-depth discussion is required for the majority of the results presented. Please take note of the highlighted issues and respond to the questions in the comments.
Additional comments: Dear respected author,

Thank you for your excellent scientific research. It is a valuable study, but it requires revisions based on the comments provided to you in the manuscript and this review form. All the issues raised should be addressed.

Sincerely,