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: IN OR OUT: RESPONSE SLOWING ACROSS HOUSING CONDITIONS AS A MEASURE OF AFFECT IN THREE WESTERN LOWLAND GORILLAS (GORILLA GORILLA GORILLA)
Review round: 1
Reviewer: 2

Output:
Basic reporting: For the most part, the article is written clearly. However, the abstract needs revision (see comments below). In addition, the article seems to be missing a large portion of background literature relevant to their hypothesis regarding the effect of housing condition on affect. See specific comments below.
The article is structured appropriately with the exception of some methodological details that should be moved from the introduction to the method section, and there are several points that need to be expanded upon in the method section (see general comments below). Additionally, the structure of the discussion should be revised to improve readability and the flow of results in the context of previous literature and alternative hypotheses. As it stands now, the discussion jumps around quite a bit between different findings, alternative hypotheses, and background literature. See comments below.
Experimental design: To me, it seems that there are two knowledge gaps that this study is attempting to address: 1) testing the validity of the response slowing paradigm in measuring ape affect, 2) whether housing conditions impact gorilla affect, as measured through the response slowing paradigm. I think this needs to be made clearer in the introduction. More importantly, the authors must include more background information regarding their second aim (the effect of housing condition on affect). Lastly, and perhaps most importantly, the hypothesis that gorilla affect will be affected by housing conditions seems to be largely confounded by weather / season, as is even mentioned by the authors. However, they do not attempt to address this. I have expanded on each of these issues below.
Validity of the findings: My biggest concerns are related to the validity of the findings. Because of the apparent confounds mentioned above, I am doubtful of the ability of the authors to make any solid conclusions about their hypotheses and about the validity of this experimental paradigm. The purpose of this study was to use the response slowing paradigm (which is dependent on longer latencies to respond to threatening stimuli) to assess the effects of available space. However, not only did the results fail to show longer latencies to response to threatening stimuli as a function of housing condition, the gorillas didn’t show longer latencies in response to threatening stimuli in general. If the paradigm is dependent on response slowing to threatening stimuli, but gorillas didn’t do this, how can conclusions be made regarding the effect of space and housing? More generally, what does this mean for the idea of “response slowing” as a measure of affect? What does this mean for hypotheses regarding available space? While the authors give several alternative explanations for their results (e.g., increased attention, dominance, vigilance, habituation to stimuli), they do not explicitly address the above issue.
A paragraph detailing the shortcomings of this study, along with ways that future research could address these issues, is needed in the general discussion, particularly regarding 1) the low N, 2) the housing condition confounds of temperature, husbandry procedures, and season. Additionally, there seem to be many alternative explanations that need to be highlighted in the discussion. Weather, environmental complexity, individual space preferences, social proximity, husbandry routines, the lack of investigation of interactions between daytime and nighttime housing, and coping mechanisms during prolonged periods of space restriction (see sources listed above) should be discussed in the context of previous research.
Additional comments: Line 21: please correct “affective” to “affect”.
Line 21-22: “response” appears twice, please revise to make less repetitive.
Overall, the abstract needs some work to improve detail and clarity. Specifically:
-The background portion of the abstract needs more detail. What is response slowing? Does “individuals” refer to humans, primates, other animals?
-Please provide more detail regarding how the housing arrangements at the time of testing were impacted by daytime and nighttime housing arrangements. This point is also difficult to follow in the body of the manuscript (Lines 165-173).
-Please specify N for the experiment.
-Please clarify whether the first sentence in the “Method” portion of the abstract refers to the general method for both experiments.
-More detail is needed regarding “three daytime and nighttime conditions” in line 26-27, and “four conditions composed of daytime and nighttime arrangements” in line 28. These are supposed to correspond to varying levels of space restriction, but this is unclear here.
-In my opinion, the claim cannot be made that the “results are somewhat consistent with those of a previous study” (Line 31-32) given that just one subject showed response slowing across two experiments.
Line 41-42 “…most elusive of the states…” please clarify what other states to which this refers.
In line 46, the authors state the judgement bias paradigms may not assess affective states at all, but then go on to say in Line 53-54 that “Previous studies have shown that judgement bias tests can be sensitive to affect changes….” Please clarify the difference here.
Line 59-60. Please expand on the theory or background behind response slowing. What does it aim to test? What does a slowing in response indicate, or why is response slowing indicative of negative affect? How does that relate to the “behavioral inhibition” that is mentioned later in the paragraph?
Line 55-58. Please remove current study procedure from this background literature paragraph. It feels like the introduction bounces around between current study aims/methods and background information (including line 101-109 as well). Therefore, it may be helpful to move all current study information to the last paragraph of the introduction and/or the method section. Specifically, line 104-109 seems that it would be more appropriate in the method section.
Line 110-113. Much more background literature regarding the effects of housing on behavior and welfare must be included here. Differences in housing are at the core of their hypothesis, but there is no background information to support how they came to that hypothesis. For example, the article is missing some critical background information regarding apes’ coping strategies in response to confined spaces. Although much of this background is in chimpanzees, I believe it is highly relevant here and I have included some sources below. The predictions regarding space also make no mention of space complexity, which, some have argued, is more important than amount of space alone. I would like to see additional detail regarding these points in the introduction, which may likely inform their hypotheses as well.
Importantly, the author’s predictions are largely confounded by differences in weather in their “housing conditions”. As they state in line 115 – 119, the hypothesized increase in positive affect could occur due to either 1) access to larger spaces, or 2) warmer weather, 3) a combination of these. The authors make no attempt at discussing this confound and how it might impact their data.
•	Aureli, F., & de Waal, F. B. M. (1997). Inhibition of social behavior in chimpanzees under high-density conditions.
•	de Waal, F. B. (1989). The myth of a simple relation between space and aggression in captive primates.
•	de Waal, F. B., Aureli, F., & Judge, P. G. (2000). Coping with crowding.
•	Duncan, L. M., Jones, M. A., van Lierop, M., & Pillay, N. (2013). Chimpanzees use multiple strategies to limit aggression and stress during spatial density changes.
•	Herrelko, E. S., Buchanan-Smith, H. M., & Vick, S.-J. (2015). Perception of available space during chimpanzee introductions: Number of accessible areas is more important than enclosure size.
•	Nieuwenhuijsen, K., & de Waal, F. (1982). Effects of spatial crowding on social behavior in a chimpanzee colony.

Line 131: Dominance struggles are mentioned several times throughout the paper. Please include dominance status of each individual if the data are available.
Line 133-135 should be included in the procedure section. I would like to see additional housing information regarding the types and amounts of spaces available to gorillas in the method section. Were these holding areas in which gorillas were tested the individual stalls where they sometimes slept? Also, what is the size of these testing areas? It would immensely helpful to include a diagram of their housing, including all areas mentioned in the method section.
Line 165-173: This section should be revised and more detail added to clarify how the housing combinations created the “conditions” that the authors tested.  As mentioned above, please include additional information from the introduction (lines 109) on the amount and type of space available in each of these housing conditions in the method section. Please provide additional detail regarding the husbandry considerations, weather, and temperature that determined these housing conditions. What affected nighttime locations? Specifically, why would gorillas sometimes be locked into the stalls instead of the dayroom? Lastly, do the authors have any information regarding social proximity in these housing conditions, specifically at night? For example, did the gorillas tend to sleep farther apart in the “accessed” condition?
What time of day did testing occur?
What are the dates of testing for experiment 1?
Approximately how long did it take the gorillas to complete their 50 trials for each session?
Line 186-187: how often did that occur?
Line 194-197: Why are there no expectations and analyses regarding interactions between the daytime and nighttime conditions? This seems like a logical next step that being locked indoors for the day and then sleeping in a stall overnight might result in the slowest response times (given a longer period of access to smaller spaces, according to their predictions). Additionally, because they did not examine interactions, they cannot rule out the possibility that previous daytime housing conditions drove the effect of what they attributed to previous nighttime housing condition (or vice versa). For example, because they collapsed across nighttime conditions to examine the effect of daytime conditions (that is, looking only at the main effect of daytime condition), perhaps the effect of slowest response time in the daytime “accessed” condition was driven solely by the nighttime “stall” condition. At the least, a discussion of these possibilities is warranted.
Line 233-243: It may be helpful to organize the discussion such that group-level effects are discussed first, followed by individual-level effects. So this paragraph should perhaps focus only on the group-level effects. Additionally, the discussion should attempt to focus on one idea at a time. For example, the first paragraph introduces increased vigilance, previous study findings, and talks about both housing condition and stimulus type effects, all of which are brought up again later in the discussion. In my opinion, each paragraph should detail a specific effect and a discussion of the relevant background info/previous studies, ideas about why the effect occurred, etc. As the discussion is currently, it bounces around between group and individual effects, and introduces new concepts without detailing them (e.g., line 253-255 talks about spacing during testing, but does not expand on why this might affect results).
Additionally, the discussion could be strengthened by including information from some of the space and complexity studies noted above. I think some of the findings may be explained in terms of findings from these previous studies.
Line 241: Please rephrase this sentence such that it is not a double-negative, and expand on what the consistency between the results of the current study and Cronin et al. may mean for the test paradigm, species, etc.
Line 246: by “in general” do you mean “descriptively”? Because not all of the gorillas showed a significant difference?
Line 298: how are the conditions random if they were also based on husbandry constraints?
Line 305: typo “M” before the parentheses?
Line 326: couldn’t this be another explanatory variable for the housing condition findings in experiment 1? Perhaps the previous day’s agonistic encounters affected response time?
Discussion:
Line 347: Could another alternative explanation be the choice of stimuli used in the studies? Could the threatening stimuli used perhaps be not as aversive as the author’s assumed? And perhaps the non-threatening stimuli had some sort of emotional valence attached, whether positive or negative?
Line 348: “It is also possible that apes are generally less sensitive to environmental changes compared to monkeys.” I don’t think the literature supports this (see citations above as well). I think a larger discussion regarding space availability, complexity, and coping mechanisms in response to confined space is needed here.
Line 355-357: This same argument has been made by de Waal and colleagues (see citations above). Again, I think these studies would help to explain the results found in the current study, and this should be elaborated upon in this discussion.
Line 371-374. This is a huge confound in the experimental design and results, in my opinion. This point should be elaborated upon: how might this have affected the results of both experiments?
Line 390-405: These paragraphs should be stated much more tentatively. Given the confounds with housing condition in the current study, low N, some null findings, subject-dependent effects, and findings that were in opposition to the hypotheses and previous research, a larger discussion needs to be included about the validity, efficacy, and utility of this paradigm in assessing ape affect (including the points I just mentioned).
The finding that each gorilla showed a different pattern of results should be discussed in the context of the validity of the paradigm. Does the fact that they were all different tell you anything about individual housing preferences? Does it tell us anything about the validity of this paradigm?
Figures:
Fig 2: Mismatch between legend and bar colors.
For all figures, please include brackets corresponding to asterisks showing significance. For example, in figure 4, which conditions are different from one another and correspond to each asterisk set?
Fig 6: Please outline the “access” condition, or use a different color as the bar is difficult to see. Additionally, how is Pende’s latency different between dayroom and access if the error bars overlap significantly? Is this typo? If not, this should be addressed in the discussion or legend.


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: IN OR OUT: RESPONSE SLOWING ACROSS HOUSING CONDITIONS AS A MEASURE OF AFFECT IN THREE WESTERN LOWLAND GORILLAS (GORILLA GORILLA GORILLA)
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is well-written, and the introduction and discussion integrate relevant literature. The potential strengths and weaknesses of the paradigm are clearly explained and the data contributed by the current study are helpful to the field. I have no concerns regarding the article structure.
Experimental design: One weakness of the experimental design (or the presentation of the experimental design) is that it is difficult to follow how the housing conditions differ from one another along the key dimension of available space. This confusion starts in the abstract, where “nighttime” and “daytime” housing are introduced as the variables of interest, rather than available space which isn’t mentioned until later. The authors do a great job of explaining how and why bachelor gorilla affect may be impacted by amount of available space, but the conditions “indoors/outdoors/accessed; daytime and nighttime” are difficult to track along this meaningful dimension. Minimally, a figure that shows the available spaces and how different scenarios / combinations relate to the cells in Table 1 would be useful (this is raised in comments below as well). Alternatively, classing conditions based on amount of long-term and short-term social space could be more intuitive, and potentially change the model structure (see comment immediately below).

Related, I think the potential impact of timeframe needs to be considered in some way. The daytime access is a long-term factor (seasonal, if I understand). The nighttime access is a recent short term factor (immediately previous night). There is a third way that social space enters in, and that is the immediate presence of a conspecific nearby (or not) while testing. Given the effects observed in previous studies, is there a logical prediction about timeframe that could be incorporated here (? Did the authors consider presence/absence of a neighbor during testing as its own effect in the model? (Unless this covaries 100% with another condition, which I may have not understood, see comment above.) I realize daytime and nighttime are different effects in the model, so statistically they are considered differently, but they are not discussed in this way. Minimally, introducing the different timeframe of these effects of interest would be helpful in the discussion, ideally with reference to previous literature and predictions.
Validity of the findings: I’m not sure the detailed individual subject analyses that follow the group analyses are warranted, or helpful. The authors are questioning the paradigm in the introduction, there are only 3 gorillas, and there aren’t corresponding behavioral data indicating differences that can aid predictions or interpretations. Related, while I appreciate the honesty of the speculative nature of the potential reasons for the individual differences (lines 321-333) they aren’t very compelling as presented. Individual differences way heavily in the discussion (starting line 375), and I think this is still relevant to include. However, it does not need to focus on these specific individuals. Instead the reviewed literature could remain and a call for more study with larger samples and corresponding behavioral or rank-related data.
Additional comments: In this study, the authors applied a response-slowing paradigm to the study of three zoo-housed gorillas from one social group, and questioned whether any response-slowing effects observed were predicted by housing conditions. The authors clearly discuss the past uses of this paradigm and the conflicting results that have accompanied it, and set the stage for this study well. Their results do not overwhelmingly support the use of this paradigm in apes, and leave open two possibilities 1) that the apes were not experiencing negative affect 2) that the apes did experience negative affect but it was not apparent in this test. Despite the complicated nature of the findings, the authors handle the discussion well. In addition to the more substantial comments above regarding the meaningfulness/explanation of conditions and the focus on individual data, I provide the following minor comments as well:

Minor:

Line 75: This is a very clear and important paragraph, but the final sentence seems to focus only on the minimal training and not the limitations explained. Would it make more sense to complete the paragraph with something like “Although there are open questions about the reliability of the response slowing task, the feasibility that comes with minimal training makes it a potentially valuable avenue to continue investigating” (or something similar)?

Line 95-98: this sentence long and a bit hard to follow.

Line 159: technically speaking, the tone was not a reward (not initially at least), suggest saying that it provided auditory feedback to the subject to indicate that the touch was successful and a food reward would be delivered.

Line 281: unclear how the gorillas never failed to select stimuli if one gorilla (Chip) stoped participating during some sessions?

Line 320; The term “dysregulated fear response” is used in the abstract and the introduced in line 320, this should be defined early.

Line 328: using the phrase “as in Experiment One” makes the meaning ambiguous, suggest rephrasing

Line 344: revise “more significant,” perhaps replace with “greater”

Line 352: comma unnecessary

Line 404: it may appear to readers that the “i.e.,” provides a definition of stereotypic behaviors, which it doesn’t. replace with definition of stereotypic behavior, or rephrase

Line 412: some more recent references regarding foraging style as in indicator of welfare may be:

Troxell-Smith, S. M., Watters, J. V., Whelan, C. J., & Brown, J. S. (2017). Zoo foraging ecology: Preference and welfare assessment of two okapi (Okapia johnstoni) at the Brookfield Zoo. Animal Behavior and Cognition, 4(2), 187-199.

Troxel-Smith, S. M., Whelani, C., Magle, S. B., & Brown, S. (2017). Zoo foraging ecology: development and assessment of a welfare tool for. Animal Welfare, 26, 265-275.

Throughout: should the threat stimuli be referred to as “threatening stimuli”? Seems more intuitive and would correspond with “non-threatening stimuli”


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: IN OR OUT: RESPONSE SLOWING ACROSS HOUSING CONDITIONS AS A MEASURE OF AFFECT IN THREE WESTERN LOWLAND GORILLAS (GORILLA GORILLA GORILLA)
Review round: 2
Reviewer: 1

Output:
Basic reporting: n/a
Experimental design: n/a
Validity of the findings: n/a
Additional comments: Overall, the manuscript has improved.
Am I convinced that this study tells us anything about affect in gorillas?  No - because the sample size is still 3, which cannot be changed.


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: IN OR OUT: RESPONSE SLOWING ACROSS HOUSING CONDITIONS AS A MEASURE OF AFFECT IN THREE WESTERN LOWLAND GORILLAS (GORILLA GORILLA GORILLA)
Review round: 2
Reviewer: 2

Output:
Basic reporting: All sections of this manuscript are much improved, and I commend the authors for such careful and well-done revisions. My comments correspond to line numbers from the PDF provided.

The language used, context, background literature, and article structure are well done and appropriate.

Minor suggestions related to Figure 4:
1) Please include in the title that the figure depicts latency across ALL stimuli types.
2) The title includes that “*** = p <.001”, but three asterisks don’t appear in the figure. Therefore, please specify the p-values to which * and ** correspond.
3) Lastly, it is difficult to tell which effect corresponds to each set of asterisks. I think the placement of these, and perhaps additional brackets, would aid in showing which asterisks correspond to which effect.

Line 107-110 now seems out of place. I think the sentence starting on line 109 would be better toward the beginning of the paragraph starting on line 129. The sentence from 107-108 might be better suited later in the last paragraph of the introduction, where the authors talk about threatening stimuli (Line 149). Lastly, editing the sentence starting on line 110 to have more of an emphasis on affect would allow it to serve as a new introductory sentence to the paragraph. Something like…“Previous studies have indicated that different housing arrangements have an effect on ape affect, as measured through behavior.”

119-120: please add a reference for this specific finding.
Experimental design: These areas are much improved. The authors have done a great job in adding methodological detail. There are just a few points regarding methodological detail that remain for me:

In the abstract, “nested conditions” are described, but they are not mentioned or explained in the method section. Please add an explanation of what is meant by nested conditions in the “Testing” section of the Procedure (starting on line 190).

Line 189-194: Please be consistent with terms used to describe housing conditions throughout the manuscript and between the text and figures (e.g., in figure 3, “Indoor” is listed as “Inside”, and “Inside” is used in L230, 242 but the condition is labeled as “Indoor”). Additionally, I understand that perhaps the authors are trying to distinguish between daytime and nighttime conditions, but isn’t the daytime condition “Indoor” the same as the nighttime condition “Dayroom” (i.e., locked in the NDR or SDR)? If so, labeling both of these consistently might aid in reducing confusion between the conditions.

Additionally, in their reply to a reviewer, the authors explain why they chose to analyze the housing conditions separately, rather than creating 9 conditions based on a combination of each daytime and nighttime condition ( “the issue of space when sleeping at night was qualitatively different than the issue of space when foraging throughout the day….”). This makes a lot of sense and explains why they did what they did. As such, I think it would be helpful to include exactly that information, perhaps at the end of the “Testing” paragraph (L189-208).

L218: Perhaps I missed this elsewhere, but can the authors add here that it took approximately 10 min for the gorillas to complete each session? I think this is important given that they were confined to a smaller space during testing.
Validity of the findings: This section is much improved, results are discussed appropriately and in-context, and includes alternative explanations.
Additional comments: In the abstract, I suggest making it clearer that the housing conditions represented varying levels of space and choice restriction. For example, a simple phrase stating that the housing conditions represented different levels of space and choice restriction could be added to the end of this sentence: “In Experiment One, they completed 48 50-trial sessions across combinations of three nested daytime and three nighttime conditions, which represented different levels of space and choice restriction.”

Line 122: Nieuwenhuijsen is misspelled.

Line 194: missing end parenthesis.

Line 300: “condition-related” rather than “condition related”?

Line 371: missing (year) for Schino et al. in-text reference.

Line 383: Please expand on this idea: to which result are you referring? What is meant by tradeoff?


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: IN OR OUT: RESPONSE SLOWING ACROSS HOUSING CONDITIONS AS A MEASURE OF AFFECT IN THREE WESTERN LOWLAND GORILLAS (GORILLA GORILLA GORILLA)
Review round: 2
Reviewer: 3

Output:
Basic reporting: I have no concerns about basic reporting in the revised manuscript.
Experimental design: I have no concerns about experimental design in the revised manuscript.
Validity of the findings: I have no concerns about the validity of the findings or how findings are discussed or interpreted in the revised manuscript.
Additional comments: In this revision, the authors have addressed my earlier concerns. The only comments that I have at this point relate to minor wording corrections/clarifications throughout. Specifically:

Experiment One, subsection Testing: there is no report of what food rewards were used. If they were the same as training (small piece of chow or produce), were these mixed/randomized to avoid inadvertent associations between stimuli type and food? Please add details.

Figure 4: What is the y-axis label?

Line 21-22: Don’t need both parentheses & commas

Line 32: Suggest rephrasing “had been accessed to” to “had access to”

Line 73: Suggest changing “previous” to “recent”

Line 83: Suggest changing “unlike” to “without”

Line 102: Suggest starting sentence with “However, Bethell…” to point out contrast with previous sentence/finding.

Line 342: rather than say these results “validate the use to some extent” suggest saying the results are encouraging, or something similar.

Line 423: rather than “it is a quick and efficient tool,” suggest “it could be a quick and efficient tool” to correspond with cautious support for the approach that has been evidenced to this point.

Are stalls and aisles synonymous?


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: ARTHROPOD IGF, RELAXIN AND GONADULIN, PUTATIVE ORTHOLOGS OF DROSOPHILA INSULIN-LIKE PEPTIDES 6, 7 AND 8, LIKELY ORIGINATED FROM AN ANCIENT GENE TRIPLICATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this study, Veenstra has shown an intriguing insight into evolutionarily origin of insulin superfamily in arthropods. He showed several important arguments to suggest that three different groups of arthropod insulin family genes called gonadulin, arthropod insulin-like growth factor and relaxin likely originated from an ancestral gene triplication.
Experimental design: I have no quibbles with the quality and rigor of the data present and do not feel that there is a need for additional analysis.
Validity of the findings: This is an elegant and detailed study that addresses an evolutionarily aspects of the highly complicated and diverged insulin systems in arthropods.
Additional comments: 1) Although the high conservation of the amino acid sequence in arthropod relaxin group, this is not the case for gonadulin group. If gonadulins indeed use Lgr3 as primary receptor, it is likely that arthropod gonadulins share high sequence similarity like arthropod relaxins. Does the author have any idea about this point? Is it possible that Lgr3 has another specific ligand other than gonadulin? or Is it possible that gonadulins have another receptor other than Lgr3?

2) I agree with the author’s opinion that aILGF and BIGFLP evolved independently. But it’s interesting that expressions of both Dilp6 and BIGFLP are regulated by ecdysteroid in the fat body (Okamoto et al., 2009a and 2009b; Slaidina et al., 2009). Does the author have any idea about this unusual similarity between Dilp6 and BIGFLP? Is it a convergent evolution? The independent evolution of BIGFLP and Dilp6 from different ancestral gene was also discussed in Okamoto et al., 2009a and Mizoguchi and Okamoto, 2013.

Other minor points as follows:

Line54: insulin-like growth factor → insulin-like growth factors (IGFs)

Line54: I don’t know why the author pick only Ins3 in here. Ins3 → insulin-like peptides (INSL) 3-5

Line56, 57, 426, 442, 444, 445, 447, 459, 482, 508: I recommend the author to use ‘IGFs’ instead of ‘ILGFs’ as an abbreviation of ‘insulin-like growth factors’.

Line89-90, 123-124: The author can cite Liao and Nassel, BioRxiv 2020 (https://doi.org/10.1101/2020.05.02.073585) instead of the Chintapalli et al., 2007 for the explanation of the expression and function of dilp8 in the ovary in Drosophila.

Line96: conversation → conservation

Line114: The author doesn’t have to cite Gontijo & Garelli, 2018 for two times in one sentence.

Line165: The ‘aILGF’ is firstly appear in here. The author needs to explain what is aILGF in the first appearance.

Line 288: I recommend the author to use ‘aIGF’ as an abbreviation of ‘arthropod insulin-like growth factor’.

Line295: The author needs to delete one of ‘sequence’.


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: ARTHROPOD IGF, RELAXIN AND GONADULIN, PUTATIVE ORTHOLOGS OF DROSOPHILA INSULIN-LIKE PEPTIDES 6, 7 AND 8, LIKELY ORIGINATED FROM AN ANCIENT GENE TRIPLICATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: Table 1: As described in the introduction, many ILPs are associated with specific neuroendocrine cells in the CNS. Why are the reads shown for the antennae but not the brain or heads of honeybees? Showing reads from the brain or heads might be better suited to identify any CNS-associated transcripts.

Figures and tables: The meaning of the grey and black highlighted residues should be explained for all figures showing sequence alignments (Figures 1, 4, and 9). In figure 6, labeling the axes rather than describing them only in the figure legend would make interpretation a bit easier. Supplemental – spreadsheet 2: what is the significance of the yellow tabs?

There are a few instances in which the language could be improved for better clarity. Notably, what is meant by the ‘first additional exon’ (Line 267)? Further, it is unclear what is being referred to regarding the ‘insulin-like growth factor described above’ (Lines 283-288). Define aILGF the first time it is used (Line 165). In the methods section, aILGF seems to refer to alternatively-spliced ILGFs. However, it is later defined as arthropod ILGF (Lines 287-288). Several typos were also found (i.e. Lines 67, 355, 433, 439, 514 and the caption for Figure 11 – ‘decpods’).
Experimental design: This is an interesting and well-written paper that seems to focus on three goals: first, to identify insulin-like peptides in arthropods, second, to determine how widespread the two general classes of insulin receptors - receptor-tyrosine kinases and G protein-coupled receptors – are among arthropods, and third, to investigate the evolution of arthropod ILPs and their respective receptors. The study also clearly addresses the lack of knowledge of arthropod ILPs outside of typical model organisms such as Drosophila and Bombyx. Analysis, and interpretation seem reasonable based on data that were presented.

I have very limited experience with the bioinformatics methods utilized in this study, but I think clarity could be improved by adding a more thorough description of the analyses conducted using the SRA toolkit and Trinity, rather than directing readers to a separate paper which also does not seem to provide sufficient detail to repeat the analyses (lines 142-149). One thing that was unclear was whether Trinity could identify orthologs or was simply used to assemble reads. Was another tool (i.e. BLAST+ as described in Veenstra 2016) used to identify orthologs once the transcriptome was assembled?
Validity of the findings: Findings appear to be consistent with the data provided.
Additional comments: I appreciate how the author stepped through the main logical arguments that support the finding that dILP8 and the gonadulins are orthologs.

The identification of decapod relaxins containing an odd number of cysteine residues is intriguing (Lines 334-336 and Figure 9). Has this been observed for other neuropeptides? This observation is mentioned briefly in the results and the author should consider expanding upon it in the discussion section.

I also found the observation of a Bombyx-specific ILP interesting and wonder if there any other evidence that BIGFLP is found in other lepidoptera. Might the same types of analyses used in this ms be used to address this? Is there evidence of other species-specific ILPs? Understanding how common this phenomenon is would help to put the observation of the BIGFLP into perspective.


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: ARTHROPOD IGF, RELAXIN AND GONADULIN, PUTATIVE ORTHOLOGS OF DROSOPHILA INSULIN-LIKE PEPTIDES 6, 7 AND 8, LIKELY ORIGINATED FROM AN ANCIENT GENE TRIPLICATION
Review round: 1
Reviewer: 3

Output:
Basic reporting: x
Experimental design: x
Validity of the findings: x
Additional comments: The author used bioinformatics approaches to analyze presence and evolution of various insulin-like peptides (ILPs) in different arthropods. This is an interesting paper which sounds more like a review. The interpretation of the results and hypotheses proposed in the paper are based on sequence similarities and available expression data from some species. These conclusions are rather speculative and need at least some experimental evidence which are beyond the scope of this paper. It is intriguing that “gonadulins” have only been found in hemimetabolan insects, but are missing in most Holometabola, except hymenopterans and flies. Similarly “relaxins” are restricted to representatives of a few non-related insect orders. Is it because these peptides and their receptors have been lost during evolution or (more likely) their sequences are very variable?
The author may consider mentioning obvious structural similarity between vertebrate IGF and some insect ILGF which are larger than regular insulins and composed of a single chain with three intramolecular disulfide bridges.


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: FROM THE INDEX CASE TO GLOBAL SPREAD: THE GLOBAL MOBILITY BASED MODELLING OF THE COVID-19 PANDEMIC IMPLIES HIGHER INFECTION RATE AND LOWER DETECTION RATIO THAN CURRENT ESTIMATES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The research utilized the Global Epidemic and Mobility Model (GLEaM) to in a posteriori approach to fit the model predictions to the observed data. Authors define the knowledge gap in COVID-19 existing models using biased data to fine tune model parameters, and I appreciate the authors stating that but it is not clear how their approach differs. This should have been made clearer to the reader, by specifically/stating why is their approach not biased compared to others who have fine-tuned their model parameters (as they did).
I also appreciate their attempt to describe findings however at times they are out of place, such as results presented in the intro. Also leaving out the objectives to be only implicitly presented rather than in a clear objectives statement, over use of the word “this” at start of many sentences which is confusing (which what?) should be reworded and addressed.
Experimental design: Lines 47-48 “Also, the existing modelling approaches often use biased data for tuning parameters or assessing models quality”
o	Could you please add more explanation on how the existing models use biased data?
o 	Could you please explain how your model will avoid the problem of using biased data?
Lines 52-53” but none of the models succeeded to describe the pandemic at the global level”
o	Could you please explain why?
o	Please add examples and references.
o	Could you please explain how your model will succeed in describing the global epidemic?
Line 60: Results presented in introduction please move to results section and continue your description from earlier introductory lines towards a clear knowledge gap that justifies your effort.
Lines 65-8: I appreciate the authors summarizing their report here but a statement of objectives of the modeling exercise is still needed
Materials and Methods:
What is the study population? There is no description of the study population? Is it only the 13 countries that you applied the model for?
Data sources:
Line 80 “The reference data about the number of SARS-CoV-2 diagnosed patients in the period from Jan 22, 2020, to Mar 26, 2020”
o	Why you relied only on the data starting from Jan 22 to Mar 26, 2020 to build your model?
o	What is your justification of using these data to simulate the global pandemic starting in November 12? As in lines 181-182.
Lines 87-89 Could you please briefly describe how the software adjusted for other data such as subpopulation selection, commuting patterns, or air travel flows?
Lines 91-92 “Below and in Table 1, we present two subsets of model parameters: 1) reliable and evidence-based derived from literature, and 2) knowledge and analysis-based estimations”
o	In Table 1 there is only one set of parameterization could you please clarify the difference between both?
o	 Which one of the two sets provide the best outcome?
Line 124 “Knowing the limitations of previous modelling attempts” what are these limitations?
Line 121: You describe tDR as “rate of total diagnosed to undiagnosed cases”, this needs to be reworded as you state it’s a rate but define it as a ratio (use of “to”), and is it the rate of diagnosed only or both diagnosed and undiagnosed (omitting the word “to”)? Later in the discussion you describe it as rate of diagnosed cases (line 231), which is it?
Lines 134-5: The arithmetic dictates the inequality but why did you choose 0.0001 greater than 0.006, and shouldn’t this have been explored as a stochastic parameter?
Line 161: The model equations are missing, providing a third party input file is not sufficient. I am unable to reconcile your model specification. This results in several issues that I am unable to reconcile, such as, in your recovered (line 176) you state that it includes fatal cases. However, you don’t talk about the role of the recovered in reducing the number of susceptible in the population, if you are, then you also need to state whether you discounted the dead (fatal cases) since they wouldn’t impact herd immunity.
Line 177: This is not a sufficient number of iterations for a sensitivity analysis. I understand the limit by the software that would require a paid subscription, and I am not affiliated or have any conflict of interest here, but to base your entire paper on 10 iterations producing very little datapoints is not useful  - case in point fig 2, there only 10 datapoints and removing the outlier on the far right may flip the association to positive instead of the authors interpretation of it being negative (Lines 211-2).  Have you tried contacting the developers of GLEAM and perhaps they can allow them a limited period with more iterations.
Validity of the findings: Lines 187-188 ” we limited the analysis of results to thirteen countries across four continents”
o	What is your justification? Could you please explain in more details with references?
Figure 2:
o	Colors are confusing
o	The confidence interval for the countries as in figures 4-16 has very wide range and by using this wide range the chances of your simulation to be in between is high. Could you please justify that?
Figure 3: why did you exclude Japan and Korea from the figure?
o	what happens when you add both countries to the figure, does the correlation result differ. I would suggest reporting both values with and without Korea and Japan.
Line 236: The argument regarding the role of school students may not be valid given that most countries implemented that, or at least go back to the dates this was implemented and specify this in the model
Line 253-4: Explain what do you mean many people remain in the high risk? Provide your model data that supports this even if it is true.
General comment for the country figures need to be plotted in a single matrix (page size) as several plots in 1 figure and please unify the y-axis, as presented it projects a biased view of what is happening in different locations to the reader.

Lines 267-268 “Our model if confirmed, could be used as a tool for forecasting and optimizing non drug intervention and policy making” this conclusion is beyond the goals of the paper, please interpret your findings in terms of the issues with few iterations after clarifying your ordinary diff. equations, role of fatal cases being accounted when your model handles the susceptibles reduction over time (see earlier comment) and the posteriori approach of fitting model output to the observed data.

Minor comments:
Please add keywords for your manuscript
Line 23 “sixteen selected countries” while in line 187 “thirteen countries” which one is correct?
Line 71 “GLEaM” and line 72 “GLEAM” are they different terms?
Line 75 “global spreading of A/H1N1” do you mean Influenza A/H1N1.
Line 151=152: Move to results section
Line 192 “two countries which fulfil the above criteria” do you mean did not fulfil?
Line 254: Explain “as seen in Italy” replace with factual account, this may be known to most of us now but in 10 years of time new researchers will not be able to follow that without seeking other papers.
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: FROM THE INDEX CASE TO GLOBAL SPREAD: THE GLOBAL MOBILITY BASED MODELLING OF THE COVID-19 PANDEMIC IMPLIES HIGHER INFECTION RATE AND LOWER DETECTION RATIO THAN CURRENT ESTIMATES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The language is OK, could definitely use some editing to improve the flow and clarity, references are not cited throughout the introduction section.
Experimental design: I struggled to locate a clear objective or research question, it appears that the authors are trying to tweak some of the model parameters and simulate the spread of pandemic so that they can mimic the case counts in some of the selected countries.

I have very serious concerns with some of the authors' approach:
1. It appears that the authors are suggesting that for every 610  diagnosed cases there are 100000 undiagnosed cases. That is a very strong assumption, there is definitely some underreporting of cases globally and a maximum of 1/3rd of cases could be asymptomatic but assuming that low diagnosed proportion without providing enough justification and supporting data may not be acceptable.
2. This is a stochastic model and the authors ran only 10 iterations of it to get the model outputs (Line 177). A stochastic model is not expected to give valid and meaningful insights with such a low number of model run. How did the authors conclude that 10 iteration was sufficent for model outputs to converge? If free version of the software was a limitation then that could not be acceptable for publishing a paper that may have significant impact on policy decisions during an ongoing pandemic.
3. On line 193 the authors state that Canada was excluded from the analysis because of its lack of coherency in reporting COVID-19 cases. I am not sure what the authors mean but this reviewer is not aware of any such incoherency in testing and reporting of COVID-19 in Canada.
Validity of the findings: It is hard to evaluate as the model have not been run for sufficient iterations and that is evident from very wide confidence intervals.
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: FROM THE INDEX CASE TO GLOBAL SPREAD: THE GLOBAL MOBILITY BASED MODELLING OF THE COVID-19 PANDEMIC IMPLIES HIGHER INFECTION RATE AND LOWER DETECTION RATIO THAN CURRENT ESTIMATES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Thank you for addressing my comments in detail and for increasing the number of iterations, certainly better than the first simulation. The revised manuscript also brings more details justifying your assumptions and modeling approach. The figure edits are also very welcome.
Experimental design: The methods are much more clear now and the additional information certainly walked me through the authors approach in detail.
Validity of the findings: I do have a couple of minor suggestions:
1) Lines 130 to 163 in the tracked changes revised manuscript word document are a great discussion of your approach and limitations, hence this entire section should be weaved into the discussion. It really has no place in the introduction.
2) I am concerned about the assumption that recoverds do not contribute to infection any further, specially that some reports are coming out about limited duration of immunity with SARS and MERS lasted about 3 years. Its up to the authors but seems like out of caution they should choose to protect themselves by adding some verbiage in the discussion that clearly states that some aspects of the disease are still unknown.
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: FROM THE INDEX CASE TO GLOBAL SPREAD: THE GLOBAL MOBILITY BASED MODELLING OF THE COVID-19 PANDEMIC IMPLIES HIGHER INFECTION RATE AND LOWER DETECTION RATIO THAN CURRENT ESTIMATES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The language of the manuscript has improved with the inclusion of citations, detailed description of sections, and presentation of a clear objective. However, this reviewer still finds the same issues with the manuscript and don't think that the authors had addressed those sufficiently.

1. The number of iterations: They increased the iteration from 10 to 20, both were arbitrary choices and not based upon any evidence that this number of iteration was sufficient for the model outputs to converge. They cite that the developers of the model felt that this number of iteration was sufficient; that is, unfortunately, no credible evidence. A stochastic model with so many sources of variation can not be expected to provide inferences with 20 iterations. The authors themselves acknowledge that "But we do not try to pretend our model is fully complete". It may have implications to publish a half baked science than the advantage it adds to scientific knowledge.

There is nothing novel in the manuscript that has mostly has not become common knowledge, except a set of parameters, that the authors are proposing, can mimic the number of cases in a few selected countries. To that end, the authors agree that any other potential combination of parameters may produce similar results suggesting such parameters may not reflect the reasonable estimates in the population.

Finally, the authors are using https://ourworldindata.org/ to obtain country-level data. The reviewer is not aware of the credibility of that source and finds it problematic that the authors without doing rigorous data retrieval efforts from individual countries appeared to slander some countries based on some global repository.

There was another minor issue that the reviewer noted, which is not using proper epidemiological terms: such as after pointing out by another reviewer the authors are still using the rate for mortality and detection which are not rates but a risk or ratio. The rate always has a time component. Similarly, the authors use "a single host" in their title, which also has an established term called "index case".
Experimental design: see above comments
Validity of the findings: see above comments
Additional comments: No additional comments


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

Input:
Title: FROM THE INDEX CASE TO GLOBAL SPREAD: THE GLOBAL MOBILITY BASED MODELLING OF THE COVID-19 PANDEMIC IMPLIES HIGHER INFECTION RATE AND LOWER DETECTION RATIO THAN CURRENT ESTIMATES
Review round: 3
Reviewer: 1

Output:
Basic reporting: The authors addressed my concern regarding discussion verbiage in the introduction.
Experimental design: The design is appropriate utilizing a modeling environment (Global Epidemic and Mobility (GLEAM) framework ).
Validity of the findings: Results are well described and authors did add the caveat statements regarding some of the model assumptions that future research may prove or disprove and that otherwise are currently acceptable.
I do understand the authors situation with the limited number of iterations, the costs required to run such proprietary modeling software etc. Also, I am glad the authors were able to go back to the software maker and gain more iterations but as I am sure the authors will agree, there is no doubt a larger number of iterations would have further increased the precision of their estimates by narrowing the 95% confidence intervals (CI) for each country's predictions. Perhaps a similar discussion point as the authors did for the permanent recovery assumption of their model, but for the number of iterations is due. I would even encourage the authors to state the reason, state:
In the methods: an initial simulation run with the trial version allowed X iterations was attempted prior to GLEAM software maker licensing a one time temporary increase in the trial version iterations to Y.
In the discussion: describe the changes or lack of in results due to increase in iterations from X to Y. Then discuss that it is expected that a larger number of iterations may have increased the precision of the country predictions, however, the 95% CI would have been expected to ever decrease in width given a larger number of iterations, indicating that the currently reported 95% CI may have coverage of the true predictions.
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: IDENTIFYING HUB GENES OF PAPILLARY THYROID CARCINOMA IN THE TCGA AND GEO DATABASE USING BIOINFORMATICS ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Article fails to meet our standards in basic reporting and no novelty in this work
Experimental design: article fails to meet our standards in experimental design
Validity of the findings: Article fails to meet our standards in validity of the findings
Additional comments: Author performed reanalyzed work.

GEO accession no GSE3467 was previously analysed, work done and published.
List of of following publication (titles) along with URL about GEO accession no GSE3467

1.   https://www.spandidos-publications.com/or/40/1/111 (Integrated bioinformatics analysis reveals that the expression of cathepsin S is associated with lymph node metastasis and poor prognosis in papillary thyroid cancer)

2. https://www.spandidos-publications.com/mmr/14/6/5041 (Identification of potential biomarkers and drugs for papillary thyroid cancer based on gene expression profile analysis)

3. https://portlandpress.com/bioscirep/article/39/4/BSR20190083/110840/Identification-and-analysis-of-genes-associated (Identification and analysis of genes associated with papillary thyroid carcinoma by bioinformatics methods)

4. https://link.springer.com/article/10.1007/s40618-018-0859-3  (Identification of key genes of papillary thyroid cancer using integrated bioinformatics analysis)

5. https://peerj.com/articles/7441/?utm_source=TrendMD&utm_campaign=PeerJ_TrendMD_1&utm_medium=TrendMD  (Identification of pivotal lncRNAs in papillary thyroid cancer using lncRNA–mRNA–miRNA ceRNA network analysis)

6. https://wjso.biomedcentral.com/articles/10.1186/s12957-017-1190-8  (Special role of JUN in papillary thyroid carcinoma based on bioinformatics analysis)

7. https://link.springer.com/article/10.1007/s12253-013-9625-1  (Candidate Agents for Papillary Thyroid Cancer Identified by Gene Expression Analysis)

8. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6182223/  (SDC4 Gene Silencing Favors Human Papillary Thyroid Carcinoma Cell Apoptosis and Inhibits Epithelial Mesenchymal Transition via Wnt/β-Catenin Pathway)

9. https://onlinelibrary.wiley.com/doi/abs/10.1002/jcp.28932  (Identification of key genes and pathways of thyroid cancer by integrated bioinformatics analysis)

10. https://onlinelibrary.wiley.com/doi/abs/10.1002/jcp.29154  (microRNA‐599 promotes apoptosis and represses proliferation and epithelial‐mesenchymal transition of papillary thyroid carcinoma cells via downregulation of Hey2‐depentent Notch signaling pathway)

GEO accession no GSE3678 was previously analysed, work done and published.
List of of following publication (titles) along with URL about GEO accession no GSE3678

1. https://www.sciencedirect.com/science/article/pii/S0014299918306812  (Identification and validation of potential target genes in papillary thyroid cancer)

2. https://www.spandidos-publications.com/mmr/14/6/5041  (Identification of potential biomarkers and drugs for papillary thyroid cancer based on gene expression profile analysis)

3. https://www.spandidos-publications.com/10.3892/etm.2018.6023  (Identification and bioinformatics analysis of overlapping differentially expressed genes in depression, papillary thyroid cancer and uterine fibroids)

4. https://www.spandidos-publications.com/ijo/51/4/1311?text=fulltext  (Anoctamin5 regulates cell migration and invasion in thyroid cancer)

5. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5650329/  (RNA-sequencing investigation identifies an effective risk score generated by three novel lncRNAs for the survival of papillary thyroid cancer patients)

6. https://www.thieme-connect.com/products/ejournals/html/10.1055/s-0035-1569289  (Identification of Genes Associated with Papillary Thyroid Carcinoma (PTC) for Diagnosis by Integrated Analysis)

7. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197007  (Preserved SCN4B expression is an independent indicator of favorable recurrence-free survival in classical papillary thyroid cancer)

8. https://onlinelibrary.wiley.com/doi/full/10.1002/ijc.29172  (A three‐gene panel that distinguishes benign from malignant thyroid nodules)

9. https://peerj.com/articles/7441/?utm_source=TrendMD&utm_campaign=PeerJ_TrendMD_1&utm_medium=TrendMD  (Identification of pivotal lncRNAs in papillary thyroid cancer using lncRNA–mRNA–miRNA ceRNA network analysis)

10. https://wjso.biomedcentral.com/articles/10.1186/s12957-017-1190-8  (Special role of JUN in papillary thyroid carcinoma based on bioinformatics analysis)

GEO accession no GSE33630 was previously analysed, work done and published.
List of of following publication (titles) along with URL about GEO accession no GSE33630

1. https://www.spandidos-publications.com/or/40/1/111  (Integrated bioinformatics analysis reveals that the expression of cathepsin S is associated with lymph node metastasis and poor prognosis in papillary thyroid cancer)

2. https://www.spandidos-publications.com/or/36/5/3005  (Elucidation of the molecular mechanisms of anaplastic thyroid carcinoma by integrated miRNA and mRNA analysis)

3. https://www.spandidos-publications.com/mmr/18/1/695?text=fulltext  (Competing endogenous RNA regulatory network in papillary thyroid carcinoma)

4. https://www.sciencedirect.com/science/article/pii/S0014299918306812  (Identification and validation of potential target genes in papillary thyroid cancer)

5. https://portlandpress.com/bioscirep/article/39/4/BSR20181616/110809/Down-regulation-of-DANCR-acts-as-a-potential  (Down-regulation of DANCR acts as a potential biomarker for papillary thyroid cancer diagnosis)

6. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6492601/  (Long Non-Coding RNA ZFAS1 as a Novel Potential Biomarker for Predicting the Prognosis of Thyroid Cancer)

7. https://link.springer.com/article/10.1007/s12253-018-0561-y  (Down-Regulation of APTR and it’s Diagnostic Value in Papillary and Anaplastic Thyroid Cancer)

8. https://www.futuremedicine.com/doi/abs/10.2217/fon-2019-0016  (LncRNAs SNHG12 and LINC00152 were associated with progression of patients with papillary thyroid carcinoma)

9. https://www.thieme-connect.com/products/ejournals/html/10.1055/s-0035-1569289  (Identification of Genes Associated with Papillary Thyroid Carcinoma (PTC) for Diagnosis by Integrated Analysis)

10. https://www.spandidos-publications.com/ol/18/5/4726  (Long non‑coding RNA small nucleolar RNA host gene 7 is upregulated and promotes cell proliferation in thyroid cancer)

GEO accession no  GSE53157 was previously analysed, work done and published.
List of of following publication (titles) along with URL about GEO accession no  GSE53157

1. https://www.thieme-connect.com/products/ejournals/html/10.1055/s-0035-1569289  (Identification of Genes Associated with Papillary Thyroid Carcinoma (PTC) for Diagnosis by Integrated Analysis)

2. https://onlinelibrary.wiley.com/doi/full/10.1111/1759-7714.13270  (Integrated analysis identifies DUSP5 as a novel prognostic indicator for thyroid follicular carcinoma)

3. https://link.springer.com/article/10.1007/s00418-018-1660-2  (Expression of serine peptidase inhibitor Kunitz type 1 in differentiated thyroid cancer)

4. https://onlinelibrary.wiley.com/doi/abs/10.1002/jcp.28793  (Integrated analysis of transcriptome data revealed MMP3 and MMP13 as critical genes in anaplastic thyroid cancer progression)

5. https://onlinelibrary.wiley.com/doi/abs/10.1002/jcp.28932  (Identification of key genes and pathways of thyroid cancer by integrated bioinformatics analysis)

6. https://onlinelibrary.wiley.com/doi/full/10.1002/cam4.1397  (Development and validation of an individualized diagnostic signature in thyroid cancer)

7. https://www.spandidos-publications.com/10.3892/ol.2015.3829  (Identification of potential therapeutic targets for papillary thyroid carcinoma by bioinformatics analysis)

8. https://link.springer.com/article/10.1007/s10238-016-0445-y  (Overexpression of teneurin transmembrane protein 1 is a potential marker of disease progression in papillary thyroid carcinoma)

9. https://www.sciencedirect.com/science/article/abs/pii/S0378111918303470  (Network-based meta-analysis in the identification of biomarkers for papillary thyroid cancer)

10. https://www.spandidos-publications.com/10.3892/ol.2018.9342  (Genetic expression profile‑based screening of genes and pathways associated with papillary thyroid carcinoma)

Re analysed work is not acceptable
Mean while, Author not provided Differential gene expression table, which is more fundamental and basic in this work.
No novelty in this work.
I recommended strong rejection.


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: IDENTIFYING HUB GENES OF PAPILLARY THYROID CARCINOMA IN THE TCGA AND GEO DATABASE USING BIOINFORMATICS ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: NO comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The manuscript entitled “Identification of hub genes of thyroid carcinoma from TCGA and GEO database by bioinformatics analysis” by Ying Wan and co-workers reports eight hub genes of thyroid carcinoma identified using bioinformatic methods and verified using clinic human THCA patients tissue samples. The authors did a good job to do bioinformatic analysis to identify the eight hub genes from four expression profiles datasets, which include thyroid tumors and normal tissues. However, there are still some issues with this manuscript.
Major concerns:
1). The authors' conclusion is not supported by the evidence provided in the manuscript. The authors tried to identify genes that could serve as the signature and potential prognostic markers of THCA. However, the eight hub genes identified in the manuscript are not unique features of THCA, but also for many other cancers. The expression patterns of those eight genes are the same in human thyroid carcinoma patients and the TCGA database (figure 5 and figure 6), which also indicate those eight genes are generally altered in many cancer types. So, the conclusion would be any single of the eight hub genes or the combination of all eight genes can not be used as biomarkers of thyroid carcinoma, though they are differentially expressed in thyroid carcinoma patients.
Minor concerns:
1). The methods should include more details. For example, the GEO datasets the authors used in the manuscript should make a clear note about the sub-types of thyroid cancer (since there are four different types) of all samples, the type of normal tissues, and also patients' genders that those samples originated. That information can help readers to determine if the current analysis has a bias in certain respects; How the authors get the “Survival” plot in figure 5i; RNA extraction and qRT-PCR analysis should revise carefully. How the samples were extracted using TRIzol, what the authors mean of all procedures were repeated three times (technique repeats?)
2. The results also can include more details. For example, where are the eight hub genes the authors identified in the manuscript located in the GO analysis, the KEGG pathway, and what are the known functions of those genes? The authors mentioned THCA pathways but did not include any details about those pathways. It would be very helpful to include more details about THCA pathways since they are directly related to the topics in the manuscript.
3. In the discussion part, the authors should emphasize how those eight genes play specific roles in thyroid cancers compared to other cancer types. In other words, why those genes can be used in the diagnosis and prognosis of THCA, but not other cancer types. The authors should explain the specificity here.
4). There are some typos in the manuscript which need to correct. For example, page 7, line 99, “dynthesis” should correct to “synthesis”; page 7, line 112, “The volcano plots of The DEG……in the form of a volcano plots” should correct to “ The DEG……in the form of volcano plots”. There are still many other places, the authors need to revise carefully.


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: IDENTIFYING HUB GENES OF PAPILLARY THYROID CARCINOMA IN THE TCGA AND GEO DATABASE USING BIOINFORMATICS ANALYSIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have identified 8 HUB genes for thyroid carcinoma using expression profiles from the GEO database.

The experimental design is rigorous and the conclusions are clearly stated.

The result section of the manuscript feels very short. Could this section be expanded?

The discussion section lists the identified genes and their background literature robustly. However, all these HUB genes have been reported to be involved in other types of cancers. Can the authors find any specific and unique connection between these HUB genes and THCA?

The figure legends should have sufficient information to understand the respective figures. For example, Fig 5 doesn't mention which one is normal tissue and which one is cancer tissue (red vs grey). Fig 5i also have hard to read words inside the graph. Fig 5a-h also failed to mention the meaning and importance of the red stars. The entire paper depends on figure 5 (and 6) for validation and therefore more attention should be given to them.

The findings of this study are very clear: the authors have identified 8 HUB gens and concluded that one of them,DCN, is a key gene for TCGA prognosis. What this paper is missing is a specific discussion on how these hubs genes are potentially involved in thyroid cancer. It is understandable that a cyclin gene or a cadherin gene (etc.) would be differentially regulated in tumor tissues. The authors should point out why do they think that these 8 HUB gens are specific for THCA and not just a bunch of genes gets differentially expressed in any tumor tissue (which this paper very robustly reviewed in the discussion section).

I think in studies like this, it is important to validate some of the negative results. This can be done very easily using RT-PCR and by picking a couple of genes that didn't get enriched but are related to the HUB genes (like a different cyclin gene as a control for Cyclin D1 and so on). Any thought?


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: IDENTIFYING HUB GENES OF PAPILLARY THYROID CARCINOMA IN THE TCGA AND GEO DATABASE USING BIOINFORMATICS ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: NO comment
Validity of the findings: No comment
Additional comments: Thanks for the authors to provide additonal information and answers my concerns. In general, the authors did a good job to identify the 8 hub genes. Since the aims of the manuscript is to apply to clinical diagnosis. I still have two concerns.
1). The authors said the 8 hub genes can be used to early diagnosis in the manuscript. However, I do not see any evidence provided by the authors. How does the authors came up with this conclusion? At least, the authors should address it more clear in the manuscript.
2). The authors verified the gene expression pattern of the 8 hub genes using two methods. One is bioinformatically analyzed the THCA datasets, the other way is to run qRT-PCR for the patients. I am convinced by the overall pattern changes of the 8 genes in throid cancer patients between cancer tissues and normal tissues. However, if look into details about the q-PCR results. I found in each gene group, there is about 15-30% patients did not show the overall trend change in the gene expression level (this even does not count cancer and normal tissue shows similar level of gene expression). For example, the overall pattern of gene FN1 is upregulated in cancer tissue, however, sample 7,13, 17, 22, 31, 47, 48 show FN1 is more abandunt in normal tissue. If the same sample ID for the q-PCR results for each of the 8 genes is for the same patient (for example, sample No.1 is patient No. 1). Then, the percentage of patients which show all trend changes of the 8 hub genes will be extrememly low.That means, even a throid cancer patient will not trypically show all 8 genes expression change, the authors should change their conclusions accordingly. The authors should analyze the data based on individual patient, and report the result in a way that a paired comparing between the cancer and normal tissue for the same patient is presented, at least for the q-PCR. Otherwise, the results will be misleading for a diagnosis purpose.


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: IDENTIFYING HUB GENES OF PAPILLARY THYROID CARCINOMA IN THE TCGA AND GEO DATABASE USING BIOINFORMATICS ANALYSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: n/a
Experimental design: n/a
Validity of the findings: n/a
Additional comments: The argument of using all eight HUB genes together as a 'whole signature' is unconvincing. Especially when a significant number of the eight HUB genes are known to be associated with tumors and are central to various cellular growth functions. I agree the idea is plausible but the authors need to use proper data and stronger arguments.

I think the responses to the first reviewer's comments are extremely poor. I agree that the reanalysis of data may be acceptable under specific scenarios. However, the authors only stated that they disagree with reviewer 1, but didn't provide specific rebuttals to the reviewer's concerns. As the reviewer specifically cited various studies, it can be expected that the authors would use this opportunity to provide proper documentation on why they believe their work contains novel findings especially under the lights of the mentioned papers.

I recommend the authors to go through the major concerns pointed out by the reviewers and try to argue on specific points.


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: IDENTIFYING HUB GENES OF PAPILLARY THYROID CARCINOMA IN THE TCGA AND GEO DATABASE USING BIOINFORMATICS ANALYSIS
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I am generally agreed to publish the manuscript as is. However, I still encourage the authors to present the raw rt-pcr data to represent each individual patient in addition to the statistic analysis. To do this, just lable the raw data like patient No.1 corresponding to each rt-pcr data for the fisrt patient, and patient No.2 to the second patient rt-pcr data, etc.


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: IDENTIFYING HUB GENES OF PAPILLARY THYROID CARCINOMA IN THE TCGA AND GEO DATABASE USING BIOINFORMATICS ANALYSIS
Review round: 3
Reviewer: 2

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: The rebuttal letter doesn't contain any response to Reviewer 3 (wrong file? uploading error?). Please go through the previous comments and respond to them accordingly.

Previous Comments:
The argument of using all eight HUB genes together as a 'whole signature' is unconvincing. Especially when a significant number of the eight HUB genes are known to be associated with tumors and are central to various cellular growth functions. I agree the idea is plausible but the authors need to use proper data and stronger arguments.

I think the responses to the first reviewer's comments are extremely poor. I agree that the reanalysis of data may be acceptable under specific scenarios. However, the authors only stated that they disagree with reviewer 1, but didn't provide specific rebuttals to the reviewer's concerns. As the reviewer specifically cited various studies, it can be expected that the authors would use this opportunity to provide proper documentation on why they believe their work contains novel findings especially under the lights of the mentioned papers.

I recommend the authors to go through the major concerns pointed out by the reviewers and try to argue on specific points.


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: IDENTIFYING HUB GENES OF PAPILLARY THYROID CARCINOMA IN THE TCGA AND GEO DATABASE USING BIOINFORMATICS ANALYSIS
Review round: 4
Reviewer: 1

Output:
Basic reporting: No Comment
Experimental design: No Comment
Validity of the findings: No Comment
Additional comments: I still encourage or request the authors to proper present the clinlical q-PCR data. In a way that the expression levels of the 8 genes for each patient are clearly labeled in this study. The authors did not provide strong argument that the combination of the 8 hub gene expression patterns is proper in thyroid cancer diagnosis. I appreciate the authors effort to try to develop a new diagnosis tool for the thyroid cancer. However, the authros have to clealy and properly report their findings. The statistic significant changes in genes expressions between the cancer tissues and normal tissues does not mean for a invidual patient you will see that change. Especially, the authors did not use paired statistic analysis to link every data point to a specific patient. That's why the minimum requirement for the authors would be proper label their q-PCR data to let future readers to have an idea how robust this diagnosis method will be and how to improve it in the future.


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

Input:
Title: IDENTIFYING HUB GENES OF PAPILLARY THYROID CARCINOMA IN THE TCGA AND GEO DATABASE USING BIOINFORMATICS ANALYSIS
Review round: 4
Reviewer: 2

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: I am happy with most of the responses the authors provided. Some of the conclusions are little stretched and could be toned down in the final proof. I still didn't get a proper reply in support of the novelty of the study, but I would leave that to the editor to decide (especially under the present lockdown scenarios).


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: ACCESS TO SCIENTIFIC LITERATURE BY THE CONSERVATION COMMUNITY
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No Comment
Validity of the findings: No Comment
Additional comments: Line 21: ~50% of the 97% or of the whole?
Line 31: Change "about" to "roughly"
Line 64: "academics AND consultants"


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: ACCESS TO SCIENTIFIC LITERATURE BY THE CONSERVATION COMMUNITY
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The manuscript “Access to scientific literature by the conservation community” describes survey results regarding the needs, methods, and barriers to access to scientific literature by the constituency of the International Union for the Conservation of Nature. It provides a quantitative assessment for an issue which is often cited but rarely assessed systematically. It found that the vast majority the constituency feel that the literature is important to their conservation work, and that about half have substantial barriers to accessing it. The studied showed that a major factor was institutional access to the literature.

The study is well-written and driven by clear research questions. The analyses are sound, and the conclusions are based on the findings. I feel that the discussion could be improved by clarifying the publishing and access options. The study may also have limitations in that it focused only on IUCN members. I discuss these aspects below along with other minor inline suggestions.

L30-32: They way the abstract is written, it could be misconstrued that this important conclusion is inferred from the 55% with access rather than direct response to a question asking about this challenge. Maybe this could be reworded to make that explicit, like in L165-167.

L53-54: Inclusion of the entire IUCN constituency is a strength of this study. There are also many conservation professionals who conduct on-the-ground conservation actions who would generally not be included here. This is especially true of small organizations and/or those whose focus is not conservation, but who have staff members who are dedicated to this work (e.g. watershed councils and city governments). It would be difficult to reach all of these individuals, so it is understandable that they were not included here. However, it would be good to mention that they are not captured and are probably even less likely to have institutional access to literature than many IUCN members.

L157: Extra “had”.

L169-186: Would this work better in the Methods section?

L214-218: It is counterintuitive that 52% is significantly different than 54% here. Is this a case where the sample size is so high that you might be getting statistically significant results which aren’t necessarily biologically significant? I appreciate reporting the factors so that we can see the overall impact, but maybe this should be discussed.

L354-355: I think the result of there being 235 journals listed out of 794 responses is in itself important. I think it speaks to the fact that most conservation practitioners often need very specific information relating to their taxon/biome/threat of focus. I think this demonstrates why buying access to a few big journals and hoping that many people will use them will likely miss the mark for most conservationists. It may be more cost effective to have a budget to pay per article where open access is lacking. I think it would be useful to stick in a sentence about the fact that so many journals were mentioned as a demonstration of this needed variety.

L364-367: This paragraph seems to be standing alone. Where are the associated questions and numbers?

L369: At some point it seems relevant to state that institutional access is a spectrum rather than a binary, and that some institutions may provide access to some journals and not others. Some of this is lost when asking the binary question of whether they have institutional access or not, and may explain why those “with” access still use other means to obtain literature.

L369: In general, I think that the discussion could benefit from a short, basic introduction to the various forms of access and options of publishing. Although it is touched on, I feel that practitioners (including myself) are often baffled by the whole process. As you mentioned, people are unfamiliar with what a “library” is, and I know that among my colleagues we are constantly confused about where publication/access fees go, what “open access” is, and just the general model of publication and access. I think it would be great if you had a short paragraph which laid these things out before getting into the specifics.

L439-441: I have not heard of “gold” (or “green”) open access before, and I’m not sure that this sentence clarified it for me.

L450-452: This sentence seems important, but I am not understanding what it means or who would be saving money.

L470: It looks like the rest of this sentence is missing.

L475-478: This is a good, clear conclusion.

Fig. 1: Spell out the languages on the figure.

Fig. 2: For all figures, I would suggest ordering the responses from most positive to most negative in a clockwise fashion starting from the top. I appreciate that the color coding is in order, but skipping around the circle is a bit confusing.

Figs. 3 and 4: These are really important results and are presented clearly.

Fig. 12: I had to stare at these for a while to find the take-home messages, but, once I did, I found them to be important. It is a matter of taste, but I might suggest flipping the ordering of the stacks (so that daily is on the bottom), getting rid of the grey (so that the bars represent responses of access), and not making the “infrequently” stand out from the others so much in color, since they are all gradients of a positive. For me, these changes would help me to quickly see that most methods don’t differ much between those with and without access (other than using the institutional access), and that most people look online for free.

Fig. 12: The result that most people get what they can online for free seems like a major finding of this study. I think it should be in the abstract.


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: ACCESS TO SCIENTIFIC LITERATURE BY THE CONSERVATION COMMUNITY
Review round: 1
Reviewer: 3

Output:
Basic reporting: I would suggest that the history of the IUCN is not needed - it doesn't add much to the main message of the paper. Instead, I would replace this information with more background on the previous work that has been done to look into lack of access to scientific papers and journals globally. There is a body of literature on this topic that the paper touches on briefly, but the introduction could benefit from a more in-depth look at this.

The authors should explain the response rate for the survey and establish representativeness, especially given the reported low number of responses in Spanish and French.

The last sentence of the conclusion seems to cut off. If indeed this is not simply an uploading error, the authors need to finish their last sentence.
Experimental design: The authors should include a definition of “constituency” for the reader. As it currently stands, it is somewhat hard to determine who was actually surveyed by the authors. Consider including a figure explaining the structure of the IUCN and its constituents for clarity.
Validity of the findings: Given the nature of this study (a surveying method), the authors should consider phrasing the results as “participants reported that…” rather than stating the results as if they are inherently correct. It’s quite possible that the survey results reflect the actual situation at hand, but changing the phrasing allows for a more nuanced view given that results stem from survey responses.

The authors should report in the text that while “region” was a statistically significant predictor when it was Western Europe/US/Canada/Australia/New Zealand, the other regions were not significant predictor levels.

The authors don’t give much information on how they did the regression in Figure 13, which makes it feel a bit abrupt. The authors also don’t report a p-value. Additionally, “perceived desirability” is an unclear way of speaking about this survey question. Does it mean participants only thought they desired the journal? Does the “perceived” qualifier pertain to the authors or the survey participants? This regression could use more clarification.
Additional comments: The authors should condense the number of figures within the article. The figures are useful but having 13 individual figures is unwieldy. Perhaps some figures could be condensed into a multi-figure panel, or some of the figures could be moved to the supplemental information?

It would be helpful to clarify what constitutes a "library" earlier in the article. The authors provide more of a background the concept of a “library” when describing their survey methods, but they introduce the concept of libraries earlier in the introduction without defining it.


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: ACCESS TO SCIENTIFIC LITERATURE BY THE CONSERVATION COMMUNITY
Review round: 1
Reviewer: 4

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: The first sentence in the Conclusions section on Lines 473-474 is the main reason this topic is so important. I think the paper would benefit from including some examples of the "numerous negative effects" that lack or difficulty of access to scientific literature has had on conservation practitioners' ability to carry out their work.
Additional comments: The paper is well written, clear, concise, and important. Conclusions are adequately and appropriately drawn from the results. However, the authors' surprise at the persistent relevace of institutional access (Lines 375-378) given the availability of free downloads on SciHub and LibGen does not take into account that the material on these sites is pirated, and therefore illegal. Some governmental organizations may preclude their employees from using sites like that by blocking internet access, and some people may have an ethical problem with breaking the law. I think including the legal status of those sites is relevant to why there is still such a reliance on institutional access and should be disclosed in the paragraph where they are referenced (Line 334).
In addition, I have three editorial comments:
Line 157: omit the word "had" betweeen "have" and "internet"
Lines 399-400: ensure the hyperlink is activated like the others
Line 471: text is missing from what was likely the last sentence of the paragraph


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

Input:
Title: MOLECULAR PHYLOGENETICS AND EVOLUTIONARY HISTORY OF THE ENDEMIC LAND SNAIL GENUS EVERETTIA IN NORTHERN BORNEO
Review round: 1
Reviewer: 1

Output:
Basic reporting: Some files of the manuscript did not complete uploaded. No Table 2, Table 3, Additional File 1, Additional File 3, and Figure S1 are not available for review.

Table 1. Please provide the meaning of abbreviation for the voucher specimens. Which museum did the specimen deposit?

Figure 7. It would be more easy to read if the annotation of lowest and highest elevation combined in a single phylogeny. Please refer to Figure 5. in https://doi.org/10.1111/2041-210X.12628. There are some free tools could do this job, such as ggtree or iTOL.

Figure 8. The calibration point should be labeled on the phylogeny.

In file S1 (addition file 2 as the author mentioned in the manuscript), no. 30 and 31 labeled as Everettia jasilini. But no. 30 and 31 were labeled as E. safriei in Table 1 and Figure 2, 7, and 8. Please check these again.

Recommend: To upload the BEAST input XML file and resulting files of Bayesian analysis.
Experimental design: Research question is well defined. In general, sampling data and methodologies could answer the research question. The research could be improved if applied more realistic clock and substitution model for divergence time estimation.

Line 152-154: The evolutionary model of each gene partition was evaluated based on AIC. What’s the results when using BIC? I have try to conduct the Partitionfinder implemented in IQ-TREE. Both AIC and BIC were suggested the combination of 1st and 2nd codon position of COI into a single partition scheme. Different best-fit substitution models were selected by AIC and BIC (as shown in the following paragraph).

Best-fit model according to BIC:
16S: GTR+F+R3
COI codon 1+2: TNe+R3
COI codon 3: K3Pu+F+R3
28S: TNe+R3
ITS: GTR+F+R3

Best-fit model according to AIC:
16S: GTR+F+I+G4
COI codon 1+2: TIM+F+R4
COI codon 3: K3Pu+F+R3
28S: TN+F+R2
ITS: GTR+F+R4

Since the partition scheme and substitution model would have impact on the inferred branch length of the phylogeny, it might have greatly effect on the divergence time estimation. I suggest the author to check the differences of estimated divergence time by using AIC- and BIC-evaluated substitution models.

Line 158: The only substitution model available in RAxML is GTR. It is over-parameterized for your data set. I would suggest to use IQ-TREE to compute the maximum likelihood phylogeny.

Line 174: Divergence time estimation using strict clock model is unrealistic for the data set. Since these snails were assumed to diverge with the ancient geological events, it would be more realistic to choose uncorrelated relaxed clock model or random lock clock model to estimate the divergence time. The application of substitution model also have impact on the estimated branch length as mentioned before. GTR model is the best-fit for the data set.
Validity of the findings: The model setting in divergence time estimation should be modified. It might greatly effect the results of estimated time.

Line 219: Lineage C is not a statistical strong supported clade due to the unstable position of Everettia sp. 7 in bootstrap analysis.

Line 250: No results of the estimated mutation rates available for reviewer.

Line 253: I do not agree with the ‘reliable divergence estimates’ because of unrealistic strict clock model and non-best-fit substitution model were applied for the divergence time estimation.
Additional comments: The article is well-written and proves informative background information of geological setting and distribution pattern of Everettia landsnails. This research provides insights into the formation of Borneo biodiversity, especially for the less researched taxa. However, the file of the manuscript did not complete uploaded. No Table 2, Table 3, Additional File 1, Additional File 3, and Figure S1 are not available for review. I suggest to reanalyze the divergence time using best-fit substitution model and more reliable clock model, rather than strict clock model.


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

Input:
Title: MOLECULAR PHYLOGENETICS AND EVOLUTIONARY HISTORY OF THE ENDEMIC LAND SNAIL GENUS EVERETTIA IN NORTHERN BORNEO
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear and unambiguous, professional English – Yes (with very minor stylistic imperfections that do not impact the clarity of the manuscript)

Introduction and background provide relevant context in clear and understandable fashion
Figures are relevant and in sufficient quality

Editor to check conformity with PeerJ standards, I don’t consider this as part of my duties
Experimental design: Research question is well-defined, relevant and meaningful

Methods used are (largely, see below) appropriate, I did not detect a significant flaw in their execution, but remark that I have no detailed knowledge of distribution modelling so can’t comprehensively evaluate this part of the manuscript.

BEAST analysis. More details need to be provided about the assumptions that went into the model. Which priors were used has an important effect on the output. Effectively, BEAST provides any tree you wish depending on chosen priors and parameters.
I am not a savvy expert myself, but believe that the birth-death model might have been more appropriate than the yule prior (either way please explain why you chose a certain prior or not). Also, a strict clock is a very risky choice since almost never its assumptions are fulfilled by a certain dataset. Again, here more detailed explanation and justification for the chosen method is needed. It might be prudent to run different scenarios, such as priors and relaxed clocks and inspect the influence on the output.

All this is likely to massively inflate the confidence intervals of the result and may result in less clarity about the dating of speciation events.However, as it stands, the molecular clock analysis pretends an unrealistic accuracy. Therefore, all conclusions based on it are also to be taken with a grain of salt.
Validity of the findings: The authors suggest that their findings support the hypothesis that uplift of the central mountains triggered initial lineage differentiation in this group and that speciation mainly occurred during Pleistocene, not later. However, I see circularity in this argument as they have used the uplift of the mountains to calibrate their chronogram. Obviously, by doing this they deprived themselves from assessing the relevance of this event for the timing of phylogenetic events in Everettia.

If the authors are interested to evaluate the relevance of this uplift, they need to find an extrinsic calibration and check for congruence. Even then, coincidence doesn’t necessarily equal causation.

Furthermore, the uplift of mountains is a slow process that took millions of years to complete (and is still on-going, I suppose). Hence, a confidence interval should be applied to the calibration. To simply use a point calibration at 10 Million years ago is an oversimplification of the facts and appears arbitrary to me.
Check Figure 8: It shows existing highlands already at 25-20 MA.

To solve this dilemma, a series of calibration points was helpful to rain in confidence intervals, but I appreciate that this is probably impossible given the lack of fossils and other suitable calibrations.

If no extrinsic calibration are available, that doesn’t mean doom and gloom. The authors can still postulate that the uplift triggered lineage diversification simply because the observed distributions and phylogenetic relationships are suggestive of this.

Under this premise, they can use their calibrations to formulate a hypothesis on the timing of speciation. This hypothesis stands and falls with the correctness of these assumptions. In the end, it is about how to fashion your argument in a logic way.
Additional comments: The claim of the authors that their study provides “implications for the evolutionary history of northern Borneo” is a bit overstated. In fact, what the authors do is using current knowledge of the geological and climatological history of the island to infer the evolutionary history of the group under study. Nothing wrong about this.

The study provides insights into the evolutionary history of Borneo’s biota only in so far as one might argue that what has been shown for the group under stduy (Everettia) might apply also to other groups. But that’s how far it goes. So, potentially I’d suggest the promise a bit down – perhaps just focus on your group as a potential model for low-agility invertebrate biota.

Overall, I consider the overall story as plausible. Its largest weakness is the molecular clock estimation as it probably underestimating or understating the error that comes with the chosen approach. This can not be easily circumvented. This error may not entirely invalidate the findings of your study, but I would argue that you should use a little more caution in fashioning your arguments by accounting for degree of uncertainty in the divergence time estimation.