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: EVOLUTION OF AFRICAN BARBS FROM THE LAKE VICTORIA DRAINAGE SYSTEM, KENYA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear language – yes
Intro and background to show context, literature well referenced: Yes
Structure conforms to PeerJ – yes
Figures relevant, high quality, etc. – mostly. The map figure needs work. I don’t know why the lake is a different color. Many of the river lines do not connect? Are they endorheic or is something missing in the GIS layer?
Raw data supplied: yes
Experimental design: Original research in scope – yes
Research question well defined – yes
Rigorous investigation performed to a high technical and ethical standard – yes
Methods described in sufficient detail – yes
Validity of the findings: Impact and novelty – largely a continuation of other small barb studies, but a welcome contribution to an area of the taxonomy that has not been well-studied.
Data is robust: yes
Conclusions well stated – yes
Additional comments: This is a good paper that continues the work on the small barbs of Africa. It is well done, and a welcome addition to the literature of the group. I have made some comments and suggestions throughout the pdf.  That said, the use of ‘Barbus’ is outdated. True Barbus is not even in the same tribe as the African ‘Barbus’. The only group still potentially advocating for ‘Barbus’ is Schmidt and Bart, and I even reviewed a paper by them recently using Enteromius. This is also confusing because, at one time, Labeobarbus was also referred to as Barbus in quotation marks and it is in yet another tribe as Barbus and Enteromius. The African Small Barbs will never be Barbus, and continuing to refer to them as such when there is a valid alternative is going backwards, not forwards. Not only is the use of Barbus in quotation marks backwards, the typesetting of it is difficult as well demonstrated in this paper. The Barbus part is supposed to be in italics, but the quotes are not. Other technical issues include: you have to spell out the genus name when using ‘sp.’ and the sp. is not to be italicized. The species listed as cf. guirali is probably aspilus. We did not find the two in the same clade, but they are easily confused. Make sure that you consult with Vreven et al. (2016) about Labeobarbus and Skelton et al (2018) about Pseudobarbus. Also note that these two reviews of African barbs by the top systematists working on African cypriniforms use Enteromius.

The map also needs some work. The lake should be the same color as the rivers, and it looks like at least some of the rivers that do not connect into anything are likely not endorheic.

Jon Armbruster

Skelton, P. H., E. R. Swartz and E. J. Vreven  2018 (2 Mar.) [ref. 35835] See ref. online
The identity of Barbus capensis Smith, 1841 and the generic status of southern African tetraploid cyprinids (Teleostei, Cyprinidae). European Journal of Taxonomy No. 410: 1-29.

Vreven, E. J. W. M. N., T. Musschoot, J. Snoeks, and U. K. Schliewen. 2016. The African hexaploid Torini (Cypriniformes: Cyprinidae): review of a tumultuous history. Zoological Journal of the Linnean Society 177:231–305.


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

Input:
Title: SPATIAL ANALYSES OF THREATS TO ECOSYSTEM SERVICE HOTSPOTS IN GREATER DURBAN, SOUTH AFRICA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is generally well-written and there are only few typos (see general comments). There are however several ambiguities concerning the names of the ecosystem services (ES): different terms are used in text, tables and figures. This needs to be corrected.

The text is less than average embedded in recent international literature on ES. Several key papers on ES hotspots are missing. Suggestions are made below. Some more background is needed for instance on what is an ES hotspot, how it is defined here, and what makes a hotspot different from an “ES area”.

The article is structured according to a basic scientific structure. Some raw data is shared, although it is hard to figure out what is being shown.

Self-contained: more details are need on the input data (ES maps), otherwise results are not reproducible.
Experimental design: The manuscript presents original primary research, despite being strongly based on results of an earlier study (Davids et al. 2016)
The findings are very case-specific, the study does not strongly relate to what has been discussed earlier within the ES field on hotspots, and the incorporation of threat in hotspots.
The analysis is a rather basic spatial overlay analysis. The assumptions on how each of the land uses poses a threat on each specific service need to be fed with more background, literature and basically a more thorough approach.
Methods are not replicable in their current form. Unclear what hotspots are, how ES maps were created, what indicators were used. Also unclear how threat and ES are linked, i.e. how potential impacts are delineated. More detail in comments below.
Validity of the findings: Given that methodology is better explained, the kind of results are robust, albeit simple in the form they are currently presented. A sensitivity analysis would be recommendable, with changing thresholds of what constitutes a “hotspot”.
Conclusions are incomplete and have little focus on the actual results.
Additional comments: This manuscript analyses spatial overlaps between spatial plans, their associated threats to ecosystem services (ES) and ES for the Greater Durban Area. The study builds on earlier ES assessment results and spatial development plans. In principle this is a solid synthesis of different data types through establishing a spatial link by potential threats. However, it is unclear how the ES data was derived, what thresholds were chosen and how the potential threats have been evaluated. There is no sensitivity analysis of the results, which hence are merely reporting percentages of spatial overlap. Furthermore, there are several inconsistencies in the use of terms. The study builds often on older references and does not adequately deal with recent developments within the field of ES, for instance on spatial priority setting through hotspots.
I will detail these points below.
Abstract: sustainable development/SDG are mentioned prominently here and in the introduction, but do not appear in the rest of the text. Please reconsider the framing, or add some analyses.
Please mention the five ES (called functions in the text) in the abstract
35-38 Please structure these examples according to standard classes (provisioning, regulating, cultural ES, or material, regulating, non-material nature’s contributions). Please refer to more recent literature, e.g. Diaz et al. 2018
39-40 the not on extinction rates is a side-line and not relevant here, please delete and focus the introduction on the relevant aspects that you aim to study (ES, threats)
62 Groot et al. 2017 – could not find this reference in the list (name would be “de Groot” I suppose)
65 beneficiaries: reference needed for each of these statements instead of a collection of references at the end of the sentence). Here appropriate is e.g. Baro et al. 2016
66 administrative boundaries: also here a reference is needed
71-81 This paragraph is rather vague and too general. It is not clear how it contributes to developing the storyline.
105 Please define what priority areas or hotspots are, how they have been defined and delineated.  Please also refer to relevant literature. E.g. Schröter and Remme 2016 (a review on ES hotspots), Schröter et al. 2017, Egoh et al. 2008
107-110 The authors distinguish different ES according to who beneficiaries (or to which type of infrastructure is protected). This is very unusual. I would argue that the five “functions” are actually the ES, and then this analysis could as well be based on just these five. Much more detail is needed on how these maps were derived, what indicators were used, and what thresholds were used to define what is a hotspot.
116 typo assess
131 EMA needs to be explained
150 D’MOSS is introduced for the second time
192 hotspot richness needs to be defined.
194 How were areas defined here? Are these any areas where there is more than zero of the specific ES provided?
How were ES measured and standardised?
210 More detail is needed on the proposed land use changes.
246-250 Much more detail is needed on how compatibility was assessed. This was probably done through expert judgement. Still, each LU change can affect each ES differently, and a critical reader would want to see a bit more background on how these assumptions were made.
250-251 Terms like “environment”, “amenity” are to unspecific and need to be explained.
259 addition of nutrients is not a decline in an ES (retention)
266-268 Names of ES are inconsistent with Table 1 and Figure 1. This is just one example. Please use consistent terms throughout.
268 “analysed groupings”: it is unclear what was done here, reproducibility not given.
270-271 “maximum values” unclear what has been done here. Please explain this thoroughly.
 279 point file is jargon, please avoid. Also here it is not clear what analyses have been done. A point has no spatial dimension, so what was assessed in relation to the ES maps?
370 typo Rockström
383 “is expected to increase” this has not been analysed here, so it seems irrelevant for the discussion.
396 Reference is inappropriate here.
397 Water is not “produced”, but rather flowing through the system.
401-403 This is only stating the obvious, could hence be deleted.
405-414 This is too general, rather specific impacts of specific threats on specific ES should be discussed (as this also would address the uncertainty in the delineation of threats to hotspots).
422 potential inaccessibility: is there any proof for this, maybe a spatial analysis?
427-439 Invasive species have not been assessed, analysed in any way as far as I could follow, so it is unclear why this is mentioned here. Suggest to delete.
456 Please mentioned that “potential” threats are meant, as there has been no assessment of the actual impact of the threat, nor provided reasonable backup by literature.
Table 1: suggest to merge 13 to five ES, and explain at least shortly methods and indicators used here.
Figure 1: phosphorous is also a nutrient, so it is not clear why this is separate here. Also, the names are not consistent with the text and the table.
Figure 2 typo hectares
Table 2: 60% and 47% (bottom row) of what?
Table 3 abbreviation EIA needs explanation

References suggested
Díaz, S., U. Pascual, M. Stenseke, B. Martín-López, R.T. Watson, Z. Molnár, R. Hill, K.M.A. Chan, I.A. Baste, K.A. Brauman, S. Polasky, A. Church, M. Lonsdale, A. Larigauderie, P.W. Leadley, A.P.E. van Oudenhoven, F. van der Plaat, M. Schröter, S. Lavorel, Y. Aumeeruddy-Thomas, E. Bukvareva, K. Davies, S. Demissew, G. Erpul, P. Failler, C.A. Guerra, C.L. Hewitt, H. Keune, S. Lindley, Y. Shirayama, 2018. Assessing nature's contributions to people. Science 359, 270-272.
Baró, F., I. Palomo, G. Zulian, P. Vizcaino, D. Haase, E. Gómez-Baggethun, 2016. Mapping ecosystem service capacity, flow and demand for landscape and urban planning: A case study in the Barcelona metropolitan region. Land Use Policy 57, 405-417.
Schröter, M., R.P. Remme, 2016. Spatial prioritisation for conserving ecosystem services: comparing hotspots with heuristic optimisation. Landsc. Ecol. 31, 431-450.
Schröter, M., R. Kraemer, S. Ceauşu, G.M. Rusch, 2017. Incorporating threat in hotspots and coldspots of biodiversity and ecosystem services. Ambio 46, 756-768
Egoh, B., B. Reyers, M. Rouget, D.M. Richardson, D.C. Le Maitre, A.S. van Jaarsveld, 2008. Mapping ecosystem services for planning and management. Agric. Ecosyst. Environ. 127, 135-140.


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

Input:
Title: SPATIAL ANALYSES OF THREATS TO ECOSYSTEM SERVICE HOTSPOTS IN GREATER DURBAN, SOUTH AFRICA
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This is a very good paper, suitable for publication in this journal. Indeed we need more like this for other large South African cities - well done.


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

Input:
Title: SPATIAL ANALYSES OF THREATS TO ECOSYSTEM SERVICE HOTSPOTS IN GREATER DURBAN, SOUTH AFRICA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript has clearly improved after amjor revisions. I have no further comments to make.
Experimental design: The manuscript has clearly improved after amjor revisions. I have no further comments to make.
Validity of the findings: The manuscript has clearly improved after amjor revisions. I have no further comments to make.
Additional comments: The manuscript has clearly improved after amjor revisions. I have no further comments to make.


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: INVESTIGATING LEAF BEETLES (COLEOPTERA, CHRYSOMELIDAE) ON THE WEST COAST ISLANDS OF SABAH VIA CHECKLIST-TAKING AND DNA BARCODING
Review round: 1
Reviewer: 1

Output:
Basic reporting: There are too many linguistic errors so the paper should be checked by an English native speaker.
Experimental design: The experimental design looks sound but lacks details. For instance, were the samples taken from disturbed habitats (secondary forests, plantations)? primary rain forest ? if yes, please add that information to the checklist and indicate whether there is any difference in species richness between different habitats?
Validity of the findings: The DNA barcoding analysis is well conducted and the presentation of the results is adequate but please see comments below:

1. The values of Table 2 on sequence nucleotide composition are shown again in the main text  (Lines 184-190). I fin dit completely uninformative and unless these results are compared and discussed with other groups I would remove it.

2.	I find Table 3 uninteresting. What does it show ? that using genbank is not the best way of identifying tropical leaf beetles ? Instead of hits to genbank I would show the BIN and ID of the nearest Neighbor and also the distance to Nearest Neighbor.

3.	 It would be interesting to have a table with the number of species/BINs per island and number of those that are shared with other islands and with the mainland.

4. The checlist only shows distribution in Sabah, but what is the geographical distribution worldwide ?

5.	I wonder about the biogeographical composition of the fauna discovered. is exclusively oriental ? what are the 4 non-unique BINs already present in BOLD come from ? do they occur in a diffrent biogeographical region ? are there any cosmpolitan/invasive/alien species (Brontispa ?)?

6. These are important phytophagous isnects yet nothing is said about their host plants. Please add any information known on host plant use from literature and new host plant records gathered during fieldwork if any.

7. Photos of adult vouchers are already available in BOLD via the DOi number of teh data set, so I wonder whether it is worth including them in the manuscript
Additional comments: The study is very interesting because it focuses on an important hot spot of biodiversity and deals with a hyperdiverse group of insects. In addition, it contributes with a significant amount of novel DNA barcodes.


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: INVESTIGATING LEAF BEETLES (COLEOPTERA, CHRYSOMELIDAE) ON THE WEST COAST ISLANDS OF SABAH VIA CHECKLIST-TAKING AND DNA BARCODING
Review round: 1
Reviewer: 2

Output:
Basic reporting: This study investigates leaf beetle diversity in 13 islands in Sabah area of Borneo. The species diversity was assessed based on morphological sorting and DNA barcoding.
The language of manuscript's main text appears fluent and good (though I am not a native English speaker), but the checklist part suffers from many small glitches (indicated below).

Introduction is quite specific and mostly deals with the history of leaf beetle research in Borneo. It would benefit much from a broader perspective to the topic. Why was this study conducted? Is it perhaps a part of a larger mission to investigate (leaf) beetle diversity in Borneo? Or globally? Please put your study to a broader context. There are large-scaler studies of tropical diversity (also in Borneo) that are not referred to. Is this study somehow related to any larger biodiversity project?

The figures are nice and informative. Table 2 does not contain, to my opinion, very relevant information and could be omitted (the meaning of these results are also not discussed).

The reference of Puillandre et al 2012 in line 234 appears incorrect. I cannot see how this reference is related to the text before it.

In the "checklist part", food plants are given for some, but not to all species. I wonder if this information was given for all taxa for which it was known.

Minor comments:
Line 203: Genbank > GenBank
Line 208: The dominant leaf beetle... > The dominant leaf beetles...
Line 275: both species not > both species have not been
Line 276: in GenBank probably > in GenBank are probably (verb missing)
Line 298: Differentiate from > Differentiated from
Line 554: Found resting on the beach Ipomoea > Found resting on the beach on Ipomoea
Line 634: Possible exhibit sexual > Possibly exhibits sexual
Similar small error are listed here are many more inthe checklist part.
Experimental design: The data is novel. Research questions are not very clearly stated and put to a larger context. Actually, in the introduction, no research questions are presented, but the authors just state that this study documents the species richness in 13 islands. It seems that large part of the observed species are new, non-described species, but this does not become very clear as the observed operational taxonomic units (OTUs) were identified by BLAST search only (?). This should be discussed more.

Material and methods should be more detailed. For example, it remains unclear how many plots were sampled in each island. Was handpicking of specimens conducted in the same plots? Were different plants in the plot systematically examined? How were the specimens "identified to the genus and species level" as most of the specimens remained un-named Do the author actually mean that the specimens were sorted into putative species (operational taxonomic units) this way? Was it really so that this sorting yielded in exactly the same delineation of OTUs as DNA barcodes, or were the final OTUs formed as a consensus of morphological sorting and DNA barcodes? It seems that the final delineation of specimens is exactly the one that was suggested by DNA barcodes, and it remain unclear of the morphological sorting conducted beforehand agreed completely with that.

The OTUs were identified based on the BLAST search (only?). I wonder why did not the authors use BOLD search, because BOLD has lots of records that are not present in GenBank and the data is cleaner (the authors acknowledge the problem of many misids in GenBank). BOLD identification tool would be a good option, and, importantly, all records (incl. unpublished) are available for identification. In the results section, the authors state: "including 68 species in 33 genera [was discovered]". However, I find that the authors should not speak about species at all, as they could not name the taxa. They should instead call them as putative species or OTUs. The authors also compiled a "species checklist", but it is not a true checklist as many OTUs were identified to the subfamily or genus level only. The inconsistent use of the term "species" is also reflected elsewhere. For example, on lines 226-228 the authors state: "...these five species are morphologically hard to distinguish from one another and thus, all these five species are...". Again, the authors should speak about putative species or OTUs. The use of the term "species" for the clusters should be revised throughout in the manuscript.
Validity of the findings: It seems that many new species were discovered, although this remains unclear, as it is not stated if the authors tried to get names for their OTUs in any other way but by BLAST search. The authors did little to assess/extrapolate how large part of the total leaf beetle diversity was discovered. The BLAST hits of less than 95% match often have little meaning. In some cases, the closest hit is a moth (Agrius convolvuli), in many other cases the closest hit is only to a subfamily level. Actually, only in a very few cases the BLAST search yielded really useful information. This calls for more discussion on the identities of the species, for example if the poor success is because the fauna of the region is so poorly represented in GenBank, or if this is because high levels of endemism in the region.

The conclusions are virtually lacking. Like introduction, the discussion part is quite specific. A broader scope here would be very beneficial for the whole paper.
Additional comments: This paper is with publication potential, but suffers mostly from a very narrow perspective to the topic. The paper would greatly benefit from wider perspective on tropical diversity and questions related to it. Also, the rationales for the study should be clarified. What motivated the authors to make this study? There are some weaknesses in the methodological part as well. They should be addressed. After a major revision, I believe that the paper would become acceptable for publication in PeerJ.


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

Input:
Title: INVESTIGATING LEAF BEETLES (COLEOPTERA, CHRYSOMELIDAE) ON THE WEST COAST ISLANDS OF SABAH VIA CHECKLIST-TAKING AND DNA BARCODING
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is a complicated case. I spent one hour just digesting the abstract and introduction, and find that this manuscript would benefit from a thorough and careful editing / rewriting process. This I cannot deliver as a reviewer. For some issues, please refer to the "General comments for the author" section. I stopped reviewing the text after the end of the introduction for these reasons.

In general, I like this study as any advance in molecular biodiversity assessment is most welcome and urgently needed to build the global database needed to address the challenges of environmental degradation and ill directed political decisions.

However the current study lacks sound hypotheses and remains of very little relevance for a broader audience - with only 101 sequences from perhaps 68 species. Yes, the study is set on islands, but all of these were very small and very close to mainland, so that I also doubt that this work has particular relevance for island biogeography. It would certainly be interesting to see how many taxa were actually island endemics, I would suspect none. This would require in depth sampling of mainland areas.
Experimental design: See above, I think this is original primary research, yes, but needs to be much better presented and integrated in a larger scale framework. Without substantial sampling from the mainland, this remains a study with no obvious hypothesis testing or scientific question.
Validity of the findings: See above.
Additional comments: Language:
Overall, the paper needs substantial proofreading. I only provide some examples in my review, but this should help to understand where more work is needed.

Abstract:

The summary starts chaotic and needs to be rewritten.

Sabah is a province of Malaysia, and part of the island of Borneo. The current study looks into the leaf beetle fauna of a few very small satellite islands off the coast of Sabah.

"Sabah, northern Borneo is one of the world’s most well-recognized biodiversity hotspots" is a bit confusing and exaggerated a lot.

First of all, please study the definiton of biodiversity hotspot: https://en.wikipedia.org/wiki/Biodiversity_hotspot

I would write something like: Borneo is one of the World's truly megadiverse areas, yet its arthropod fauna remains largely unknown.  Here, we present a study.....

"Plenty of studies...." better say: Several studies... Plenty is a lot and not appropriate wording here.

"In this study, we collected leaf beetle fauna from 13 islands..." better: We sampled leaf beetles from 13 islands....

"when the species names could not be determined" better: when the samples could not be identified to species level using the existing literature

"In addition, DNA barcodes – mitochondarial COI gene – of the species were sequenced." better: In addition, we generated DNA barcodes for the speciemens.

"A total of 68 species from 31 genera and 5 subfamilies were collected with 12 species name being determined." better:

"We collected 68 putative species from 31 genera and 5 subfamilies according to morphology, 12 of these could be identified to species level."

"In addition, DNA barcoding also reveals phenotypic variation
in leaf beetle species, particularly in the case of the subfamily Galerucinae"

Was that not known long before?

"This study provides baseline knowledge and information about the DNA barcodes of leaf beetle species on Sabah’s island habitats for use in future studies."  This is not very appropriately in wording.

The study provides the first DNA barcoding data for leaf beetles from this part of the World, with the next steps being larger scale sampling across a wider geographic area in order to better understand large scale patterns of tropical arthropod diversity.

Introduction:

Generally, there are too many references.

"Although Borneo is recognized as one of the world’s biodiversity hotspots," see my comments above.

"taxonomic research on Borneo leaf beetles has been limited to a few publications,"  In the abstract it is said there were "plenty" of studies.

"possibly due to logistical difficulties and the inaccessibility of forest habitats" I am not entirely sure what this means. Yes, Bornean forests are in decline, but it should be really easy to local researchers to access forest, even in these days and time and even outside national parks I would suspect.

"Although Sabah has the highest number of islands in Malaysia, leaf beetle species diversity on islands has been little explored. In view of the fact that island habitats are generally known to have high species endemism"

Yes, but most of the islands studied here are very small and very close to the mainland. The most remote island visited here is Mengalum Island and only 50km offshore Kota Kinabalu. The scale in Fig 1A does not seem to be correct.

"To date, there are 73 records of leaf beetles with 15 Barcode Index Numbers (BINs) registered in the Barcoding of Life Data system (BOLD), but none of these records are from Sabah or Borneo."

I checked. http://www.boldsystems.org/index.php/Taxbrowser_Taxonpage?searchMenu=taxonomy&query=Chrysomelidae&taxon=Chrysomelidae

Specimen Records:	40,613
Public BINs:	2,034

"For all the reasons stated above, this study (1) documented the species richness of leaf
beetles from 13 selected islands on the west coast of Sabah, and (2) sequenced DNA barcodes of the leaf beetles to provide phenotypic polymorphism information and baseline DNA barcoding knowledge for future taxonomy study."

This conclusion of the introduction says all and nothing. I would suggest to also reword this to better reflect the aims of the study and hypotheses to be tested.


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: RECENT ADVANCES AND CHALLENGES ON APPLICATION OF TISSUE ENGINEERING FOR TREATMENT OF CONGENITAL HEART DISEASE
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a nice review article on the treatment of congenital heart disease using tissue engineering approaches, including describing the different types of stem cells that could be used.

I have the following suggestions to improve the article:

1. Line 83, the authors state that 'Several materials and biomaterials have been used in the surgical management of CHDs...' Could a couple of examples be described?
2. Line 86, write Tissue Engineering in full the first time it is mentioned
3. Line 101, 'This evidence shows the existence of Lin c-kit+ cells which confirm cardiomyocyte proliferation and cardiac regeneration'. Considering the confusion over the phenotype of a cardiac stem/progenitor cell, I suggest to re-write this sentence as 'This evidence shows the existence of cardiac stem/progenitor cells which can differentiate into new cardiomyocytes and participate in cardiac regeneration'.
4. Line 102, Could the authors give the original reference (Porrello et al. 2011, Science) and describe in more detail that the neonatal 1 day old Mouse heart can regenerate after partial surgical resection, but this capacity is lost by 7 days of age, and in adulthood.
5. Line 134, define Cyanotic and Acyanotic
6. Line 125, 3.0 Congenital Heart Disease: Types, malformations and surgical intervention. It would be good if a Table could be inserted that summarises the types of CHD, and their manifestations.
7. Line 170. Reword the sentence beginning 'The main challenge....' to 'The main challenge with artificial materials is alterations in the size and function of the heart from neonate, infant to adulthood, which current artificial material cannot adjust too.'
8. Line 225, provide clarity on '...about the actual function of cells following citation on the clot'.
9. Line 309-311, explain why PLLA combined with BM-MSC has demonstrated good results for vascular tissue engineering, and in which context - in vitro or in vivo, and which type of CHD?
10. Line 372, write in full SC first time it is written.
11. Line 373-374, describe in more detail the results of study reference 110. Which in vivo disease model was this tested in?
12. Line 436, change great to good
13. Figure 1 shows the different stem cells and their differentiation into cardiomyocytes, however what about endothelial cell and smooth muscle cell differentiation? And also this cartoon of differentiation is not reflective of the text. More literature needs to be described on ESCs, iPSCs, UCB, AFSCs, BM-MNCs or MSCs, CPCs ability to differentiate into cardiomyocytes in vitro and their cardiac regenerative potential in in vivo animal models, and preferably CHD models.
14. Please state for each study described whether the work was carried out in vitro or in vivo
15. Line 538, state the phenotype of CPCs and the markers they express as well as c-kit, which are Sca-1+, PDGFr-alpha+, CD31-, CD34-, CD45-, tryptase-. See and reference Vicinanza et al. 2017, Cell Death & Differentiation.
16. Line 538, change 'proliferating' to 'self-renewing'
17. Line 539, re-write 'characteristics; differentiating into all three cardiogenic cells types, which are cardiomyocytes, smooth muscle cells and endothelial cells'. Note the change of epithelial cells to endothelial cells.

18. Line 74, CDH should be CHD
19. Line 202, insert 'the' between repairing and heart.
20. Line 203, change have to has
21. Line 210, change was to is
22. Line 236, change by to from
23. Line 252, change contacted to carried out
24. Line 283, remove s
25. Line 353, change myocardial to myocardium
26. Line 362, should hemostasis be homeostasis
27. Line 416, 'write An equally important point in choosing a suitable biomaterial....
28. Line 418, abbreviate cardiomyocyte to CM not CMC
29. Line 468, change 'were able to make a' to 'showed'
30. Line 485, change form to from
31. Line 526 change 'in lamb of animal models' to 'in a lamb animal model'.
32. Line 557, Figure 1 legend, change will to could
33. Line 558,  Figure 1 legend, change This to These
34. Line 559, Figure 1 legend, remove 'a' and 'growth'
35. Line 569, change lays to lies
36. Line 573, remove 'with'
37. Line 583, remove 's' from where is written 'the best SCs candidates..'
38. Line 585, insert the word 'enable' between 'to' and 'deeper'
39. Line 586, change understand to understanding
40. Define abbreviations used Figure 1 in legend
Experimental design: Not applicable as it's a review
Validity of the findings: Not applicable as it's a review
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: RECENT ADVANCES AND CHALLENGES ON APPLICATION OF TISSUE ENGINEERING FOR TREATMENT OF CONGENITAL HEART DISEASE
Review round: 1
Reviewer: 2

Output:
Basic reporting: This review article is timely, and of broad and cross-disciplinary interest. It is written clearly and unambiguously, using a professional English mostly throughout (some minor phrase structure revisions are advised). In addition, the manuscript's structure should be revised to comply with the publication's format (add "Survey Methodology" section, use the correct section numbers).
However, in its substance, this manuscript does not represent a meaningful addition to other relatively recent literature reviews, which have approached the exact some subject very thoroughly and with a clear point of view.
Experimental design: no comments
Validity of the findings: As this literature review is limited to briefly state the recent advances on the application of stem cells and tissue engineering approaches towards CHD treatment, with minimal critical analysis of the previous art, it is difficult to impress the reader the huge potential (and corresponding challenges) of the field, now and in the future.
Additional comments: In the manuscript entitled 'Stem cells, tissue engineering and congenital heart disease', the authors aim at providing an introduction to the pathology followed by a throughout overview of the many approaches using stem cells and tissue engineering methods to develop feasible therapies. The authors provided an ample array of such examples, however there is an inherent lack of structure to this review that hampers its potential impact. Importantly, the authors tend to dump information with little historical or methodological context, making very difficult to the reader to understand why a particular approach was developed and used, what are its strengths and weaknesses, and where to go from there (i.e. what possible future it might have in the clinic).

Moreover, the authors tend to exercise only a minimal critical analysis of the many studies they refer, going so far as to cite verbatim without further insights (e.g. '...It was concluded that heart “ECM could promote proliferation, specific cell differentiation and myofilament formation” [113].') A more carefully prepared review should have a personal point of view to highlight, using the available literature to support their position and guide the reader; as it is, this review is limited to be just an enumeration of previous studies.

Fortunately, these issues can be addressed by improving the structure of the manuscript, e.g. by presenting the work in an historical context, and/or by analyzing the described advances/innovations more critically, e.g. to highlight those showing better outcomes/higher accessibility to the clinic/more robust and diversified applications/better promise considering their specific challenges and limitations. The addition of a table for 'Frequent types of CHDs, their main challenges and corresponding therapeutic approaches so far', as well as a revised table 2 compiling a more exhaustive list of examples, grouped by type of engineered tissue or by type of biomaterial or stem cells used.


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

Input:
Title: RECENT ADVANCES AND CHALLENGES ON APPLICATION OF TISSUE ENGINEERING FOR TREATMENT OF CONGENITAL HEART DISEASE
Review round: 2
Reviewer: 1

Output:
Basic reporting: NA
Experimental design: NA
Validity of the findings: The authors have responded to my comments and made changes, except 1.

When the authors describe the Cardiac progenitor cells starting line 517, the CPCs are CD34-negative, tryptase-negative (please correct, line 519), and they should reference the paper by Vicinanza et al. 2017, Cell Death & Differentiation, which is the most up-to-date description and characteristics of the CPCs. The authors should also describe the in vivo experiments in Vicinanza et al. 2017, Cell Death & Differentiation which show the regenerative potential of clonogeneic CSCs following transplantation after MI.
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: RECENT ADVANCES AND CHALLENGES ON APPLICATION OF TISSUE ENGINEERING FOR TREATMENT OF CONGENITAL HEART DISEASE
Review round: 3
Reviewer: 1

Output:
Basic reporting: I am happy with the revisions.
Experimental design: n/a
Validity of the findings: n/a
Additional comments: n/a


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

Input:
Title: MOLECULAR ASSESSMENT OF POCILLOPORA VERRUCOSA (SCLERACTINIA; POCILLOPORIDAE) DISTRIBUTION ALONG A DEPTH GRADIENT IN LUDAO, TAIWAN
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript Molecular assessment of Pocillopora verrucosa (Scleractinia; Pocilloporidae) distribution along a depth gradient in Ludao, Taiwan by De Palmas et al. present basic and important evidence of vertical distribution of different haplotypes belonged to P. verrucosa and other two species with similar morphology. Additionally, this study documents the frequency of misidentification of Pocillopora species according to morphology. Although the manuscript is well organized in sections, also had some issues that should be addressed before publication (see below).
Experimental design: Authors identified Pocillopora species identity using mtORF and RFLP methods of Histone region. Overall the methods and techniques used are suitable for addressing the question they are asking. Although the amplification of ORF is well validated for identification of Pocillopora species through the Indo-Pacific, Histone region has been only tested and validated in Hawaii. I recommend at least have one or two histone sequences from each sample identified by using ORF marker. This will help in future studies comparing Pocillopora species in several regions.
Validity of the findings: This study is particularly important to untangle the ecological differences of Pocillopora species and the method that is described in the manuscript might be replicable in other regions.
Additional comments: Minor comments
Line 71. Most of the recent genetic studied divide the genus Pocillopora in similar genetic lineages and species. However, the denomination of each group change depending of the author. Please specific the publication that you are citing. For example: According to Schmidt-Roach et al. (2014), Clade 1 is composed of . . .
Although the authors indicate the identity of the clades with the haplotypes of ORF in the manuscript. I suggest use the current valid name of the species identified by molecular methods along the manuscript, figures, supplementary material, etc. This will help to avoid confusion in the scientific literature. For example P. verrucosa (Clade 2), complex of species P. meandrina and P. eydouxi (clade 3) and Pocillopora sp. (Type 8).

Line 90-92 Some changes in the colonial and branch morphology are associated to light and high or low current conditions. Please review the following papers: Kaniewska et al. 2008 (Mar Biol 155:649–660) and Paz-García et al. 2015 (Oecologia 178: 207–218).

Line 129 Change incubation by extension.

Line 275-276 Please indicate the meaning of DRRH in the first mention in the text.
Line 289	Please indicate the meaning of MCEs
In figure 2 use name of the Pocillopora species and in parenthesis the number of the clade


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

Input:
Title: MOLECULAR ASSESSMENT OF POCILLOPORA VERRUCOSA (SCLERACTINIA; POCILLOPORIDAE) DISTRIBUTION ALONG A DEPTH GRADIENT IN LUDAO, TAIWAN
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment (see general comments)
Experimental design: no comment (see general comments)
Validity of the findings: no comment (see general comments)
Additional comments: Review - Molecular assessment of Pocillopora verrucosa (Scleractinia; Pocilloporidae) distribution along a depth gradient in Ludao, Taiwan

General comments:  This study examined the depth distribution of Pocillopora in Taiwan, focusing on P. verrucosa- like morphology. The genus is notorious for morphological plasticity, therefore molecular markers (specifically a mitochondrial marker and nuclear marker RFLP assay) were used to confirm species identity and examine haplotype differences across the sampled range. The findings indicated that P.verrucosa is distributed across a broad depth gradient, with no clear genetic differentiation across depths, implying that for this species deeper waters may serve as a refugia. P.meandrina was also rarely misidentified in ~10% of the samples and appear to be more restricted to shallow waters, although additional studies are needed to confirm this.  Overall the manuscript is extremely well written, very concise and very well done. It is rare for me to not be able to make suggestions to improve the manuscript, but I think this paper is suitable for publication after some minor suggestions.

Specific comments:

I don’t think the subheadings in [] are necessary. I would leave them out.

Line 76: P. cf. effusus not effuses. Autocorrect always changes it to effuses so watch out.

Line 77: an unclear position into the Pocillopora phylogeny

Line 98: the degree of overlap

Line 108: Subsamples were collected from each fragments

Line 168: which were targeted in this study.. Delete extra period.

Line 169: Haplotypes H4 was found at 23-30m in depth, and H5 was found at 23-30m

Line 204: How about “However, they have since been identified at other locations such as Reunion Island…”

Line 207: more cosmopolite than previously though. Use thought, not though.

Line 219-220: Haplotype H8 was not found at 15m, but we suspect that it could be present at all depth since we found it at all other depths.

This is a little confusing. How about “In our study haplotype 8 was not found at 15m. However, we suspect that this haplotype could be present at this depth given that it was collected at all other depths.”

Line 233: Our data corroborate the presence of P. verrucosa in the mesophotic zone of Ludao  (Denis et al. In press).

Line 248: deep community composition has been shown to

Line 251: species that contributes to this community overlap

Line 280: the ability of an offspring from deeper habitats to survive in the shallows, and the fecundity of deeper organisms are premises to this hypothesis.

What do you mean here about the fecundity of deeper organisms? It is not clear how this fecundity contributes to the deep reef refugia hypothesis.

Line 286-287: The recovery of this species could potentially then rely on the recruitment of coral larvae

Lines 308-310: In this case, a molecular approach is required to correctly and quickly delineate coral species and  provide a better understanding of coral species biology and ecology.

Lines 314-315: …to evaluate the contribution of shallow and deep populations to recruitment and population maintenance.

Line 315: While  molecular approaches have been used

Figure 2:
  It would be more useful if the color of the circles surrounding the clades was the same color as the writing. For me it looks like the writing “Type 8” is in brown but the circle around the haplotype is grey.

Table 1:
 Summary of haplotype diversity per site, corresponding to literature references, and
 their geographic  locations previously collected.

It would also be nice to have the species name following Schmidt-Roach et al. (2014) in the table so that people less familiar with all the various types could more easily understand what species these haplotypes belong to.

I do not feel that Table 2 is needed because it presents the same data as Figure 2 but without the genetic distances.


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: PHYLOGENOMICS PICKS OUT THE PAR EXCELLENCE MARKERS FOR SPECIES PHYLOGENY IN THE GENUS STAPHYLOCOCCUS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Some minor spelling and grammatical issues, but English was clear and understandable. Raw data was shared.

Fig. 1 - do the authors have left and right the correct way around here? They say the ML phylogeny with the scale bar is on the right, but it is shown on the left. And that on the left hand side, numbers by the nodes reflect number of SGF trees having that partition, but the tree with the numbers by nodes is on the right. Also, what are the coloured branches supposed to signify?

Fig. 2 - the authors refer to RB distance in the legend, rather than R-F.

Supplementary table 3 would benefit from gene names (or TW locus numbers) for readers trying to find an individual gene in that table.

Supplementary table 4 lists FDR and P-values - it would be useful if the authors stated how many homology groups belonged to each GO.
Experimental design: The research question was well defined. The phylogeny provides an interesting update on previous studies, given the expansion of the number of described staphylococcal species in recent years.

However, the authors fail to justify their exclusion of a large number of orthologous genes from inclusion in the dataset used to generate the final concatenated "species" super-alignment phylogeny, that individual  genes are tested against. Just because the individual genes' phylogenies differ significantly from the "species" phylogeny should not be sufficient on its own. Genes may evolve at different rates - if they are orthologous and single copy with no evidence of recombination, they shouldn't be excluded just because their phylogenies disagree from the species phylogeny. It's fine not to select them when considering individual genes to use as markers, but they should be included in the species phylogeny. On lines 164-165, the authors state "it is remarkable that both the super alignment and the consensus tree produced the very same topology" - but is it really remarkable, if you've removed all the genes whose phylogenies disagree with the super alignment phylogeny?
Validity of the findings: Lines 169-171: The authors state "as expected many of the enriched biological processes and molecular functions have to do with housekeeping functions..... Taken together, these results demonstrate that the 275 seem to be real orthologous groups". What gene ontologies did the homology groups that were discarded (because their phylogenies differed significantly) belong to? Do they differ to the "real" orthologous groups?

Lines 180-181: The authors state "every single orthologous group has its own topology - not shared by any other orthologous group", but there didn't seem to be any data presented to support this.

The authors also seem to contradict themselves in places, depending on the argument being made at the time. Line 209, they state "these two genes show a rather low genetic diversity". One of those genes, dnaJ, was amongst their top 17 markers. Lines 185-186, the authors had stated: "these 17 orthologous groups present very decent values of nucleotide diversity" and line 253, again seemingly referring to the same 17 orthologous groups: "these few genes have good values of nucleotide diversity" (although it's not entirely obvious here whether they're referring to the 17 genes, or the smaller subset of 4 genes, as they just refer to "these few genes". However, in the next sentence, they do refer to 17).

Lines 218-219: when discussing the four staphylococcal strains that were sequenced as part of the study, the authors state "in 2 of these cases, namely INIFAP 009-16 and INIFAP 002-15, the initial classification was incorrect". What is this based on? Are the authors just assuming that their phylogeny is correct, rather than the 16S RNA classification? Or have they performed any biochemical testing to determine the species type independently (also, how had the authors initially determined that these isolates were staphylococci, in order to know to sequence them and include in this study?)?

Lines 256-257: the authors state "furthermore, the particular combination of just three of them gave the exact same topology as the PSTT", but do not state which three genes they are referring to.

The maximum-likelihood phylogenies would benefit from bootstrapping, especially if one of the criteria being used is nodes shared by two trees. If these nodes are not supported, should they be perhaps collapsed? The authors could have used a number of other metrics in addition to just RF distance and nucleotide diversity. How many of the sites were informative? How many homoplasies were present for each gene? Which measure of nucleotide diversity was used, and have they used a correction (and did they use the DNAbin or haplotype options from the nuc.div function?)

In the discussion (lines 282-283), the authors state that "previous studies have shown that S. aureus and other species from this genus have not endure that many recombination events in the core genome". However, both references refer only to S. aureus. References for the "other species" would be beneficial given that some coagulase-negative staphylococci are thought to be quite recombinant.
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: PHYLOGENOMICS PICKS OUT THE PAR EXCELLENCE MARKERS FOR SPECIES PHYLOGENY IN THE GENUS STAPHYLOCOCCUS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall basic reporting is satisfactory.

However, the manuscripts need correction in english wherever required for example -

line no 58 -
within a giving lineage - within a given lineage (given to giving)

line 273

it is very likely that many of the markers here found  - it is very likely that many of the
markers found here..  (found here to here found)

line 276 -

some other evolutionary that prevent the history of the gene to reflect
the speciation events.   - some other evolutionary....?  - incomplete sentence?!

number of orthologous genes there found. For instance, very recombinogenic species,
very - highly...
there found?
Experimental design: Overall the experimental design is satisfactory
Please see General comments to author
Validity of the findings: No comment.
Please see general comments to author
Additional comments: The authors propose a novel approach to identify powerful markers to infer robust phylogenetic relationship of bacterial strains from a particular genera. This is a major work in the field of phylogenomics and will be a reference for similar work other bacteria of importance in basic and applied research. The finding have not only implications in taxonomic or inter-species research but also in multilocus sequence typing and analysis studies.

However below are specific comments that need to be addressed before any decision is taken

1) Why no reference or type strains of species taken in to consideration?

2) Why no correlation with phylogenomic criterion for groups like Average Nucleotide Identity (ANI)?

3) What is the GC content of SFG and 17 markers genes?  sign for HGT

4) There may be miss-classification in genera that are closely related to Staphyloccoccus. Why authors have considered using genera that are closely related to Staphylococcus...or can these makers help in identifying miss-classified and correct genus status of strains that are outlier or closely related to genus Staphylococcus?

5) How is the performance of 4 or 17  marker genes in case of MLST of important species like S. auerus and S. epidermidis? If not 5, query 5 is related and important and need to be a part of this present study.

5) Splits tree of the marker genes identified in the present study is not presented or discussed.


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: PHYLOGENOMICS PICKS OUT THE PAR EXCELLENCE MARKERS FOR SPECIES PHYLOGENY IN THE GENUS STAPHYLOCOCCUS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The wording in the paper seems casual in many places and could be more concise.
The structure of the paper is fine however there are sections in the results that would be better suited for the discussion.
Species need to be in italic both in the actual paper, tables and references, as do gene names.
Scale bars on trees should be bigger, see Figure 1 left as an example.
Experimental design: The research question is interesting, however, the paper struggles between its two aims, none of which are clearly outlined in the abstract itself. Was S. aureus chosen because of the number of genomes available or because of its clinical significance?
The idea of determining representative genes to illustrate true phylogeny is interesting however their second aim was to present methods for non-WGS laboratories for proper species determination. Whereas the paper properly identifies such targets, it would have been suitable to define how to amplify these by PCR. See line 217-222 as an example where WGS should be applied despite this paper to obtain proper species assignment,
Figure 1 represents the alignment on the 275 SGFs, however, no information is presented on the pairwise distance of these, and additionally what is the diversity of these genes within each species? As mentioned, there are a vast amount of genomes available however these are not used to understand within-species diversity which could be important depending on inter-species distances. also, in refseq, I believe genome data of close to 50 species of Staphylococci are now available.
4 isolates are mentioned in the method section, but not why these were included? For validation?
Line 128: from what are the SGFs significantly different and what is the true tree to compare to?
Line 155-160: were the 663 trees manually compared? And what is the true history of the species for comparison?
Line 185: very decent value?
Line 237: phylogeographic studies?
Line 257: which 3?
Validity of the findings: The aims are too wide in my opinion - what is the true species tree for comparison, why is it useful to know this from a few genes from a clinical perspective. And the markers then decided to represent that is presented as a marker for non-WGS labs, but no methods like PCR primers are presented to allow that.
The paper needs to be more concise in language and larger sections in the Result part could be moved or incorporated into the discussion.
As for the markers, it would be interesting to get a basic knowledge of their intra-species diversity among the many available draft genomes of staphylococci to emphasize their stability (recombination) and lack of overlap between species.
Additional comments: See above.


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

Input:
Title: PHYLOGENOMICS PICKS OUT THE PAR EXCELLENCE MARKERS FOR SPECIES PHYLOGENY IN THE GENUS STAPHYLOCOCCUS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Some minor spelling and grammatical issues, but English was clear and understandable. Raw data was shared.

Good use of literature, but the authors do seem to switch between referencing styles on occasion (e.g. lines 187 and 249).
Experimental design: The authors have significantly strengthened the manuscript in this version, both in terms of the number of genomes analysed, and through the changes to their methodology.
Validity of the findings: No comment.
Additional comments: Minor Comments:
1. Would lines 111-119 (the biochemical results) be more appropriate in the results section?
2. Lines 124-5:"to trim the reads". What parameters were used here? What Q value? Reads greater than or equal to what length were kept?
3. Line 201: rather than 'the same applies to most of the other species', I think readers would appreciate a specific value here, e.g. 'the same applies to X% of the other species'
4. Line 205: 'see green labels in Figure 2'. There were lots of different green shades in this figure - the authors should find another way of referring to the relevant species in this figure.
5. Line 225-226: as for point 3, the authors should be more specific here. Rather than 'This figure also shows that most of the OGs...', use 'This figure also shows that X% of the OGs...'
6. Lines 231-232: include units for nucleotide diversity values 0.2 and 0.26.
7. Lines 236-237: 'the bold candidates from Table 1'. There are no bold candidates in table 1.
8. Line 265: 'the initial classification was incorrect' according to the PSTT. It would still be interesting to know what these species are assigned as by biochemical test.
9. Line 277: 'over 45 species'. The authors could just state the actual number here.
10. Line 304: 'almost exact same topology as the PSTT'. Are the authors over-stating this a little? Is 79% similarity (Figure 4) really almost exactly the same?
11. Figures 1 and 4 - the tree scales need units.
12. Figure 2 - why are there white boxes in the ANI for the unknown strains?
13. Figure 2 - would benefit from the phylogenetic tree on the left hand side of the figure also being labeled with colours for species - would make it much easier to interpret.

Spelling:
Line 78: Should be 'schleiferi', rather than 'scheiferi'.
Line 87: 'misidentification', rather than 'miss-identification'
Line 137: 'consistent', rather than 'consistency'
Line 169: 'python', rather than 'phyton'
Line 220: 'OG', rather then 'OGs'


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: SPECIES DIVERSITY OF DRIFTING FISH EGGS IN THE YANGTZE RIVER USING MOLECULAR IDENTIFICATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this study Liu et al. analyzed an incredible high number of freshwater fish eggs of the Yangtze River of China using a mitochondrial gene fragment (Cyt bB). I think that the analysis and results are interesting and likely helpful for other researchers in this research field, but I favor a somewhat more broader and comprehensive discussion of the application of molecular data for the identification of fish eggs. Take a look at Hofmann et al (2014) (Journal of Applied Ichthyology 33: 925-932) and other similar studies. The authors should also include a discussion of modern molecular approaches in order to assess species biodiversity of a habitat, e.g., the use of high-throughput sequencing technologies (meta-barcoding), proteomics (MALDI-TOF), and others. In this context I miss a comprehensive discussion of possible pitfalls of using mtDNA in terms of species delineation and specimen identification, e.g., the existence of incomplete lineage sorting, heteroplasmy etc. as part of the introduction or discussion. Is it possible to differentiate the analyzed fish eggs by the means of morphological traits? Currently, no information about this important aspect is given.
Acceptable English is used throughout, except for some minor grammatical mistakes. However, the current phrasing of the abstract should be rephrased and improved. In addition, please check the manuscript carefully for typos, e.g. line 16 (identified instead of identify) or 42 (Hypophthalmmichthys nobilis instead of Hypophthalmmichthys, nobilis). There are various other typos existing, including missing blanks or double blanks.

Detailed comments

Line 40: Please provide name of first descriptors of the studied species here and in the following

Line 108: Change “Cyt b” to “Cyt b (b in italics)” here and in the following

Line 108-115: I feel that the space lining of this part is not correct

Line 109: Did you have published the used primer before? In such a case you should only provide the primer name and reference instead of listing up all nucleotides. If both primers are new, however, you should re-arrange them in a triplet code: GAC TTG AAG …

Line 111: Just a comment: Think about using a smaller PCR mix (25 µl instead of 50 µl) in order to save resources.

Line 116: Have you checked your sequences for the presence of pseudo genes?

Line 118: Which company was employed for sequencing your amplicons?

Line 119: Currently, no accession numbers are provided. However, this is essential for publication.

Line 122: Even if you have analyzed protein-coding gene sequences of more or less closely related species and not taxa from different phyla, Clustal W and Clustal X should not be used to align sequence data any more. Other programs, e.g. Muscle, MAFFT or Clustal Omega are more useful and adequate

Line 123: Provide a reference for the K2P model

Line 124: Think about updating your MEGA version; the actual version is 7. It has also implemented Muscle (see above)

Line 124-125: Please give references for the neighbor joining approach (> Saitou & Nei 1987) and the non-parametric bootstrap analysis (> Felsenstein 1985)

Line 139: Replace “Arliquin” by “Arlequin”

Line 144: The total number of analyzed eggs was 8983, but only 7933 eggs were studied. Why?

Line 144 following: Whereas you mentioned reconstructing a NJ topology in the M&M paragraph, no figure is shown. Why?

Line 144 following: In think you should highlight your results a little bit more than refer on table 1. This is also true for the discussion. For example, you can provide percentage values for the most important values

Line 173: Replace “molecular methods” by “mitochondrial sequence data”

Line 186: Culters in italics

Line 207-209: Rephrase this sentence

Line 270-274: Whereas you did not use DNA barcoding, you cite two studies of Hebert and co-authors. However, they do not appear within the manuscript. This is also true for Hajibabaei et al. and various others. Check your reference paragraph carefully.
Experimental design: The research question is relevant and well explained. In terms of using DNA sequence data to identify specimens, however, the analysis of a partial fragment of the CO1 gene has become the most popular approach by far (DNA barcoding sensu Hebert). This is also true for fish species. So, can you explain why you have used cytochrome b instead of CO1? Furthermore, I miss the NJ topology of the analyzed taxa.
Validity of the findings: I think that the authors should highlight their results a little bit more than to refer on table 1. This is also true for the discussion. For example, they can provide percentage values for the most important values. What about inter- and intraspecific distances? Such information will help to improve the manuscript.
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: SPECIES DIVERSITY OF DRIFTING FISH EGGS IN THE YANGTZE RIVER USING MOLECULAR IDENTIFICATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well written in English and clear throughout the text. The background/context provided in the manuscript for the study conducted is good, and the figures and the tables, containing relevant data and that allow the authors to reach their main conclusions, were provided. The raw data is also shared. Literature references provided are enough. The article followed a clear structure with standard sections and sub-sections. Although the subject is not completely new (fish species identification through barcoding of DNA extracted from eggs), it is highly relevant and provides a clear example where species identification greatly benefits from the use of DNA-based tools.
Experimental design: The authors conducted a very interesting study about the molecular identification, based on DNA barcoding, of fish species using the eggs spawned and collected from the Yangtze River and its tributaries (China), during a period of 2 years. The research question is well defined in the manuscript and the background and context provided reveals its high relevance. The methods were adequate to address the questions of the current study, as well as the data analyses conducted, and enough details were provided. The permissions needed to sample the eggs were also obtained before the study. There is just two points for which the authors should give more details: 1) why did the authors used Mitochondrial (mt) cyt b gene marker instead of Mitochondrial COI marker, since COI is the reference DNA barcode used for animal species identification (thus reference databases would be more complete with COI barcodes belonging to fish species) and 2) when the authors state in the data analysis sub-section that all sequences were trimmed to the same length; the authors should provide the final length of the sequences. One negative point of the study is that there were stations that were sampled only in 2016.
Validity of the findings: The authors are very straightforward in presenting their results, which is very good. The discussion is well conducted, but please verify again the nomenclature of the fish species since I have found some errors in the names of some species. The conclusions were appropriately stated and the importance of using DNA-based tools was proved by using this particular case. However, one point that is not very clear is if the samplings were just conducted once, during the period of May to July, or more than once. The results would be more reliable if the eggs counts provided were based in different sampling points between May and July (replicates), for each station.
Additional comments: Minor comments:
In Table 1 correct "total" by "Total number of eggs"; "speices" by "species" and "Shonnon J" by "Shannon H"
I would suggest that you change Table 2 legend to "Fish Jaccard’s similarity index (JI) and river-way distances among sampled stations in the Yangtze River (Mantel test, R=-0.9003)"
Other minor edits were directly made in the document that I send attached (edits made are in changetrack).


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: SPECIES DIVERSITY OF DRIFTING FISH EGGS IN THE YANGTZE RIVER USING MOLECULAR IDENTIFICATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: It is nice to see that the authors accepted all comments and/or remarks. However, some minor concerns still remain. In this context, I still miss a broader and comprehensive discussion of the application of molecular data for the identification of fish eggs. Which methods have been used? Furthermore, the authors should carefully differentiate between the use of DNA barcodes (sensu Hebert et al. 2003) and the application of other markers for identification. Currently, all is mixed without structure. Furthermore, there are still various typos existing (e.g., missing blanks). I tried to get most, but please check the whole manuscript carefully. I also still miss a summary and discussion of the observed inter- and intraspecific molecular distances of the analyzed fish species. This is quite important.

Abstract: Whereas some work has been done here, I still think that current phrasing of the abstract should be rephrased and improved.

Line 36: Please provide name of first descriptors of the studied species here and in the following (e.g., Ctenopharyngodon idella Valenciennes, 1844). They are still missing but should be added.

Line 117-120: I still think that the space lining of this part is not correct.

Please see some specific comments made via sticky notes on the PDF file of the manuscript.
Experimental design: As already mentioned, the research question is relevant and well explained. As part of thier reply the authors clearly explained why they used cyt b instead of CO1. The (detailed) NJ tree is part of the electronic supplement. However, it would be nice to add a somewhat simplified NJ tree with collapsed species (see MEGA tool: compress/expand subtree after a phylogenetic analysis) to the manuscript.
Validity of the findings: No more comments (see above).
Additional comments: No additional comments


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

Input:
Title: SPECIES DIVERSITY OF DRIFTING FISH EGGS IN THE YANGTZE RIVER USING MOLECULAR IDENTIFICATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: This is a revised version of a manuscript that I have previously revised. The authors have carefully addressed the two reviewers’ suggestions and concerns, yielding an improved version of the manuscript.
Experimental design: In the first version of the MS, there were some experimental issues that arose, but that the authors have clarified in this revised version.
Validity of the findings: The authors have clarified less clear issues found in the previous version of this MS.
Additional comments: Other minor edits were directly made in the document that I send attached (edits made are in changetrack).


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: INTEGRATIVE ANALYSIS OF MICRORNA AND MRNA EXPRESSION PROFILES IN FETAL RAT MODEL WITH ANORECTAL MALFORMATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: No Comments
Experimental design: No Comments
Validity of the findings: No Comments
Additional comments: In this study, entitled "Integrative analysis of microRNA and mRNA expression profiles in the fetal rat model with anorectal malformation" authors interrogate the miRNA and mRNA expression profiles in terminal hindgut tissue of 20d old ARM rat embryos. Based on the data provided, authors suggest that they identified the differentially expressed miRNA and mRNA in terminal hindguts of ARM rat samples compared with control and list the various pathways and other genomic features involved in such samples. The study is significant in determining the miRNA expression in ARM samples, however following things need to be addressed:

1: Authors should be descriptive about qPCR in method section – like How much cDNA was used? What was the PCR conditions? How many amplification cycles used? Whether melt curve analysis was performed? If yes, mention the same in the method section. Also, provide a relevant reference for qPCR analysis.

2: Authors should provide PCA plot for every 3 replicates of case and control microarray. It would be really helpful to distinguish the case and control replicates.

3: Authors have mentioned that they identified ARM by light microscopy. Include those images as a part of Figure 1.

4: Do authors determined the sex of embryos used for study? Mention it in the method section.

5: Why authors have chosen 20d gestation age? No citation provided. Must reference it.

6: Also, authors should provide the reference for ETU usage for inducing ARM. Presently, it is missing.

7: Provide the list of primer sequence used for qPCR analysis as a supplementary table.

8: Authors should be consistent in writing miRNA throughout the manuscript. At many places it is MiRNA. Same should be corrected.

9: There are grammar and spelling errors in the manuscript that should be addressed.


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: INTEGRATIVE ANALYSIS OF MICRORNA AND MRNA EXPRESSION PROFILES IN FETAL RAT MODEL WITH ANORECTAL MALFORMATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The author described an integrative analysis of microRNA and mRNA profiles in  rat model of anorectal malformation by miRNA array, 33 miRNAs and 772 mRNAs were then screened between ARM group and control group,providing a basis for further research on miRNAs and mRNAs in the pathogenesis of ARMs.
Experimental design: There was a severe flaw in the experimental design. All the fetal samples used in this study were obtained on the gestional gay 20, on this time point, development of  the hindgut has finished as we know that the gestional day 13-14 was the key time point for anorectal development,  microRNA and mRNAs can only play their roles during this period, after that, parts of microRNA and mRNAs may lose their activity. therefore, author should choose the fetal samples on the gestional day 13-14 but not 20.
Validity of the findings: the results were valid but not scientific.
Additional comments: The author described an integrative analysis of microRNA and mRNA profiles in  rat model of anorectal malformation by miRNA array, 33 miRNAs and 772 mRNAs were then screened between ARM group and control group,providing a basis for further research on miRNAs and mRNAs in the pathogenesis of ARMs. Although the results were attractive, there was a severe flaw in the experimental design leading the results were not scientific. there was so many spelling and grammatic errors throughout the paper making it difficult to read


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: INTEGRATIVE ANALYSIS OF MICRORNA AND MRNA EXPRESSION PROFILES IN FETAL RAT MODEL WITH ANORECTAL MALFORMATION
Review round: 1
Reviewer: 3

Output:
Basic reporting: Needs work. I believe that the article uses clear English to describe the study design and setup but could use some work on the following aspects
1) There are a lot of spelling and grammatical errors that can be resolved with a spell check and some minor proofreading. (Some of these are marked in my copy of the manuscript, but I have not marked it up thoroughly)
2) Additionally, the authors switch between present and past tense frequently especially in the discussion making it harder to understand distinguish statements of fact from speculation.
I believe that the authors have not given sufficient background of the ETU induced ARM model.  Is this a well-established model in the field? Are there other models available to study ARM? If so, the authors should cite relevant studies and describe their reasons (and pros/cons) of selecting this particular model. Additional details including dose of ETU used and days of gestation before analysis will help flesh out the model in more complete details.
The authors have done a good job of sharing all the raw data. Are the planning to upload the microarray data for public access on NCBI GEO or other suitable servers?
Additionally, I believe that figure 1 can be replaced by a more informative study design. The number of animals used for analysis for mRNA vs miRNA analysis for example remains unclear to me. This would help in clearly conveying the work rather than a workflow which is fairly standard and easily grasped by perusing the materials and methods.
Experimental design: I believe that the current work is original primary research that is within the aims and scope of the journal.
I commend the authors for clearly defining the research question at hand and explaining its relevance to the understanding of ARM
I believe that the authors have attempted to clearly explain the methodology used although i am confused by the number of animals used per arm of the study. The animal section of materials and methods states that 20 mice were used for the ETU arm of the study and an unknown number were included in the control group. Additionally of the 20 mice, 12 were used for total RNA and 6 for miRNA. What were the 2 unreported mice used for? Also, how were the numbers (12 and 6 chosen for the mRNA and miRNA analysis respectively? Did you run power calculations? If not, what was the basis for this division?) My recommendation to clarify this would be to create a schematic depicting the study design along with animal numbers

I believe that the authors have thoroughly investigated the mRNA and miRNA profiles obtained by the microarrays and subsequent analysis. However, the genes they chose for qPCR validation are not the top changed genes on their list. For example, mir-598 is the lowest gene on the up regulated miRNA list while mir-3084a is somewhere in the middle. What is the rationale behind this specific choice of miRNA for validation? Why not just pick the top 5 per category? The choice of mRNA for validation is similarly puzzling.
 I would also like for the authors to provide the primer sequences that they used to investigate each gene by qPCR. This will help others who investigate the same genes to contextualize the author’s findings.
Validity of the findings: I believe that the findings in this study are valid. The data used for this study is robust although i believe that additional validation either in the form of protein expression or in vitro analysis on cell lines would certainly bolster the findings. The authors have focused on the RNA expression levels and have done preliminary validation of a handful of genes by qPCR. However, mRNA expression levels without the corresponding impact on the protein levels does not lend itself to drawing meaningful conclusions about the biological question under study.
Additionally, the authors spend a majority of the discussion speculating about the modulated miRNA and their potential targets and attempting to connect it to known biology without much data to support this speculation. While some speculation is clearly justified to create a complete picture, their use of it is excessive and tends towards overstating their findings.
Additional comments: I believe that the authors have made a strong attempt to understand the RNA expression underlying ARM development in the ETU model.
While the authors have characterized the changed genes in the ARM model extensively using KEGG and GO analysis, I believe that more meaning could be obtained by validating some of the changes at a protein level as well.
 Additionally, while correlating the miRNA changes with their targets, the authors often use qualitative terms instead of quantitative. Some options to improve this would be by running correlations on the miRNA and their targets to quantify the relationship. Similarly, the authors state that “there is a high consistency between the microarray and qPCR data” (lines 216-217). This can also be quantified.
Overall, I think that the readability and applicability of the paper will be greatly improved by focusing the discussion on the key findings of the paper and its implication. There are several instances where the authors have gone into an extensive discussion of the biology of signaling in ARM although they have personally not investigated any signaling related aspects at all


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: INTEGRATIVE ANALYSIS OF MICRORNA AND MRNA EXPRESSION PROFILES IN FETAL RAT MODEL WITH ANORECTAL MALFORMATION
Review round: 1
Reviewer: 4

Output:
Basic reporting: In this manuscript the authors evaluated the microRNA and mRNA profile of anorectal malformations (ARM) in fetal rats. The authors for the first time evaluated the microRNA profile in ARM and adds valuable information to the field.

The language should be once proof read for vocabulary and grammatical mistakes.

The discussion part appears to be redundant and should be shortened to make it clear and to the point.
Experimental design: 1.	Number of Rats given ETU treatment is confusing. As total 20 females were selected for experiment. How many are given ETU treatment and how many are control rats should be made clear.
2.	For validation experiments, why particular miRNA and mRNA were chosen should be described? Why highly upregulated and downregulated miRNA and mRNA were not checked with qRT-PCR. miRNA and mRNA showing high connectivity should have been chosen for validation.
Validity of the findings: No comments
Additional comments: The following should be considered to improve the manuscript.
Language should be checked
Line 54: Day 20
Line 55: Spelling “terminal”
Line 76: group “of”
Line 79: correct “pathology”
Line 89: “to the best of my knowledge” should be removed
Line 93:  correct “data”
Line 113: RNA isolation and quantification should be described in detail. Homogenization step, RIN number details etc should be provided.
Line 129: internal control for miRNA should have been smaller RNA. Any justification
Line 149: what was the total number of miRNA scanned, upregulated and downregulated should also include % of total in form of vein diagram to make it more clear and representable.
Line 196: there is no English word “Resultly”
Line 330: target prediction cannot be done based on downregulation of rno-miR-381-3p and Fgf16 as they are downregulated among other differentially expressed genes and miRNA. Please change the sentence. This can be done experimentally on one to one basis or predicted bio-informatically.
Fig 1 “Dephosphorylation”
Fig 5: How normalization was done in both cases. Are they comparable??


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: INTEGRATIVE ANALYSIS OF MICRORNA AND MRNA EXPRESSION PROFILES IN FETAL RAT MODEL WITH ANORECTAL MALFORMATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: No comments
Additional comments: Authors have done a good job in addressing this reviewer concerns. No more questions.


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: INTEGRATIVE ANALYSIS OF MICRORNA AND MRNA EXPRESSION PROFILES IN FETAL RAT MODEL WITH ANORECTAL MALFORMATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: I believe the authors have taken effort to correct a lot of the errors previously highlighted in this section. A few errors remain but can be fixed by a thorough proofreading of the the final manuscript. a few examples (but not all) of errors listed below
1) recent not rencent on line 72
2) figure 2 legend: black and NOT white means no difference
Experimental design: I believe that the authors have taken effort to correct all the errors previously highlighted in this section. No further comments
Validity of the findings: I believe that the findings in this study are valid.
Additional comments: I have one main request of the authors: Please rework your discussion to only the pertinent points and shorten it dramatically. Currently about half the discussion is redundant to the results section or is irrelevant. The authors have spent several lines purely restating the results from the results section and increased the length of the discussion quite a bit (see lines 288-295, 299-303,319-341 etc). Additionally, the authors have gone into an extensive discussion of the biology of signaling in ARM and into details of micro RNA mediated RNA regulation of several transcripts which i find unnecessary. I believe that this section needs real effort to focus it solely on the findings and implications of the authors findings to prevent it from being a review of the signaling and biology related to ARM.


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: INTEGRATIVE ANALYSIS OF MICRORNA AND MRNA EXPRESSION PROFILES IN FETAL RAT MODEL WITH ANORECTAL MALFORMATION
Review round: 2
Reviewer: 3

Output:
Basic reporting: Authors have included more information and made the manuscript crisp and clear.
Experimental design: ok
Validity of the findings: no comment
Additional comments: We thank the authors for improving and providing additional data to the manuscript for easy understanding.


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: EFFECTS OF INCONSISTENT REPORTING, REGULATION CHANGES AND MARKET DEMAND ON ABUNDANCE INDICES OF SHARKS CAUGHT BY PELAGIC LONGLINERS OFF SOUTHERN AFRICA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English is generally good, clear and unambiguous. There are minor grammatical errors throughout, some of which I’ve marked in the pdf, e.g. at lines 60, 65, 73, and 75.

The introduction provides appropriate background information to show the context.

The analysis code has not been provided. The raw data have been provided but are incomplete, lacking the ‘observer present’ variable. Figures are provided in the MS but not as separate files.

The data are aggregated but the data file reports a precise location for each row of data. Is this a median or average location or simply the location of the first set?

Acknowledgments include funders, which should instead appear in a separate funding statement.
Experimental design: The research appears to be suitable for the journal, and the research question addresses an important issue that is clearly identified in the MS.

The methods are not sufficiently well described to reproduce the analysis. The model equations should be supplied in the text, and the analysis code should also be provided. I loaded the data into R and ran gamms with lme4, following the reported methods as closely as possible. The delta models all failed to run.

More information should be provided about the error back-calculation and error propagation methods, rather than just giving references.
Validity of the findings: I was puzzled by the decision to aggregate the data before analysis, given that the set by set data were available. In aggregated data, the uncertainty associated with each row is affected by the number of sets aggregated, which breaks the assumption that the data are iid. This is not a major problem but could easily be avoided.

More serious is that in a binomial model, effort (hooks) should be included in the gamm as a predictor, because effort affects the probability of non-zero catches, i.e. a cell with more effort will be more likely to catch at least one shark. However, Tables S1a and S1b suggest that effort was not included in the binomial model, which will bias the standardization results.

The standardization formulae appear to include ‘- 1’ (see Table S1a), which forces the model through the origin. This is unusual and may be problematic, and should at minimum be discussed and justified.

I would like to see more data exploration, perhaps in the supplementary material, to show that data are being modelled adequately. For example, I found that for N_BSH the seasonal relationships seem to be different in each region, though they are quite small effects so probably not problematic. There is no need to fit season as a categorical variable, when month could be fitted as a continuous variable.

Please explain why all the covariates are fitted as random effects. What is the benefit? It would be useful to fit them as fixed effects, and show the parameter estimates, which could be compared with the scale of the inter-annual changes. In general, the analysis would be stronger if the CPUE standardization was done more carefully. The same covariate model seems to be applied to both species and to the binomial and continuous components, but may not be the best approach in all cases.

There are sufficient data to analyse each region separately, and doing so would strengthen the argument if each region shows the same pattern in 2010, which seems to be the case for blue shark.

The statement is made that the observer CPUE declined 2000-2005. It would be useful to plot these estimates, and compare the observer catch rates with the logbook catch rates.

The recent increase in Japanese standardized shortfin mako CPUE may also be affected by changes in reporting, so may not support the hypothesis of increased shark biomass. See chapter 1 of Hoyle et al 2017 below.
-	Hoyle, S.D., Semba, Y., Kai, M., Okamoto, H. (2017) Development of Southern Hemisphere porbeagle shark stock abundance indicators using Japanese commercial and survey data. New Zealand Fisheries Assessment Report 2017/07. WCPFC Scientific Committee 13th regular session WCPFC-SC13- SA-IP-15: 64.

The discussion is generally well argued and consistent with the evidence, apart from the concerns identified above.
Additional comments: No additional comments


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

Input:
Title: EFFECTS OF INCONSISTENT REPORTING, REGULATION CHANGES AND MARKET DEMAND ON ABUNDANCE INDICES OF SHARKS CAUGHT BY PELAGIC LONGLINERS OFF SOUTHERN AFRICA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The basic reporting of the paper seems OK. But see general comments to authors and Editor below.
Experimental design: The experimental design seems to have considerable problems, as for example the use of only log-book data aggregated by month for this type of analysis. But see all details in the general comments to editor and authors below.
Validity of the findings: Given the issues identified in the validity of data, and other issues in the methods as for example not being clear why the GLMMs all variables (except year) are used as random, I have serious doubts about the validly of the results. But see the general comments to authors and Editor below.
Additional comments: Main issues:
Data: The data used seems to come exclusively from logbooks. While this is valuable data, there may be issues with reporting, species ID, recording (or not) of discards, etc. As observer data is also available in some cases (mentioned in the text), a comparison should be made between the two sources and discussed.

Representativeness: The fleets analyzed operate exclusively in the South Africa EEZ, while the species analyzed (blue shark and shortfin mako) are highly migratory (South Atlantic and whole Indian Ocean stocks). Therefore, the trends described for those local operating fleets cannot really be extrapolated to population levels. Such data could potentially provide some interesting local indicators, but even those need to be carefully discussed given the source of the data (logbooks) with all its problems and limitations.

Methods: The methods used (Delta method for combing GLMs) seem standard for CPUE standardization. However, there are issues not well explained as for example how the data was filtered and also the % 0 (I assume it was the reason why the Delta approach was chosen, but if that is not specific the reader does not know). Also, it seems from the text and the available dataset on the additional materials that the data was grouped by vessel/month which is also not clear, and can have issues of losing variability associated with set-specific data. Finally, the models used were GLMMs but the only fixed effect was "year", and the reason for formulating and using this approach is not clear. Typically, a random effect would represent variables for which only the overall variation is needed (e.g., vessel effects) while variable that are interesting to quantify directly (e.g, spatial/seasonal) should be used as fixed effects.

Further detailed comments are listed below:

The criteria used for any data filtering/cleaning should be clearly described.

At some point, and even though it seems that set-by-set data is available, this data was then grouped for the analysis. This can be a problem, as much o the variability associated with the fishing activity can be lost with such data aggregations.

Could the authors please refer if there are any possible ID issues between short and long fin makos? Isurus oxyrinchus vs Isurus paucus? I would assume that especially in logbook data this can be a serious problem.

Were interactions tested and/or used in the models? I cannot find deviance table and it would be important to have and analyze those.

The GLMMs seem that only the year was used as fixed while other variables were used as random. I would assume that variables such as spatial/seasonal effects should also be used as fixed, as it might be important to study their effects in the CPUE rather than only take into account their variability.

Were the % 0s similar, i.e., a similar issue for the 2 species? Where other models for dealing with 0s considered? As the CPUEs are considered in n/1000hooks, models for count data (e.g, Poisson, NB, Zero-inflated Poisson or Zero-inflated NB, depending on data characteristics and dispersion) could have been tested and compared.

The method used for getting the year effect is not clear, and how the coefficients were used. Typically, for CPUE standardization we want to extract the marginal means (ie.LS means) for the year effect. Was this what was done? But as it seems that the only fixed factor used was year, then in this case only the year coefficients are considered? Either way, the methodology used is not clearly described and raises questions on if it was applied correctly.

Any possibility that the discarding practices (or under-reporting rates) may have changed over time. If the discarding/reporting was consistent through time then it is only a problem in the scale. But if there are changes through time, then the entire results/trends will be biased.

The increases do not seem to be biologically possible to be explained as abundance trends. So this likely means that there are other effects (e.g, targeting, reporting, discarding vs retention, etc) that are not being taken into account in the standardization. In that case, the resulting standardized index should not be used as a proxy for abundance indicator. This is discussed alter in the discussion of the paper, but as it is not clear what is causing those trends, then the utility of the results is also not very clear.

As those increases are seen for the local fleet, it must really be either targeting and/or reporting issues that are affecting the results, which will be masking with any abundance trends in the species.

It would also be important to analyze some type of mosaic plot (or have a table with N observations per category) so that the reader can understand if there are actually data for all explanatory variable combinations. This is especially important in cases as the Delta method that has a binomial component, with assumptions in terms of samples per category.