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: UNIMODAL HEAD-WIDTH DISTRIBUTION OF THE EUROPEAN EEL (ANGUILLA ANGUILLA L.) FROM THE ZEESCHELDE DOES NOT SUPPORT DISRUPTIVE SELECTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper is well written and the language and grammar are good.  I have marked some edits on the manuscript.
The paper addresses one of the longstanding topics of eel biology, their headshape.  The debate has long focussed on shape and diet and competitive advantages, adaptation vs survival.
In my view, this paper doesn’t make much more progress in untangling the cause effect of diet and headshape.  The additional information on maturation, condition and migration are interesting, but it is admitted in the paper that the differences are small or absent and sample sizes are in places too small.  However, the title is Unimodal head width distribution….. even though the tests on lines 205-216 are largely inconclusive, one pointing to unimodality and two pointing to bimodality.  The authors then visually “chose” unimodal since there is strong overlap.
Experimental design: Project Design: on line 224 it states that more data was needed and on line 252-254 it says …as more data was taken into account…..  What does this mean? It wasn’t mentioned in the methods or results and if more data could be taken into account to strengthen the result, why wasn’t it?
Validity of the findings: the title is Unimodal head width distribution….. even though the tests on lines 205-216 are largely inconclusive, one pointing to unimodality and two pointing to bimodality.  The authors then visually “chose” unimodal since there is strong overlap.
In lines 220-222, uni and bimodality were equally supported between eel stages of eel.
My experience is that eel headshape distributions are a continuum, but are often heavily skewed in one direction or the other, varying widely between locations and habitats, even quite proximate ones. This paper picks a single eel population from the Schelde Estuary.
The abstract starts off with a categorical statement in line 26-27 and then concludes in line 31 that the distribution is unimodal and in lines 33-34 that it’s a continuum from narrow to broad, even though the statistics were a bit ambiguous. Lines 241 and 242 state equal support for uni and bimodality but then go on to say that uni is more “plausible”.
Section 3.3 relationship between HW and body condition, as defined by condition factor, was signif p<0.01 but with an r2 of .02, so only 2% of the variation in condition factor (body weight for length) is explained by HW – hardly convincing.  Barry et al. and others have shown significantly lower lipid reserves in eels with boarder headwidths which is at odds with this finding.
The Migration speed section should be more clearly described and linked to the downstream migration of maturing silver eel (if that’s what it is).  It is not clear to the more generalised reader.  Especially when it is compared to home range movement of foraging immature yellow eels (i.e. Barry et al), which is a different thing.
Additional comments: I find it difficult to recommend this for publication without major revision.  The context needs to be set to the Schelde Estuary and the title need to change.  A more appropriate title might be something like: Distribution of headshape  in European Eel in the Schelde Estuary,(with reference to body condition and migration speed?).  The abstract needs to show the equal support for the two models and state that it is the authors visualisation that selected the unimodal form. Body condition increased with HW but only 2% of the variation was explained.  Downstream migration speed was not influenced by head width.


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: UNIMODAL HEAD-WIDTH DISTRIBUTION OF THE EUROPEAN EEL (ANGUILLA ANGUILLA L.) FROM THE ZEESCHELDE DOES NOT SUPPORT DISRUPTIVE SELECTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: It is my view that the current, second manuscript has been improved over the first and now fulfills the criteria and standards of the journal.


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: UNIMODAL HEAD-WIDTH DISTRIBUTION OF THE EUROPEAN EEL (ANGUILLA ANGUILLA L.) FROM THE ZEESCHELDE DOES NOT SUPPORT DISRUPTIVE SELECTION
Review round: 2
Reviewer: 2

Output:
Basic reporting: See below Note to Editor
Experimental design: See below Note to Editor
Validity of the findings: See below Note to Editor
Additional comments: I have reviewed this second version almost without reference to the first and I haven’t made any edits on the manuscript.  I appreciate the suggested changes being taken into account.
1.	Variation in headshape in eel is clear.  This varies between eel sizes, locations, habitats and maybe even by sex and is linked with diet, niches, growth rates, lt/wt and other body conditions such as lipid levels. However, cause and effect is still strongly debated.

Much of the published literature, and the discussion by observers, fishers etc refers to them as an “either or” narrow or broad leading to this debate about bimodality.
Head shape is a continuum from an extreme pointy narrow head to an extreme blunt broad head with widely vary distributions along this continuum, sometimes leading to apparent bimodality within sites (such as Ide et al) or to heavily skewed distributions in one direction or the other.  The combinations of these sites, and or the generalising in discussions, has perpetuated the view of “bimodality”. I’m saying this because I’m trying to decide what this current paper is actually trying to say.

In this paper, there is still a tendency to generalise to the whole population by stating that other authors show have shown bimodality (Torlitz, Ide & Kaifu). These examples are sampling eels from multiples of habitats and locations and then discuss a pooled bimodality. Your study is from one specific location within one specific river.

I reiterate my statement “that eel headshape distributions are a continuum, but are often heavily skewed in one direction or the other, varying widely between locations and habitats”.  This does not mean there is no selective pressure in the Schelde.  There are weak tails to your normal distributions which you ignore in line 216 and then you refer to them in lines 304-305. By analysing residuals, it is difficult to tell where your eels lie on the continuum.

Line 35, while correct, implies that there are no selective pressures on head shape, which in your case could be unidirectional selection pressure rather than disruptive pressure. This makes the point being made on line 98 confusing; yes biomodality can arise from disruptive selection, but equally unimodality can derive from unidirectional selection pressure. (And don’t discount the idea that eels can be quite choosey and selective in what they choose to eat – regardless of prey abundance)
I would suggest that if many of the references to bimodality were exchanged for “variations in head shape” the paper would read more accurately.
So for example, line 98 could read: From an evolutionary point of view, variations in headshape may arise from different selective pressures at many locations, or even disruptive pressure such as observed on the Frome (Cush…).
Line 306 repeats this bimodal statement, or absence of a clear bimodal distribution, but ignores that you were looking @ 1 niche, in 1 habitat type in 1 location. Line 308 states that this shows no selective pressure – on what basis? Or do you mean no disruptive selection pressure? You didn’t examine the diet in these fish and you don’t state where your eels lie on the continuum of the distribution of narrow to broad.
2.	Following on from that, the discussion about body condition (Wt for length) supporting the lack of disruptive selection I don’t understand.
If there is a unimode, slightly skewed, there is likely to be some selective pressure towards that mode, but we don’t know where your eels lie on that distribution. Line 316 says that body condition increased with head width (body condition also increases with eel length). How does increasing K with headshape support a lack of disruptive selection when it such a weak relationship. And then the very low contribution (2%) actually supports it even more??
Written another way, body condition rises with bigger heads which means there is no selection, but because the relationship is so weak it really support this – that doesn’t make sense to me).
Related discussions on body weight, condition, energetics, and amount and size of prey in Lines 320-325 are quite speculative and don’t really move the debate further forward.

The statement in line 68 has not been supported: The consumption of different prey items not only leads to different head morphs, but can also influence habitat selection…..I suggest delete this – we don’t understand the cause and effects here.

Line 284-288  This doesn’t always apply to eel which can become quite specific to one food item, such as Asellus, in spite of an abundance of chironomid larvae, for example. And the size of eel can also influence the food types they prefer. So line 288 can conclude, but not always in eels.
Line 288-291 You haven’t related the diet of the individuals to their headshapes. You didn’t look at diet in your eels.

Line 319. You again apply cause and effect by stating that the eels with the broad heads occurred “as a result of feeding on hard prey,” – change this.
Line 320.  They may be capable of eating a wide range of prey but I’m not sure they do.

3.	4.4 Swimming
There was no relationship with head width. So the rest of this paragraph is almost redundant.  You are inferring something on the ocean migration from an upper estuarine observation of downstream movement, which is very different; flow rates, tidal flows, changes in salinity eel physiology and osmoregulation are all likely to influence swimming speed in the estuary, none of which are so relevant in the oceanic phase – we see similar differences in the speed of salmon smolts for example.

Line 344.  This sentence is not supported and should be removed How does swimming speed (which wasn’t different between narrow and broad in the estuary) in the ocean explain how narrow heads have less wt for length – even though we know that narrow heads have more lipids (I acknowledge that this obs on lipids isn’t in the paper).

4.	Conclusion
Line 344: Does it? You say dimorphism isn’t observed so why are you trying to explain it? You mean the absence of clear dimorphism in the Schelde estuary indicates a lack of evidence for disruptive selection, but doesn’t discount unidirectional pressures on variation in eels head shapes. Unimodal headshape in this location does not appear to subject to disruptive selection, but may have developed as a result of unidirectional selective pressure.

Line 348 Please remove this sentence (Bimodality is not a general phenomenon…..). Its not true.

The conclusions need to be reworded in the light of these comments (opportunism, one direction selective pressure, where are your eels on the distribution etc).

Line 353-354 also needs rewording.
Line 356 – delete “yet different morphs…..  as this is not a conclusion of your study.

5.	A few other bits
The catch location doesn’t say whether it was above or below the tidal weir.  And a salinity range would be interesting if they were captured below the weir.

The Title: The title should say in the estuary of the Schelde – not “in a single river drainage”.  This is exactly the issue under debate.  They were from one location in one habitat in one river.

Methods: is there, or was there recently, any fishery in the estuary.  Fishing pressure can influence the distribution of sizes and head shapes in eel stocks.  Different growth rates, head shapes, dietary habits etc can all lead to selective harvest (though bait selection, mesh size etc) and a different distribution of head shape.  This needs to be declared.

I still find it difficult to recommend this for publication without further major revision.  I remain to be convinced that this paper as it is written has advanced our understanding much further.


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: SEXUAL DIMORPHISM IN THE ARACHNID ORDERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: See "General comments for the authors".
Experimental design: See "General comments for the authors".
Validity of the findings: See "General comments for the authors".
Additional comments: In this manuscript, the authors revise the forms of sexual dimorphism in arachnids, a major group of arthropods with an enormous diversity of body plans and behaviors. Although there is scattered information on sexual dimorphism of isolated species and also of some specific clades, there is no comprehensive review of the subject spanning the whole class. As someone that works with arachnids and is interested in sexual selection, I thus think the manuscript is very welcome. However, I have three major concerns about the manuscript. These three concerns are more or less interconnected and I will elaborate each of them below.

1) It is not clear to me if the manuscript intends to be a systematic review or simply an overview of sexual dimorphism in arachnids in which the authors select some emblematic examples. Both goals are valid, but it would be important to properly explain to readers what kind of review authors want to provide. I think this point is important because there many cases of sexual dimorphism in arachnids that were not addressed in the manuscript. In what follows, I mention examples for some orders.

1.1) Acari

- I am conducting a huge review of parental care in arthropods and, based on my own experience, I know that is very hard to compile information on Acari. Although there are several books covering different aspects of their biology, detailed reviews on specific subjects are basically non-existent. In the manuscript, the authors provide general data on sexual dimorphism in Acari, focusing on oribatids. In fact, this is the most tractable group within the Acari because it is more intensively studied than other groups. However, even in oribatids there are some reports of sexual dimorphism that were not mentioned in the manuscript. For instance, there is at least one case of sexual dimorphism in leg length in oribatids. In Ameronothrus lineatus, adults are sexed by the gender-specific size difference and also the longer legs relative to body size of the males. Please see: "The biology and life history of arctic populations of the littoral mite Ameronothrus lineatus (Acari, Oribatida)" -- G. Søvik (Experimental and Applied Acarology 34:3-20, 2004).

1.2) Araneae

- There is a very conspicuous case of sexual dimorphism in the linyphiid Oedothorax gibbosus. Males have a huge gland on the cephalothorax that provides a nuptial secretion to females. Curiously, the presence of this gland is also intrasexually dimorphic, so that a male morph without the gland is also found in natural populations. Please see: "Male dimorphism in Oedothorax gibbosus (Araneae, Linyphiidae): a morphometric analysis". -- S. Heinemann & G. Uhl (Journal of Arachnology 28:23–28, 2000).

- Among the Mygalomorphae, there are several cases of sexual dimorphism in the length of urticating setae. Please see review in: "Morphology, evolution and usage of urticating setae by tarantulas (Araneae: Theraphosidae)" -- R. Bertani & J.P.L. Guadanucci (Fortschr. Zool. 30:403–418, 2013).

- Elongated legs in males may have different functions and I think these different functions could be better explained and explored in the manuscript. In some species, males use elongated legs in intrasexual contests. Leg length is probably used in the evaluation of the opponent as an indicator of his size and strength. Thus, the main selective pressure leading to leg elongation in these species is intrasexual selection. In other species, legs are used as sexual display to attract or court females and the main selective pressure is intersexual selection. Although some examples of leg elongation probably promoted by intersexual selection were mentioned in the manuscript, I think the authors should provide more information and examples of leg elongation probably promoted by intrasexual selection (i.e., male-male contests). Example: "Behavior and ecology of four species of Modissimus and Blechroscelis (Pholcidae)" -- W. G. Eberhard & R. D. Briceño (Rev. Arachnol. 6:29-36, 1985).

1.3) Opiliones

- There is a well-reported case of sexual and intrasexual dimorphism in the length of the second pair of legs in the harvestman Serracutisoma proximum. The second legs are used as whips in male-male fights for the possession of reproductive territories. This example has not been mentioned in the manuscript, and considering that we know much more about the biology of S. proximum than we know about Phalangium opilio, I think the attention devoted to this later species is unbalanced in the manuscript (which also includes a figure about sexual dimorphism in P. opilio). Please see: "Resource defense polygyny shifts to female defense polygyny over the course of the reproductive season of a Neotropical harvestman" -- B. A. Buzatto & G. Machado (Behavioral Ecology and Sociobiology, 63:85-94, 2008). / "Conditional male dimorphism and alternative reproductive tactics in a Neotropical arachnid (Opiliones)". B. A. Buzatto et al. (Evolutionary Ecology, 25:331-349, 2011) / "Male dimorphism of a neotropical arachnid: harem size, sneaker opportunities, and gonadal investment" -- R. Munguía-Steyer et al. (Behavioral Ecology, 23:827-835, 2012) / "A sexual network approach to sperm competition in a species with alternative mating tactics" -- D. G.Muniz et al.(Behavioral Ecology, 26:121–129, 2015) / "Experimental limitation of oviposition sites affects the mating system of an arachnid with resource defence polygyny" -- D. G. Muniz & G. Machado (Animal Behaviour, 109:23-31, 2015) / "Devoted fathers or selfish lovers? Conflict between mating effort and parental care in a harem-defending arachnid" -- L. M. Alissa et al. (Journal of Evolutionary Biology, 30: 191-201, 2017).

- There is a review of intrasexual dimorphism in harvestmen: "Male dimorphism and alternative reproductive tactics in harvestmen (Arachnida: Opiliones)" -- B. A. Buzatto & G. Machado (Behavioural Processes, 109:2-13, 2014). Although the focus of this paper is on intrasexual dimorphism, I think it can provide useful information for your manuscript on sexual dimorphism. In general, minor males are similar (but not identical) to females, whereas major males have exaggerate traits that make them different from both minors and females. The paper provides a table with a review of the known cases of intrasexual dimorphism in harvestmen and also indicates which cases are supported by statistical evidence and which cases are only "observational".

- In the family Neopilionidae, there are several cases of extreme male morphologies related to cheliceral size and shape. In a recent paper on this family, there is the first description of male trimorphism in arachnids. As I said above, the same traits that are intrasexually dimorphic are sexually dimorphic in neopilionids. Please see: "Multiple exaggerated weapon morphs: a novel form of male polymorphism in harvestmen" -- C. J. Painting et al. (Scientific Reports, 5:16368, 2015).

- In a recent paper, there is a macroecological analysis of the magnitude of sexual dimorphism in harvestmen. We first showed that the length of the breeding season in harvestmen as a whole is strongly determined by the number of months with proper climate conditions (mean temperature above 5 °C and high precipitation). The length of the breeding season, in turn, strongly influences the type of mating system of each species: when the breeding season is very short, males rely mostly on scramble competition polygyny; when the breeding season is long, males rely mostly on resource defense polygyny. Finally, the type of mating system is correlated with the magnitude of sexual dimorphism in harvestmen: in species with scramble competition males do not bear weaponry and are usually smaller than females; in species with resource defense, males bear weaponry and are usually larger than females. I really think this paper provides nice information for your review. Please see: "Macroecology of sexual selection: a predictive conceptual framework for large-scale variation in reproductive traits" -- G. Machado et al. (The American Naturalist, 188:S8-S27, 2016).

2) My second criticism is tightly related to the first one: based on the information provided by the authors, it seems to be impossible to replicate the result of the literature search. In the last years, there is growing concern about reproducibility of scientific research and people working with any kind of systematic review proposed the PRISMA (Preferred Reporting Items for Systematic reviews and Meta-Analyses) protocol (http://www.prisma-statement.org/). This protocol allows the readers to understand how the literature search has been conducted, how many papers have been initially found, how many have been discarded, and how many papers were ultimately included in the review. Following this protocol has a great advantage: the authors can provide both qualitative and quantitative information for the readers. I am not proposing the author conduct a meta-analysis, but they can add numbers to provide a thorough general picture. The following topics could be addressed: (1) how many species were detailed studied in each order?; (2) For how many species do we have statistical data on sexual dimorphism?; (3) What is the percentage of species in each order for which we have reports of sexual dimorphism on body size, legs, chelicerae, pedipalps, etc.? In the current format, we have an interesting collection of examples and a poorly-supported attempt to detect broad patterns. I am sure that numbers would make the review much more useful and appealing to a broad audience.

3) Overall, there is extensive information on the types of sexual dimorphism in different arachnid orders, but the functional meaning of these types of dimorphisms is not properly explored in the manuscript. According to my view, the review could be greatly benefitted from more behavioral information related to how males and females use sexually-dimorphic structures. Even for poorly-studied groups there are behavioral data for some species describing how males use their sexually-dimorphic structures. This is the case of pseudoscorpions, which are nicely explored by the authors in the manuscript. For other groups, such as whip-spiders and harvestmen, there is also information on the behavioral role of sexually-dimorphic male traits that could be better explored in the review.

Moreover, the selective pressures that may have promoted the evolution of some types of sexual dimorphism could be expanded. Although there some brief comments on this subject along the manuscript, they are not evenly distributed among the orders (and here I am only talking about the more intensively studied groups, such as spiders, scorpions, harvestmen, pseudoscorpions, and perhaps camel-spiders and whip-spiders). As the authors state in the introduction, the evolution of sexual dimorphism may be the result of both natural and sexual selection acting on males and/or females. In my opinion, the manuscript would be more useful and informative if the authors devote more space in the discussion to explore the role of these selective pressures. More specifically, the following topics could be addressed: (1) Is there any evidence that niche partitioning promotes sexual dimorphism in arachnids? If so, which sex seems to be modified and why?; (2) Is there any evidence about fecundity selection acting on females in the different orders? I am sure that there are correlations between female size and fecundity for many different species; (3) When males have sexually dimorphic structures, are they used mainly as weapons or ornaments? In other words, what selective pressure seems to be more pervasive, intra- or intersexual selection?; (4) Scramble competition seems to be the most common mating system in arachnids and, according to the theory, sexual selection should favor male traits that increase the chances of finding a receptive female. These traits would include longer legs and more developed sensory structures (Andersson, 1994: book "Sexual selection"). Does this prediction hold for arachnids as a whole?

Besides the three major concerns mentioned above, I also have numerous small comments and suggestions, which are presented below.

I hope my comments help the authors to improve the manuscript. In case of any doubt, please contact me.

Glauco Machado
Department of Ecology
University of Sao Paulo
E-mail: glaucom@ib.usp.br

= = = = = = = = = = = = = = = = = = = = = =

Minor comments/suggestions:

Line 40: "(Shine, 1989; Andersson, 1994)"

Lines 40-46: please consider the following suggestion:

"Documenting and describing the occurrence of sexually dimorphic traits can provide important insights into the evolution of morphology. Amongst vertebrate groups, for instance, the occurrence of SD is well-documented. In mammals, SD has been compared in 1370 species, representing around 30% of known mammalian species (Lindenfors, Gittleman & Jones, 2007). Datasets of similar size have been used to quantify SD in reptiles (Cox et al. 2007) and birds (Owens & Hartley, 1998)."

Line 65: "...asymmetry (Proctor, 2003), extreme size..."

Line 84: "...across eleven modern..."

Lines 103-112: please see the following paper: Kilmer JT, Rodríguez RL, 2015. Do structures with sexual contact functions evolve negative static allometries? A case study with the harvestman Leiobunum vittatum (Opiliones Sclerosomatidae). Ethol. Ecol. Evol. 29:64–73.

Line 121: "(e.g. Moya-Laraño et al. 2002; Foellmer & Moya-Larano 2007; Grossi & Canals 2015)"

Line 133: "...on the posterior part of the opisthosoma in schizomids..."

Line 166: "Garwood et al.’s (2017)"

Line 170: "...with this clade being the sister group..."

Line 174: "(e.g. Behan-Pelletier & Eamer 2010; Behan-Pelletier 2015a, b)"

Lines 175-176: please merge these two paragraphs.

Line 185: "...some mite species."

Line 198: "Setal arrangement also varies between sexes..."

Lines 200-203: "Male pedipalps are enlarged relative to female conspecifics and have branches unseen in females in some species of Astigmata, and in the most extreme cases appear antler-like (Proctor, 2003). Chelicerae are also enlarged in some male feather mite species (Proctor, 2003)." -- Thus, in this group, pedipalps and chelicerae are not fused to each other forming the gnathosoma, right? Please explain it.

Line 221: "Research into SD among mites and ticks has thus far been..."

Line 281: in the arachnological literature, the expression "true spiders" is used mostly to the Araneomorphae and not to the order as a whole.

Line 282: "(Platnick & Raven, 2013)" -- I suggest to change this citation for the most recent version of the World Spider Catalogue (https://wsc.nmbe.ch/).

Line 284: "...and the ability to produce silk" -- at least two other arachnid groups contain species that produce silk: pseudoscorpions and mites. Thus, you should to re-phrase this sentence.

Line 288: "Spiders are typically characterised..."

Line 292: are you really referring to the "Tetragnathidae" or to the "Nephilidae"?

Lines 293-294: another well-reported case of reversed SSD in spiders occurs in the genus Allocosa from Uruguay. In species of this genus, males are larger than females and there are several behavioral traits that indicate sex-role reversal.

Lines 306-318: I suggest to remove this paragraph because pedipalps in male spiders are sexual organs, thus you are talking about primary and not secondary sexual dimorphism. If you decide to mention all cases of primary sexual dimorphism in all arachnid orders, you will need to talk about spermatophore and penis morphology. As far as I understand, this is not the main goal of the manuscript. Thus, for the sake of consistency, you should not include sexual dimorphism related to pedipalp morphology in spiders. The only exception, according to my view, is the example of Allocosa males, which have sexually dimorphic digging structures on their pedipalps.

Line 350: "conflict" -- I think the best word here is "contest". Please change it throughout the entire manuscript.

Line 351: " conflict" -- I suggest to change this word for "male-male competition" or "intrasexual selection".

Line 360: "and" -- non-italic.

Line 387: "Parisitiformes"

Lines 424-425: "The metric..."

Lines 427: "Cranaidae and Oncopodidae"

Lines 456-457: "...suggesting that chemical secretions may also play a role in warding off rival males" -- Although it is possible, I have never seen males releasing secretion during intrasexual fights.

Line 465: " (Metasarcidae, Cranaidae and Oncopodidae..."

Line 467: "During contest, males..."

Line 476: the word "ornaments" is inadequate because there is no empirical evidence supporting that sexually-dimorphic structures in harvestmen function as ornaments. Most of them unequivocally function as weapons.

Lines 481-482: "Identifying a reliable proxy for overall body size and statistically testing SSD should be a priority" -- According to my personal experience, there is no general proxy of overall body size in harvestmen. Perhaps the best candidate would be the width of the cephalothorax, but this body segment is highly fused with other body segments. Thus, in many groups, it is difficult to recognize the cephalothorax and measure it.

Lines 507-598: "It is not uncommon, for example, to find both strong male-biased and female-biased SSD in claw size within a genus (see figure 9, Zeh, 1987b)" -- Please note that the phylogenetic studies of pseudoscorpions begun only in the late 1990's. I would not be surprised if the species used in the study by Zeh (1987b) now belong to different genera. Please try to check this information in the excellent website by Mark Harvey (http://www.museum.wa.gov.au/catalogues/pseudoscorpions).

Line 515: " (Weygoldt, 1966, 1969)"

Lines 531-532: in the section on Palpidradi you say: "They are also the least speciose order, with 78 extant species (Harvey, 2003)" (line 379-380). Here you say that "Ricinulei, or hooded tick spiders, are a small arachnid order comprising only 58 described species (Prendini, 2011)". Based on the numbers you presented, Ricinulei is the least speciose order.

Line 535: "No consensus exists..."

Line 555 and 678: "leg play" -- What does it mean? Is it a type of copulatory courtship?

Line 558: "...and longer (Pittard, 1970) in male..."

Line 603: "...Furthermore, the male pedipalp..."

Line 657: "In Heterometrus laoticus the telson is found..."

Lines 658-659: "(Booncham et al., 2007)"

Line 660: "Other structural modifications can be found (?) in males of Anuroctonus

Line 660: "Phaiodactylus"

Line 685: "sp.)" -- non-italics.

Line 763: "manus" -- What is it? Please explain.

Line 821: "near the abdomen" -- Please be more specific.

Line 839: "...and the patella..."

Line 842: "tibial apophysis" -- On the pedipalp? Please make it clear.

Line 845: "(Gravely, 1915; Yoshikura, 1973; Weygoldt, 1972, 1978; Rajashekhar & Bali, 1982)"

Line 848: "T. indicus" ("and" -- non-italics)

Line 858: " Klingel, 1963; Weygoldt, 1971, 1972"

Line 889-890: "...female scorpions may engage in sexual cannibalism of males (see Peretti et al. 1999); this could hypothetically to lead to the rare male-biased SSD within this group" -- According to Peretti et al. (1999), "no post-mating cannibalism was ever observed in [scorpions]". Thus, any hypothesis to explain sexual size dimorphism based on the assumption of sexual cannibalism should be revised or eliminated from the manuscript.

Line 892: "hypothesised in some species of the spider family Linyphiidae (Lang, 2001)" -- This example was not mentioned in the topic about spiders.

Line 900: "...leg length, shape, armature..."

Line 917: "...uncertainty. In arachnids..."

FIGURES:

- I suggest to merge figures 1, 2, 4, 4, 6, 8, 10, 11, 13, 15 and 16 into a single plate for comparative purposes. Please see the suggestion below:

"Fig. 1 – Patterns of sexual dimorphism in arachnids: (a) Acari, (b) Amblypygi, (c) Araneae, (d) Palpigradi, (e) Opiliones, (f) Pseudoscorpiones, (g) Ricinulei, (h) Schizomida, (i) Scorpiones, (j) Solifugae, and (k) Uropygi. In all schemes body parts coloured red indicate male-biased SSD, green indicates a female bias, and purple mixed sex bias. Legs are numbered 1-4, chelicerae are marked “C” and pedipalps are marked “P”; male (♂) or female (♀) symbols denote sexual size dimorphism in overall body size."

- If you accept the suggestion above, please remove the text from lines 146 to 151 (this information is now presented in the legend of Fig. 1) and from lines 1469 to 1472 (this information is already presented in the main text).

- Fig. 17 is in fact a table.


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: SEXUAL DIMORPHISM IN THE ARACHNID ORDERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript entitled “SEXUAL DIMORPHISM IN THE ARACHNID ORDERS” is aimed to provide an overview of the research to date on sexual dimorphism in individual arachnid orders, using these data to analyse trends driving the evolution of sexual dimorphism across Arachnida.

This is a generally well-written paper, with an appropriate use of academic English throughout, except for small typos (see comments to the authors).

The paper does not always include an adequate framework where to place its aims, and some relevant references are lacking. For example, the authors emphasize that spiders are the only arachnid group in which some broad revision of SSD has been done. However, the section devoted to the Order Araneae is extremely limited. Authors summarise main findings in this group by saying that research has probed the degree to which spiders follow Rensch’s Rule (L. 57), but they do not provide a single reference to support this view, nor do they document that the opposite, i.e., that spiders do not fulfil predictions from the rule, is generally accepted to be true.

Structure generally conforms PeerJ standards. However, having to review patterns of sexual size and shape dimorphism in such a morphologically diverse taxonomical group requires a huge amount of information and a large extension, making the paper excessively long and fragmented.

Figures are relevant to illustrate the morphological diversity of the analysed taxa.

No new raw data are provided, as the paper only reviews already published data.
Experimental design: This is a literature review article within the scope of PeerJ.

The main contributions of the paper are supposedly related to the range of taxonomic groups included, the use of type II regression to analyse patterns of sexual size dimorphism and the choice of appropriate estimates of body size. However, the real contributions of this paper to filling these gaps is questionable. Actually, the paper mostly summarises common trends of variation described up to date and lacks any new proposal for data analysis or size estimates. For example, figure 3 represents results from a type II regression analysis, but there is not a single reference in the text confronting this with alternative techniques.

The use of methodological terms is obscure. One of the authors’ claims is that shape should be considered together with size in the analysis of sexual dimorphism. However, throughout the paper, authors mostly refer to sexual size dimorphism, and they use size and shape as two interchangeable terms. For example, they talk about sexual size dimorphism in Acari’s glandular system (L. 208), when referring to the arrangement of the dermal porose areas.

The Methods section is extremely short and undetailed. Particularly, the authors give no clue on the criteria used to select the papers eventually included or excluded from their revision.
Validity of the findings: Spiders have increasingly become a model group in the evolutionary analysis of sexual size dimorphism, whereas information about other arachnid groups is scarce. Enlarging the range of taxa analysed looks extremely promising. However, at its present state, this paper mostly describes patterns of variation and does not make any new or original contribution to the analysis of their evolutionary origins. Probably, the research question is not appropriately addressed, and the paper should be better addressed to a taxonomically oriented audience.

Though this is not a primary research, a lot of information needed to support the author’s claims is lacking. Particularly, there is a lack of essential information about ecology and phylogeny, limiting the paper scope in the analysis of evolutionary trends. Indeed, one of its main conclusions is that hypotheses about the evolutionary history of arachnid sexual dimorphism are limited by the uncertainty in arachnid phylogenetic relationships.

Conclusions are not always clearly drawn from the data. For example, the main conclusion of the paper is the driving role of fecundity selection (L 885) when explaining the general F>M trend of sexual size dimorphism across arachnids. However, only two arachnid orders are acknowledged to follow this pattern when referring to body size, whereas the opposite (relatively larger males, M>F) is true across the Class, as shown in Table 1.
Additional comments: L. 84: typo (elven)

L. 91-102: there seems to be a confusion of terms. Assessment of Sexual dimorphism in vertebrates is diverse, and it is uncertain to say that it is generally based on body mass. Similarly, there is an agreement to use carapace width as an indicator of spider body size, whereas the width/length ratio of the carapace should be used as a shape, and not size, estimator

L. 97 typo (thought to indicative)


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: SEXUAL DIMORPHISM IN THE ARACHNID ORDERS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised version of the manuscript has been substantially improved and all my major comments were properly addressed by the authors. I have several small comments and all of them were directly inserted in the text. Please consult the attached file.
Experimental design: Please consult the attached file.
Validity of the findings: Please consult the attached file.
Additional comments: I'm really glad to see how much energy you put in the revised version of the manuscript. I think the text has been greatly improved and I'm sure that this review will be an important contribution to the field and a highly cited paper. Congratulations!


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: GENETIC POPULATION STRUCTURE OF THE PELAGIC MOLLUSK LIMACINA HELICINA IN THE KARA SEA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English is not yet perfect. I have added a list of examples (not exhaustive) to my general comments.
Experimental design: This is a relevant addition to what was already known about an abundant pelagic mollusk. The authors have gathered data in a difficult to reach Arctic Sea and connected the new data to data published earlier.
The interpretation of results can be improved and elaborated; for suggestions see me general comments.
Validity of the findings: There is a small difference in sample size between what is mentioned in the paper and what I found in the data files provided. For details see General Comments.
Additional comments: Major comments
L106-107 you report on Fst values from the AMOVA and not Phi-st which would be more standard. The latter takes into account nucleotide differences between haplotypes. Are you certain you used the haplotype frequencies only? In that case the use of Fst throughout the manuscript is OK. Please add that to the lines 106-107 and provide arguments why you chose to disregard the sequence information. If you did use the sequence information (preferable), you should add that at lines 106-107, and change Fst to Phi-st throughout the manucript.
L109 reports 57 sequences but the data file 'limacina-helicina-kara.txt' contains 65 sequences.
L112, in contrast, does report on 236-179=57 sequences (236 is the number in 'limacina_helicina_kara_pacific_svalbard_236seq.txt' and 179 is the number of sequences used from other sources as reported here in L112)
L137-138 Contrary to what is written here, Table 2 does not indicate which values significantly depart from 0. This should be added.
About Fu's Fs: throughout the manuscript you interpret the significantly negative values as evidence of a bottleneck (e.g., L157), with or without recent expansion (e.g., L41-42, L159). To my knowledge this is not correct. A negative Fu's Fs means that there are more haplotypes in the sample than expected on the basis of the number of pairwise nucleotide differences and the sample size. This happens (among others) after a recent population expansion. A bottleneck is not necessary to cause the low Fs and so you should not say that.
Within Kara Sea. You sampled at three locations in Kara Sea but do not report on (the absence of) structure among these samples. Only L109-110 reports on one comparison, while the other two other pairwise comparisons as well as the overall Fst for the three samples are missing. It seems obvious from the haplotype frequencies that there is no structure but in my opinion you should report on it nonetheless.
L45-48 (and L204) on the basis of what do you conclude that the origin is Pacific and not Atlantic? Just the Fu Fs analyses seems limited evidence (and the interpretation needs to be reconsidered as mentioned above). Your data do not refute a scenario in which the species originates from the Atlantic, colonized the Pacific quite a while ago with no traces of a bottleneck there, while Pleistocene reductions in the Atlantic cause the bottleneck signature here. Also, the fact that both haplogroups are star-like (as you note yourself in L119-120) makes it likely that both have a similar demographic history. Limacina's do have a fossil record which I suggest you dive into to add more credibility to this claim. Burridge et al 2017 (Plos One) may provide some insights into the location of origin of Limacina species and they also reference fossils.
L185-186 'indicate an exchange between these two populations' This is not correct. The observation is consistent with ongoing exchange but it does not indicate it. It is also possible that population subdivision is recent, and an absence of exchange would not yet show in the haplotype frequencies. I suggest to change 'indicate' to 'is consistent with' and mention the alternative explanation as well.
L192-195 and L200-203 continuing from the previous point, here you contradict yourself. You say that different water bodies do not fully mix in the Kara Sea and that this may underlie (non-significant) genetic differences within Kara Sea. If you just concluded that there is exchange between Kara and Svalbard, but (some) isolation within Kara, that sounds contradicting to me. You need more data to have more certainty.

Minor comments
L27 you analyzed 65 haplotypes of which 19 Kara Sea. Better would be to report how many new individuals you sequenced (Kara Sea), how many haplotypes were among them, and how this compares to the number of haplotypes found previously by other authors.
L31-32 'While the other one, named Pacific, is widespread and found throughout all L. helicina populations.' Also at L122-123. It seems strange to choose a regional name to describe a haplotype group that is found everywhere. I suggest to use a different name.
L32-35 the description of the structure that you found is not precise. Please change it so that the reader knows exactly at what level you did detect structure and at which level you did not.
L45-46 what do you mean by 'the form' here? (same at L166-168)
L157 'by populations between the Pacific and the Arctic' it is unclear to me what you mean here
L159-160 'The observed wide diversity of haplotypes indicates a large population size' also not clear what is meant here
L179 'an isolated Atlantic haplotype along with the Pacific haplotype (Fig. 3)' I do not see what exactly in Fig. 3 you mean here
L179-181 'The main northward currents from the Pacific to the Arctic region are nowadays passing through the Bering Strait' I suppose you mean the main northward currents going into the Arctic, because what other routes into the Arctic from the Pacific are there except the Bering Strait?

Spelling/grammar etc.
L20 delete 'the data'
L32-33 sentence is not grammatically correct
L39 with 'samples' do you mean 'numbers'? (same for L154)
L39-41 is a repetition from what you write just before it (same for L153-155)
L41 delete 'the' in 'indicate the directed selection'
L42 change 'the passage of the bottleneck' to 'a population bottleneck'
L114 delete 'the' from 'the Svalbard'
L132 change 'Atlantic haplogroup' to 'The Atlantic haplogroup'.
L143-144 consider to rephrase 'suggestion about genetic similarity of L. helicina populations from the Kara Sea and around Svalbard' to something like 'expectation that L. helicina from the Kara Sea would be genetically similar to those near Svalbard'.
L147 delete 'the' in 'to the haplotype group'
L148 delete 'is' in 'minor part is to'
L158 I do not think 'redundancy' is the right word here
Table 2 and elsewhere: decimal in English is done with a period not a comma (so 1.5 not 1,5)
Figure 3 caption: please provide more information, e.g. size of circle indicates sample size.
L192 change 'Tis' to 'This' and 'according' to 'accordance'


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: GENETIC POPULATION STRUCTURE OF THE PELAGIC MOLLUSK LIMACINA HELICINA IN THE KARA SEA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is pretty well written (but I am not a native speaker either).

References are fine.

Good structure.

Well presented research, only one "problem":

I consider (very) confusing the fact that authors chose to name the two haplotype clades as Atlantic and Pacific, and the also the regions Atlantic and Pacific. I can follow it (since I am familiar with haplotype networks), but I would recommend maybe just calling them I and II, or something like that. Especially when you have Pacific haplotypes in the Atlantic sample, but not Atlantic haplotypes in the Pacific sample. This phrase, as you can read, can be very confusing. If you name the two clades I and II, then you have no problems: “haplotypes from clades I and II were present in the Atlantic, meanwhile only Clade I ones were present in the Pacific”. Much easier to understand.
Experimental design: Few comments regarding methods:

Did not see any ANOVA done. There are sample by sample Fst distances, not AMOVA. So, methods have to be changed to that, like” Between samples Fst distances were calculates in Arlequin…”

Some results are not shown – distances between the samples from the authors. “We obtained 57 sequences from the Kara Sea. The highest Fst value found between the southern and northern parts was 0.023 (p < 0.5)” but they have three samples.  They can add there a “data not shown”

In my opinion, using only one marker for testing for Tajima and Fu’s is not appropriate/informative for planktonic species with such hugh population sizes. They ALL show negative values. See Wares 2010 Evolution 64: 1136-1142.
Validity of the findings: Nice paper regarding population structure in the Arctic, a hot topic nowadays due to climate change.

data is robust and well analyzed.
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: GENETIC POPULATION STRUCTURE OF THE PELAGIC MOLLUSK LIMACINA HELICINA IN THE KARA SEA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article is written in unambiguous, professional English, but it might benefit from last correction by a native speaker. I am not a native speaker myself but I would e.g. consider a definite article necessary in the title: Genetic population structure of the pelagic mollusk…
All relevant literature is cited and provides sufficient background for the understanding of the manuscript. However, the formatting of the references contains several mistakes and needs to be revised carefully. E.g. most of the species names in the titles are not in italic (see number 3,13,16,23 etc.). Sometimes the Journal names are misspelled (i.e. capital letters missing in 16, 27…) Identical articles appear twice (e.g. 16 and 17, 26 and 27) and/ or lack list of authors, citation 35 is incomplete.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The manuscript entitled „Genetic population structure of pelagic mollusk Limacina helicina on the Kara Sea“ (# 23753) by Abyzova et al. presents novel genetic data on the population structure of an ecologically important pelagic gastropod form the Siberian Arctic Sea. It fills (part of) the gap in population genetic data between previous studies from the Svalbard area and from the subarctic western North Pacific, presenting novel insights into haplotype diversity and connectivity. Limacina helicina plays a key role in studies on ocean acidification and climate change, thus I believe that the presented data is of general interest and presents a valuable contribution in documenting current population patters. I clearly want to recommend the manuscript for publication, but would like the authors to consider and review the following questions/ suggestions below prior to publication:
One question: is it correct to speak of different “habitats” (see Abstract and Line 79 and further on) in regards to your sampling sites? In my understanding a habitat provides specific abiotic or biotic factors. Which factors/ parameters differ between your sampling sites that justifies calling them different habitats? In the Conclusions you refer to different “biotopes” – please explain in more detail how the three localities differ.
I would recommend rearranging your results: why not present first the analyses on your Kara Sea samples alone (maybe also adding an additional haplotype network including only Kara Sea specimens)? It could easily be included in Fig. 2 as A and B and would offer the opportunity to show the differences between your three sampling localities and how the haplotypes are spread among them. Once you presented your own data, you could then continue by analyzing your data in the context of the published sequences from the different geographic areas.
Minor corrections in the text:
Line 53: locally
Line 55: there is no Hunt 2008 in the references, should probably read Hunt et al. 2008
Line 76: Limacina in italics
Line 96: Out of curiosity: how was the percentage of successful COI sequences? I.e. how many specimens were available to the study and how many did you sample to get your sequences?
Line 106: Excoifer
Line 112: In total, 179 L. helicina …
Line 117: Layton et al., 2017
Line 143: maybe replace “suggestion” with “hypothesis”
Line 168/170: Sromek et al, 2015
Line 174: A similar spread…
Line 175: Laakkonen et al. 2013
Line 177: during the glacial period between…
Line 192: This is in accordance with
In the tables you refer to Spitsbergen – which is only called Svalbard throughout the main text.

My apologies for the delayed review!


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: GENETIC POPULATION STRUCTURE OF THE PELAGIC MOLLUSK LIMACINA HELICINA IN THE KARA SEA
Review round: 2
Reviewer: 1

Output:
Basic reporting: 1.	The authors did an overall good job of incorporating the reviewers' comments. I have a number of additional suggestions, but they are mostly minor.
2.	L17-20 these two sentences make it sound as if two different data sets were collected to test for a) difference among locations, and b) difference among habitats. But they are the same comparison among the same three samples, aren't they? So this should be rephrased to reflect that.
3.	L28 'combined into two significantly different groups'. The word 'significantly' here is not based on a statistical test and should be omitted.
4.	L62 do you really mean per square meter here, or per cubic meter instead? It is a pelagic organism after all, so measuring their density in 2D seems unlogical.
5.	L66 you mention here 'Limacina' but because you mean the species here I suggest to change it to 'Limacina helicina' (in italics).
6.	L79-81 The salinities lack a unit.
7.	L106-109 It is not common practice to write p < 0.5. Instead, I suggest to write n.s. for not significant. The same for L137.
8.	L106-109 please refer here to your Table 3.
9.	L168 H1 and H2 are haplogroups (or haplotype groups) but not haplotypes
Experimental design: 10.	L162-164 Not clear if this sentence refers to your own data or analyses, or if it refers to a general pattern for other taxa. Please make this more clear. If it refers to a general pattern, please provide references. If it refers to your own data/analyses, I am not convinced that this can be concluded; you did not do analyses that show that diversity increased at that time.
11.	L168-172 This is still not explicit enough after revision. I would suggest to change 'The northward currents from the Pacific to the Arctic region are currently passing through the Bering Strait (Palumbi & Kessing, 1991; Questel et al., 2016), which is reflected by the wide distribution of the haplotypes H1 throughout the Arctic and by the absence of haplogroup H2 south of the Bering Strait in the Pacific Ocean.' to: 'The currents flowing between the Pacific and the Arctic through the Bering Strait have a predominantly northward direction (see references in Questel et al., 2016). This lends support to a hypothesis that Limacina helicina may only effectively migrate from the Pacific into the Arctic and not the other way around, which is consistent with our observation that the H1 haplogroup has reached the Arctic, while the H2 haplogroup appears to be absent from the Pacific (Fig. 3)'.
12.	L169-170 Palumbi & Kessing, 1991 is not a good reference here.
13.	L187-188 Indeed, this paper makes an important contribution to zooplankton phylogeography by providing data on this large Arctic sea, which is not easily accessible.
Validity of the findings: 14.	all is good
Additional comments: Grammar and spelling:
15.	L27 change 'around Arctic' to 'around the Arctic'
16.	L38 delete 'due to'
17.	L53 change 'for' to 'studies on'
18.	L64 change 'shelf Siberian' to 'shelf for the Siberian'
19.	L79 change 'this station affected' to 'this station is affected'
20.	L119 change 'includes majority of' to 'includes the majority of'
21.	L125 change 'haplogroup also' to 'haplogroup was also'
22.	L127-129 I am not a native speaker but I think it is better to use 'diversity' instead of 'variety'
23.	L143 change 'North of Atlantic' to 'North of the Atlantic'
24.	L154 change 'for alternative explanation' to 'for an alternative explanation'
25.	L162 change 'in Pacific refuge' to 'in a Pacific refuge' and 'H2 - in Atlantic' to 'H2 in an Atlantic refuge'.


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: GENETIC POPULATION STRUCTURE OF THE PELAGIC MOLLUSK LIMACINA HELICINA IN THE KARA SEA
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comments
Experimental design: Again – there was no AMOVA done. I do not see any analysis of the partitioning of the variance. The only think I see there is pairwise ϕST distances between samples or regions, not an AMOVA. Methods should then read “Pairwise ϕST distances were calculated in Arlequin between samples within the Kara Sea and between seas”. That IS NOT an AMOVA analyses. An AMOVA will give you three statistics (ST, SC, CT), about how the genetic variance is distributed within samples, between samples within groups and between groups. It is true that they the same screen in Arlequin, but... Results explain properly what the authors did though.
Validity of the findings: no comments (after previous review)
Additional comments: Lines 114 and 115: “A total of 65 haplotypes was found” should read “A total of 65 haplotypes were found”. The noun is still “haplotypes”. Only singular if “total” is the noun.

Substitute “Phi-st” by ϕST (the greek letter) in the text


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: GENETIC POPULATION STRUCTURE OF THE PELAGIC MOLLUSK LIMACINA HELICINA IN THE KARA SEA
Review round: 3
Reviewer: 1

Output:
Basic reporting: The authors did a good job of incorporating reviewer comments. I have no further issues.
Experimental design: No further issues.
Validity of the findings: No further issues.
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: GENETIC POPULATION STRUCTURE OF THE PELAGIC MOLLUSK LIMACINA HELICINA IN THE KARA SEA
Review round: 3
Reviewer: 2

Output:
Basic reporting: All my questions have been answered / solved
Experimental design: All my questions have been answered / solved
Validity of the findings: No comment
Additional comments: All my questions have been answered / solved


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: ALL YOU CAN EAT: THE FUNCTIONAL RESPONSE OF THE COLD-WATER CORAL DESMOPHYLLUM DIANTHUS FEEDING ON KRILL AND COPEPODS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well written and clear,  and professional English have been used throughout.
Literature references have been well selected and they provide good background and context for the manuscript.
The manuscript has a good structure and all figs/tabs/supplementary material have been well cited and provided. Nevertheless, Video S1 has low resolution and Figure and Video captions of supplementary material are missing.
Hypotheses are well expressed and results are well presented and relevant to the hypotheses
Experimental design: The manuscript is an original primary research and reflect the aims and scope of the journal.
Research questions are relevant and well defined. Authors have clearly identified the knowledge gaps and the method to develop an experimental design able to answer the question and fill the scientific gaps.
The research investigation has been performed with good technical & ethical standard.
Method and materials are well described with good level of details and information. However, in order to facilitate the understanding to the reader, further clarifications are needed:
Line 131. In the incubation experiments is not clear if corals are in the glass bottles with one of the prey type and in two different days as suggested in table 1. See my comments in table 1
Line 142. Back at HSFS Lab, It is not clear an aspect of the experiment. At the beginning of the section/paragraph it should be said the samples are moved from HSFS lab- acclimation tanks to HSFS jetty- in situ experiment.
TABLE 1
If date is the incubation date, following the protocol explained in the test it means that e.g :
09/09 dive day with 20 coral specimens
10-11/09 acclimation days (48h)
11/09 incubation day
If this is correct, you should have (in theoy) in the table"
11/09 EV N 10
11/09 CP N 10

Since there is a difference between the theory and the info in the table, you should explain (at least in a note in the table)
- why are there two different days for zooplankton (one for ev and one for cp) . Couldn't be same day with different bottles for each prey type?
- different number of coral specimens than expected and for each prey type
- there are repeats for each experiment (same month, same prey type, same day) but not replicates. Is there any reason why replicates have been not considered in the experiment design?
Validity of the findings: The novelty of the results will provide good impact to the scientific knowledge. Data are robust and statistically sound providing conclusive results.
Conclusion are well stated, linked to original research question & limited to supporting results.
Additional comments: INTRO
Line 47. Taxonomic nomenclature rule is not applied. It should be Desmophyllum dianthus (Esper, 1794)
Line 51. Taxonomic nomenclature rules is not applied. It should be Desmophyllum pertusum (syn. Lophelia pertusa).
See WoRMS update, L. pertusa is not accepted anymore
http://www.marinespecies.org/aphia.php?p=taxdetails&id=1245747

New combination reference Addamo et al (2016)
Addamo, A. M.; Vertino, A.; Stolarski, J.; García-Jiménez, R.; Taviani, M.; Machordom, A. (2016). Merging scleractinian genera: the overwhelming genetic similarity between solitary Desmophyllum and colonial Lophelia. BMC Evolutionary Biology. 16(1)., available online at https://doi.org/10.1186/s12862-016-0654-8

Line 53. It is not clear why the sentence starts with "while”. It should be shallow coral banks
Line 55. Shrink to deeper or shallower communities?
Line 57. Please use the same format for geo-coordinates should be used, see line 99
Line 62. Space should be included 7 m

Line 87. Taxonomic nomenclature rule is not applied. It should be Calanoides patagoniensis (Brady, 1880)
Line 88. Taxonomic nomenclature rule is not applied. It should be Euphausia vallentini Stebbing, 1900

MAT&MET

Line 93. It should be Desmophyllum dianthus
Line 96. Capital letter for official name of ministry, it should be Ministry of Economy, Development and Tourism
Line 97. Case means season or month or dive/survey? It needs to be clarified
Line 97. It should be circa or max twenty specimens. See my comments in table 1
Line 99. Please use the same format for geo-coordinates see line 57
Line 132. It should be Desmophyllum dianthus
Line 135. I have a doubt about Turbulence? if the corals are inside glass bottles, the ins situ conditions of turbulence is not respected the real condition, because they are affected by glass bottle, which works as closed cage. Isn't it?
Line 143. The remaining zooplankton that have been not captured by corals (?). it will be helpful clarify it.
Line 157. Month or season?

DISCUSSION
Line 230. Just an observation: since corals has been moved from their natural substrate, to lab, and finally to jetty, shouldn’t be considered as semi-naturally or slightly manipulated naturally conditions?
Line 231. It should be Desmophyllum dianthus
Line 235. It should be Desmophyllum dianthus
Line 245. It should be Desmophyllum dianthus
Line 272. It should be Desmophyllum dianthus
Line 276. It should be Desmophyllum dianthus
Line 297. It should be Euphausia vallentini


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: ALL YOU CAN EAT: THE FUNCTIONAL RESPONSE OF THE COLD-WATER CORAL DESMOPHYLLUM DIANTHUS FEEDING ON KRILL AND COPEPODS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors make a convincing case that Desmophyllum dianthus is a generalist zooplankton predator capable of exploiting dense aggregations of zooplankton over a wide prey size-range – i.e., a type I functional response.  This does not preclude that the species may also employ the food sources of particulate or dissolved organics.  Their experimental methodology appears to be sound and they have exploited this species at an upwelling location, which makes it more accessible than most other deep-water coral species.  I appreciate the allusion of D. dianthus as a “ghost predator”, a fitting analogy.  Feeding behavior is not my specialty, but it seems to me that the authors have made their point.
Let me just make a few small remarks:
I think the plural of prey is prey, not preys.  I may be wrong.
Line 56: Why would global warming affect deep-water corals?
Supplemental video 1 is of zooplankton swimming.  What is the purpose of this video.  I don’t think it is referenced in the text.
Line 307: replace implicates to implies.
Experimental design: See above
Validity of the findings: See above.
Additional comments: The authors make a convincing case that Desmophyllum dianthus is a generalist zooplankton predator capable of exploiting dense aggregations of zooplankton over a wide prey size-range – i.e., a type I functional response.  This does not preclude that the species may also employ the food sources of particulate or dissolved organics.  Their experimental methodology appears to be sound and they have exploited this species at an upwelling location, which makes it more accessible than most other deep-water coral species.  I appreciate the allusion of D. dianthus as a “ghost predator”, a fitting analogy.  Feeding behavior is not my specialty, but it seems to me that the authors have made their point.
Let me just make a few small remarks:
I think the plural of prey is prey, not preys.  I may be wrong.
Line 56: Why would global warming affect deep-water corals?
Supplemental video 1 is of zooplankton swimming.  What is the purpose of this video.  I don’t think it is referenced in the text.
Line 307: replace implicates to implies.


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: OPTIMIZATION OF THE CYTOGENETIC PROTOCOL FOR PANGASIANODON HYPOPHTHALMUS (SAUVAGE, 1878) AND CLARIAS GARIEPINUS (BURCHELL, 1822)
Review round: 1
Reviewer: 1

Output:
Basic reporting: a. Authors made several unsubstantiated claims .e.g lines 46-49; 52-53 and line 57-59. these generalized claims appears to reflect the authors assumptions. So please clarify.

b. Authors claim ...... 'that the use of intramuscular injections delivered the spindle poison directly into the blood stream' may not be the case as you will need to inject the blood vessels to achieve this assumption (Line 127-129).

c. Based on the sample micrographs presented (i.e figures 3 A and 3 B), the authors assumption that the micrographs represents chromosome spread at metaphase  is unproven. the micrographs may just be artifactual changes in the tissues following the treatments.

d. The article appears to have been written by one of the authors only. In my opinion, there is a need to improve on the use of English and this can be attained if the other authors spend some time to assess the article for grammatical errors.
.....see line 77-78 ......(do the authors mean to say "as" or "has" as written?
Experimental design: a. No comment
Validity of the findings: a. In this type of studies, the pictorial evidence (i.e micrographs) are diagnostic (See Purushothaman et al 2016 (DOI 10.7717/peerj.2377);  and the statistics presented are derived from the scoring emanating from the data generated from the micrographs. Authors failed to provide a scoring format for their study.

b. Sample micrographs presented (i.e 3A and 3B) are not readable and hence not quantifiable. The reviewer advice the article is not suitable for publication in this journal.
Additional comments: 1. Based on the running title, the active word appears to be the word "optimization". So, the article would be better presented with pictorial evidences of the chromosome spread at metaphase showing the various methodologies used in the study.
2. Please provide evidence of how the scoring was made from the micrographs
3. it may be better to label the micrographs presented


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: OPTIMIZATION OF THE CYTOGENETIC PROTOCOL FOR PANGASIANODON HYPOPHTHALMUS (SAUVAGE, 1878) AND CLARIAS GARIEPINUS (BURCHELL, 1822)
Review round: 1
Reviewer: 2

Output:
Basic reporting: Figure 3 is not of good quality. It has a lot of background, it is not possible to see the chromosomes well and the two images are not in the same color scale. I recommend choosing figures that present their results in the best possible way.
Experimental design: Line 95: Some fish chromosome staining protocols use Giemsa 5% for 5 minutes. In the present protocol, you used for 1 hour and in figure 3 we can observe highly colored chromosomes. Would not it be possible to use a coloring for less time, thus improving the preparation time of the material?

The method of preparing slides by dripping 1 meter away can cause the loss of chromosomes, in addition to being difficult technically. Other methods, such as the short-distance dripping on the slide with the vapor layer should be suggested.

Line 86: I could not observe a justification in the introduction for the test of the best time for the preparation of the cellular suspension. The preparation method before hypotonic treatment is accepted and explained in the literature. So, I suggest to justify this test better in the introduction.

Line 104: "..evaluate the interactions.." does not address the objective of ANOVA analysis. The purpose of this analysis could be to verify if there is a difference between treatments or a better explanation.
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: OPTIMIZATION OF THE CYTOGENETIC PROTOCOL FOR PANGASIANODON HYPOPHTHALMUS (SAUVAGE, 1878) AND CLARIAS GARIEPINUS (BURCHELL, 1822)
Review round: 2
Reviewer: 1

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


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

Input:
Title: IMGLAD: ACCURATE DETECTION AND QUANTIFICATION OF TARGET ORGANISMS IN METAGENOMES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall the manuscript is fairly well written, although in parts it is made more difficult to read by an overabundance of commas. There are also several instances of incorrect tense being used.

The introduction is too short, and the history of approaches to this problem is not adequately cited. It would also be helpful for the reader to provide a brief description of the problem as it currently approached outside of a purely bioinformatic perspective (e.g. with PCR-based approaches). Then in the discussion it would be helpful to discuss how imGLAD fits alongside these approaches.

The introduction should also briefly describe the presented algorithm in the last paragraph, before the more detailed description is provided in the materials and methods section.

Overall, the problem being approached does not appear to be well defined. The problem is to decide a binary answer to the question “is organism X in sample Y, where Y has a metagenome?”. However, if a very closely related organism is present in the sample (say 2bp different to the target organism across the entire genome), then should the answer be “no”? If the answer should be “yes”, then where is the threshold?

The impact of library size on detection limit is effectively ignored in this manuscript when it is clearly central to this problem. It isn’t clear, for instance, whether the training dataset size and thresholds should be adjusted somehow in accordance with library size. The relationship between genome size and relative cell abundance is also not discussed, when it should be.

In silico is not hyphenated.

The term “sequence-discrete” is neither defined nor widely used in the field, so a description should be given or alternate wording used.

Line 104: “high sequence conservation”: Presumably the authors mean amino acid sequence conservation - this should be stated.

140: The choice of default as being only based on sequencing breadth was surprising given the text and figures, which often described the depth-based approach.

153: extra comma after e.g. (among other occurrences throughout the manuscript)

154: “filtered version”: it is not clear how the filtering was/is done.

194-197: The tools and libraries described should be properly referenced.

201: The inoculation experiments should be briefly described since readers often do not read the methods in detail before the results section.

201: “>99.9%”: A more exact description of the genome’s similarity should be given e.g. the number of SNPs between the two genomes.

216: Spiking in cells rather than DNA makes these experiments more convincing. However, it would be helpful to know the relative abundance of E. coli in this community in percentage terms.

234: These are genome equivalents not genomes, since DNA was spiked in, not cells.

285 and others: depth and breadth should be reported with units.

421: Grammar error. Perhaps “As the number of studies..”

Fig 2: form => from typographical error.
The meaning of the dashed line should be explained.
In (A), the red star which presumably is the negative control invites misunderstanding as it is denoted in the same way as the positives.

Fig 3: Specificity should be presented too.
Experimental design: The sequencing data for the E. coli spike-in experiment should be deposited into a public database such as SRA.

MyTaxa is based on amino acid sequences, but for closely related genomes comparison of nucleic acid sequences would presumably be more discriminating.

It is unclear why BLAST+ or BLAT is used for read mapping, instead of more common read mappers such as BWA or Bowtie that are likely to (1) be much faster and (2) use paired-end information to help correctly place reads. The thresholds for read mapping are also not reported in the manuscript, when they should be.

Runtime information should be included.

While not a prerequisite for publication, the tool would benefit from a setup.py script so that it can be published in PyPI and installed with its Python dependencies without having to manually install each of them.

The tool also seems to require “awk”, and presumably will not work with Python 3 even though the README suggests Python-2.7+. In order to reduce installation issues it may be advisable to use subprocess.check_call rather than os.system so that failing commands are immediately recognized as such.

230: The exclusion of Bacillus genomes seems odd. Inclusion of closely related genomes (but not those that are target genomes) in the training negative set would presumably be of utility to a prediction tool.

233: The relative abundance should be stated.
Validity of the findings: The approach should also be compared with the popular tool described in the following citation because the tool described attempts specifically to reduce the false positive rate incurred by mapping to conserved regions.
Tracey Allen K. Freitas, Po-E Li, Matthew B. Scholz and Patrick S. G. Chain (2015) Accurate read-based metagenome characterization using a hierarchical suite of unique signatures, Nucleic Acids Research (DOI: 10.1093/nar/gkv180)

241-2: The dataset from Be et al was derived from whole genome amplification, and thus concentration of reads in particular areas of the genome is to be expected. Therefore interpreting this as evidence that insufficient sequence was present in the sample is unwarranted. The authors should also mention that whole genome amplification was used for this dataset as this is helpful for interpreting these results.

242-244: The amount of sequencing estimated to be required to sample all diversity within a soil community does not seem necessarily relevant - the abundance of the target organism in the community and depth of sequencing undertaken is all that matters. The authors should give a description of

293: This appears to be an incorrect reference. Further, at such low breadth, it seems intuitively unlikely that sequencing breadth and depth exhibit a significantly non-linear relationship.

325: Strain-level resolution is not assessed in this manuscript meaningfully as it currently exists, and thus a conclusion that it is achievable by imGLAD is not warranted.

398: As discussed above, the richness of a community does not seem specifically relevant.

400: These numbers are entirely speculation, and I would suggest that the choice of reference genomes would be more relevant than their quantity.  This is particularly true for soil and environmental genomes, because random choice of genomes is likely to be biased towards medically important lineages.
Additional comments: The use of sequencing breadth is a natural choice as a useful input, even if it is a relatively simple metric. However, depth is more vexed. For instance, in the models described, a low breadth combined with high depth would be regarded as a positive, when this is more likely to indicate incorrect mapping. Intuitively I would expect a model to be less confident when depth is high, not less, after breadth is already taken into consideration.


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: IMGLAD: ACCURATE DETECTION AND QUANTIFICATION OF TARGET ORGANISMS IN METAGENOMES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The submitted manuscript by Castro et al., introduces "imGLAD", a computational tool for quantification of organisms in metagenomic datasets. The manuscript is well written and the literature is sufficient as well as referenced appropriately throughout. The authors provide convincing data to show that their method works as expected with the tested genomes and datasets.
Experimental design: The manuscript meets the aims of PeerJ, and is a solid piece of work with a specific research question that is clear from the title. The background and research methodology are clearly described, however, their introduction needs revised to better describe the scientific problem that imGLAD aims to address. The authors do clearly lay out the existing problems with detection and quantification of microorganisms in metagenomic datasets and point out several limitations that imGLAD is meant to address:

	1) emperical approaches are computationally expensive and specific to the system
	2) existing profilers rely on SNP pattern differences against a target genome which are hard to determine accurately and are computationally intensive
	3) existing tools do not detect organisms that are absent from reference databases
	4) existing tools are not easily adaptable to include new organisms (they require recomputation steps)
	5) It is not clear how existing tools handle co-occurence of related organisms and if strain-level resolution can be achieved

Unfortunately, imGLAD suffers from some of the same limitations and therefore, it is not really honest to claim that these limitations have been addressed. For instance, "computational expense" is highlighted several times in their introduction as a limitation to existing methods, yet in their discussion (lines 343-349), the authors explicitly state that iMGLAD is also computationally expensive. Furthermore, the argument that emperical approaches are too specific (limitation 1 above) and that existing tools are not adaptable to new organisms because they require new calculations (limitation 4), apply to imGLAD as well. This introduction as-is, unfortunately sets up unrealistic expectations for imGLAD, that ultimately are not met. I would encourage the authors to remove the negativity in the introduction, describe what the existing tools specialize in and then simply describe the specific goal of imGLAD. This would make for a more useful, less combative introduction and would more plainly describe the existing knowledge gap.
Validity of the findings: I find the results to be generally valid, and appreciate the adaptability of the method to discriminate between two closely related and known strains of a similar organism.

My only criticism is that the overall evaluation of their method needs to include receiver operating characteristic (ROC) curves. Reporting Sensitivity alone (Figure 2), "accuracy" (Figure S3) or single point sensitivity / False positive rate (Figure S4 and S5) is not sufficient. Instead, the authors should evaluate the false positive rate and false negative rate over different sequencing breadth to show how the different methods stack up against one another. This is the only fair way to compare the different methodologies, would elegantly replace Supplementary figures S3-S5 and would perhaps be preferable to the existing Figure 2 as well.
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: IMGLAD: ACCURATE DETECTION AND QUANTIFICATION OF TARGET ORGANISMS IN METAGENOMES
Review round: 2
Reviewer: 1

Output:
Basic reporting: See general comments for the author
Experimental design: See general comments for the author
Validity of the findings: See general comments for the author
Additional comments: The authors have addressed many of my previous concerns, doing further bioinformatic work and most importantly defining the problem more specifically.

On the other hand, I was unable to understand what the results were precisely for this new section, as it is too vague. In the sentence starting on line 356 onwards, for instance, summarises the results when no results have been detailed. Figure 5 provides little help because either the image or legend seems to be incomplete (see below). Also, I was unable to see any methods section corresponding to these results. So while I do not foresee that the summary presented is incorrect, I must reluctantly recommend major revisions in anticipation of a more clear representation of these results.

I apologise for the incomplete sentence left in first referee report.

123: “even richness” is not a correct term, as richness is a whole of community measure of alpha diversity and does not refer to specific lineages.

>> The term “sequence-discrete” is neither defined nor widely used in the field, so a description should be given or alternate wording used.

> We have incorporated a clear definition of sequence discrete to help readers understand the instances where it is mentioned with the appropriate citations.

Abstract, 133, 238: Despite the author’s assertions, I still could not see where “sequence-discrete” was defined.

>>194-197: The tools and libraries described should be properly referenced.

>We have double-checked all instances where a bioinformatics tool or computer language libraries is mentioned and properly included references the first time the tool/library was mentioned.

It appears this effort was at least partially incomplete. The “Bio” i.e. BioPython is not referenced.

>>The tool also seems to require “awk”, and presumably will not work with Python 3 even though the README suggests Python-2.7+. In order to reduce installation issues it may be advisable to use subprocess.check_call rather than os.system so that failing commands are immediately recognized as such.

>Thanks for this suggestion. We have modified the tool to have better interaction with the system and be safer for users.

The README on GitHub still refers to Python 2.7+.

237: “The training datasets included closely related (but distinct) species of the same genus with ANI around 95% or lower (Figure 1; see also below for within-species resolution), which corresponds to the frequently used standard for species demarcation (Goris 2007) and encompass the sequence-discrete populations”. I found this sentence unclear for several reasons. First, if the target ANI threshold is 95%, then why does the training dataset include genomes “around” 95%? Does that set of genomes include any that are >95%? Presumably it should not. Secondly, it appears to imply that a species level is the one and only sequence-discrete taxonomic level when e.g. genera are also that way.

>>285 and others: depth and breadth should be reported with units.

>This is provided now.

The figures remain without units.

Figure 2 legend: form => from (mistake occurs twice).

Figure 2 legend: “Positive datasets (crosses) are separated form negative datasets (dots) through a logistic function (solid line) based on in-silico training datasets.”. Given imGLAD’s default is to use only sequencing breadth and not depth, it isn’t clear why depth+breadth is ascribed as a solid line, where the breadth is only given the less eye catching dashed line. The dashed line is also not described in the legend.

Line 240-243: It is unclear whether the 0.03 figure came from application of the training set, or is just an arbitrary number, as it is not introduced.

256: It is not clear why depth is discussed at all, given it is not a part of the final imGLAD algorithm. This discussion of depth is what led to my original (perhaps incompletely stated) comment:

>>140: The choice of default as being only based on sequencing breadth was surprising given the text and figures, which often described the depth-based approach.

>Indeed, it was a bit surprising for us as well in the beginning, but upon deeper thinking of the problem, this result does make sense and it is well explained in the main text. Accordingly, we have taken no further action to respond to this comment.

If depth is not useful as an input into the algorithm, then why does is it appear prominently in the text, figures and even abstract: “imGLAD achieves high accuracy because it … models both the sequencing breadth and depth to determine relative abundance and limit of detection”.

337: “For instance, filtering lowered
the minimum sequencing depth required for robust detection from 0.123X (no filtering applied) to 0.061X
(p-value < 0.05) in the training datasets for E. coli.” Do these cutoffs refer to the thresholds provided by the training phase at p-value 0.05? It is not clear so that is why I am guessing that.

Figure 5: the legend appears to describe a different figure i.e. it describes 3 lines when only 2 appear to be present in the image.

>>400: These numbers are entirely speculation, and I would suggest that the choice of reference genomes would be more relevant than their quantity. This is particularly true for soil and environmental genomes because random choice of genomes is likely to be biased towards medically important lineages.

>Probably the reviewer has a good point, but we do not know what else we could do about this; neither the reviewer provides any specific suggestions. Accordingly, we have taken no further action to respond to this comment.

The authors might simply wish to tone down their speculation. Perhaps it should be noted I do not feel that a reviewer’s role is to provide constructive suggestions for each and every issue identified in a manuscript, rather, it is the author’s responsibility to find solutions for deficiencies in the manuscript or to dispel the reviewer’s concerns.

417: When using imGLAD to detect species in a new sample, there is a computationally expensive BLAT step. Therefore, I do not see how the runtime could be 0.5-1s as stated. Perhaps the authors mean the application of the logistic model could be applied to a metagenome where the sequencing breadth has already been calculated for a target genome, but in my view that is misleading - the runtime should include the mapping step.

432: “imGLAD can
provide reliable prediction even in such cases, although at the expense of a lower detection limit,
assuming the close relatives are known and available and, hence, can be used as part of the training step
as exemplified in the E. coli case above.” This is overstated - by the authors’ own analyses imGLAD does not provide reliable predictions when the genomes >98% ANI.

484: MethaPlan is misspelled.

The authors use the term “sequencing breadth” to refer to the threshold found in the training step. However, the term is also confusingly used as the unit of depth for simulation-based studies e.g. in Figure 3. This is confusing to a reader - if possible it would be better to only use it in the former circumstance.

The “default settings” are discussed in the results and discussion, but are not defined except in the methods, as far as I could see. Since readers often only briefly read the methods, it would be good to restate what these are in the results section as it is explained what is motivating their choice.


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: IMGLAD: ACCURATE DETECTION AND QUANTIFICATION OF TARGET ORGANISMS IN METAGENOMES
Review round: 2
Reviewer: 2

Output:
Basic reporting: No revisions were necessary.
Experimental design: The revisions were acceptable.
Validity of the findings: I am unsatisfied with the authors' presentation of the ROC curve (S fig 8). They chose to show only imGlad and Pathoscope and it remains unclear why other tools and conditions were omitted. They mention p-values as a prerequisite, but the only numbers really necessary are sensitivity and specificity, both of which are already be calculated. After all, "Accuracy" is defined as the harmonic mean of sensitivity and specificity (legend to Fig S3).Therefore it should be straightforward to display those same values in the form of the ROC curve.This is the standard way to evaluate new methods.
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: EVALUATION OF SOIL INTERVENTION VALUES IN MINE TAILINGS IN NORTHERN CHILE
Review round: 1
Reviewer: 1

Output:
Basic reporting: In light of the clear amount of work that has gone into this study, I'm afraid I must highlight some terminal inadequacies.

The English throughout the manuscript is relatively good, however there are sections of poor grammar and instances of duplicated text (e.g. L304-309 in the methods).

The number of tables (17) and figures (10) is extraordinarily high. Most of these should be relegated to appendices, omitted completely, or condensed into one or two much more informative tables/figure. E.g., Figures 1-9 could be presented as a single plate.

Much of the results section belongs in the materials and methods, e.g. everything up to L353.

I cannot find a single statistical test anywhere.

I understand that this is a methodological paper primarily, but there are no hypotheses presented or tested and the results are extremely repetitive with no data presented or analysed outside of tables and figures. Metal by metal might be interesting, but more informative would be a meta analysis of all sites - e.g., of the 696 tailings dams, how many by your measures require intervention?

The first chunk of the discussion (up to L453) is literally just a table by table description of the data, while the following section is more results than discussion (almost entirely results, actually). There is not a single reference in the entire "discussion", and other than stating the method has been successfully applied there is absolutely no discussion of the implications of the requirement for intervention e.g. ecological impacts, human health impacts, costs, abandonment issues.

There is great data and the potential for significant research findings out of this study, but I'm afraid in its current form it is little more than a rambling simplistic overview of a regional intervention assessment with very little significance either to the international community or to tailings management more broadly. There needs to be much, much more critical thought behind this if it's to be published in the international literature.
Experimental design: The methods are described (in great detail), but there is little originality or rigorous experimental practice. There is potential, but little rigorous examination short of fitting the applied model to a dataset and reporting the output.
Validity of the findings: See comments in section 1. In its current form, this paper has very little impact, poor communication of results and conclusions, and is woefully lacking in international context.
Additional comments: This paper doesn't do justice to the data and effort behind applying the model, for which the authors should be commended. But I'm afraid it requires very significant revision (shorter, concise methods; rewritten concise results; competely rewritten discussion that addresses the context of the study; conclusions in an international context; revised tables and figures).


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

Input:
Title: EVALUATION OF SOIL INTERVENTION VALUES IN MINE TAILINGS IN NORTHERN CHILE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear and unambiguous, professional English used throughout.

The paragraphs are too short. Hence, two or more paragraphs must be joined together.
There are repetitions in the texts (e.g. Section 3.1.)

References are alright. Sufficient field background has been provided.

Article structure along with Figure and Tables is ok.
Experimental design: Original primary research within Aims and Scope of the Journal.

Research question well defined, relevant & meaningful.
Validity of the findings: Data is robust, statistically sound, & controlled.

Conclusions are well stated and linked to original research question.
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: EVALUATION OF SOIL INTERVENTION VALUES IN MINE TAILINGS IN NORTHERN CHILE
Review round: 1
Reviewer: 3

Output:
Basic reporting: Dear editor

Thank you for the invitation to review "Evaluation of soil intervention values in mine tailings in northern Chile".

Although I request more days to review this paper, is too long to review in 10-20 days. I tried to review it although I do not sure if I did a good review.

In my opinion, the submitted paper has a high value for their publication in Peer J. In general, I have not got a great number of issues about them. I think that should be accepted after minor changes.

I have not opinioned about English quality because I am not English native.
Experimental design: Research is well-designed.
Validity of the findings: Submitted results are very interesting for other researchers.
Additional comments: Abstract

- I suggest a reduction in materials and methods and improve results or conclusions. Authors only wrote about their method but not about their findings.

Introduction

Lines 63-77.

I suggest that authors give some data about mining and their impact on Chilean environment. I know that Cu is the most valuable element in Chilean mining, but it is missing in this section. Also, if possible give an approximate number around mines in Chile. For example, "Chile has more than XXXX mining sites where Cu, Pb and XXX are the most valuable elements".

Please, add a reference for lines 76-77.

Line 89-94. I think that you can reduce some references or merged some of them.

Regarding to material and methods and results sections, I have not several comments about them. They are very well.

In my opinion, figures quality should be improved.