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: RABBIT HINDLIMB KINEMATICS AND GROUND CONTACT KINETICS DURING THE STANCE PHASE OF GAIT
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript is clearly written and has been improved by modifications in response to both reviewers.  The provided dataset is much more clearly presented.
Experimental design: The methods and experimental design are clearly articulated and sufficient detail is provided to replicate the experiment.
Validity of the findings: The findings appear to be valid based on the limited experiment conducted.  There are surely limitations due to skin motion, but these are clearly acknowledged and are not likely to change the general conclusions.
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: POPULATION STRUCTURE IN QUERCUS SUBER L. REVEALED BY NUCLEAR MICROSATELLITE MARKERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: I think this study could be a valuable contribution to expand and complement the existing knowledge about the population dynamics of Q. suber. The introduction is well written and guides the reader towards the necessity of Q. suber population genetic studies and the value of different insights from different datasets.
My main concerns relate to the presentation and interpretation of the results. In my opinion, the present manuscript could be greatly improved by adjusting some of the analysis, simplifying the presentation of the results and by modifying figures. In addition, the wording in some paragraphs should be revised carefully.

You find my specific comments in the sections “Validity of the findings” and “Additional comments”.
Experimental design: The dataset with 17 populations and 22-30 samples per population is a solid basis for the study presented here and is sufficient to address the research questions of this manuscript. The statistical data analysis of the microsatellite markers appears to be sound and the methods are generally well described, with some exceptions (further details in the sections below).
Validity of the findings: Genetic markers used for analysis:
The test for marker neutrality is a valid approach to identify outlier loci. Using only the eleven neutral loci as identified by the BAYESCAN approach would improve the interpretability of the downstream analysis of genetic diversity and population structure. In the main figure 3 for example, the authors present the PCA of the neutral loci whereas the population genetic statistics in table 2 and pairwise FST-values are based on all 13 loci (if I understood correctly, not very evident from the text). I think it would be worthwhile to move analysis performed on the other sets of markers to the supplementary information or justify the inclusion of non-neutral markers in the analysis of neutral genetic structure.

Inference of genetic clusters:
The claim of the authors to have identified three genetic clusters is not enough elaborated in my opinion. It is not very clear to me, why the mavericK analysis (which does not include location information and correlated allele frequencies) is weighted more heavily than the structure analysis to infer genetic clusters. In my view, the identification of the number of K genetic clusters in this manuscript is relying too much on the methods for the inference of K, which are known to have their biases. I can very well imagine that, if there is substructure in these populations, comparing structure results for K=2-3 using the 11 neutral loci would also reveal a third cluster similar to the mavericK results pop up for K=3. Independent of the number of clusters identified by the Evanno method, this would provide evidence for further substructure in Q. suber populations. Instead of showing the plots for different numbers of loci and different software used in figure 2, I would recommend to show structure results for e.g. K=2-4 (or until no more new clusters pop up) for the analysis of 11 neutral loci. The existence of three or more genetic cluster could be further undermined with e.g. a neighbor-joining tree of pairwise Fst estimates and the PCA that is already presented.

Diversity statistics:
In the discussion, the authors claim a heterozygosity deficit of the markers because of a deviation of expected and observed heterozygosity. Table 1 shows locus-wise mean heterozygosity estimates. Because this analysis was not performed on the population-level (or it is not stated in the methods), it Is actually expected to find a heterozygosity deficit because of the Wahlund effect. These statistics, in my opinion, cannot be used to make statements about general heterozygosity levels of the species. Observed heterozygosity here is rather a characteristic of how polymorph a certain locus is. It would make more sense to show these statistics on a population level in table 2 or per locus with further information about population estimates (see e.g. Hoarau et al. 2002). The FIS estimates per population in table 2 indeed suggest that there is an incongruency between observed and expected heterozygosity but absolute numbers of heterozygosity should also be presented and are of interest.
Additional comments: Specific comments

Line 66: Do the authors mean increased drought frequency? Please specify.

Line 72: In this context Dauphin et al. 2021: “Genomic vulnerability to rapid climate warming in a tree species with a long generation time” could also be cited.

Line 120-121: I would suggest to delete the part about SNPs, they are particularly useful for many more approaches.

Line 143-144: Do the authors know anything about how the seed sources have been sampled for the provenance trials? Could this have an impact on levels of inbreeding or heterozygosity?

Line 151-154: Information about the Bulgarian populations should be deleted, as it is not very relevant for this study.

Line 179 & 189: Were FST-values tested for their significance? If not, this should be added.

Line 182-184: What do the authors want to test with a FIS calculated for K=3? Please specify.

Line 254-261: Please present the values of heterozygosity here.

Line 258-259: The abbreviations FST and FIS have been introduced before, no need to write it out. This is also true for the other abbreviations throughout the manuscript.

Line 259: It should rather be “..the mean FIS when considering the populations separately was..”.

Line 267-268: Which are the island populations? The levels of Ar are low compared to what, below the median, among the lowest 5 populations? Please specify.

Line 269: What is high in terms of numbers? Please specify.

Line 275: I think the authors should rather show FST-values of the neutral set, since there is an interested in neutral genetic structure.

Line 274-277: To which type of FST-values are you referring to here? Nei87 272 or WC84? Please specify.

Line 278-280: This belongs, if at all, to the discussion section. Otherwise, the authors should just mention the numbers. To which heterozygosity deficit are the authors referring to, FIS? I think population-wise heterozygosity estimates are not presented here.

Line 294-295: The interpretation about the type of selection should be moved to the discussion section.

Line 300-337: How do you define the term “admixture”? I am not sure if the authors should use the term “admixture” here or rather something like “showed mixed coancestry coefficients”. Admixture already implies genetic admixture between two groups which does not need to be the case if there is e.g. a third, different genetic cluster with K=3.

Line 311 & 329: “significant”: Did you test for significance? Otherwise remove.

Line 334-337: It is not clear to me why Corsica should be separated from the remainder. They individuals of the Corsica population cluster at least partly within the main cluster in the scatterplot.
How would you explain the outliers from the Sintra and Catalonia population in Figure 3?

Line 331: “13 loci PCA scatterplots” should be replaced with something like “The PCA using 13 loci…”
Line 335: “The CA” should be replaced with something like “The CA of the neutral data set shows…”

Line 344-354: This paragraph should be revised according to my comment on the diversity statistics. In addition, the Wahlund effect can also be responsible for a heterozygosity deficit.

Line 393-477: In my opinion, this section should be revised under the consideration of my comments related to diversity estimates and the inference of genetic clusters. I don’t think the authors can speak of the existence of three genetic clusters with the current evidence that is presented in this manuscript. Generally, I like the different aspects that are brought up in the discussion.

Line 395-400: I think this paragraph should be removed. Nowadays SNP genotyping can be achieved relatively cheap with a high number of samples and SNPs per populations. E.g. pooled sequencing is a cost-effective method to get genetic diversity estimates from a high number of SNPs. Furthermore, the first sentence does not make sense, SNPs can also be in low numbers.

Line 404: “SNP” instead of “SNPs”

Line 405: Which analyses, are you referring to the studies mentioned before? Please specify.

Line 462-466: Long and complicated sentence, please revise.

Line 474-477: The authors should also acknowledge that genetic diversity estimates based on microsatellite markers are not necessarily correlating with whole genome diversity (see e.g. Väli et al. 2008).

Legend Table 1 Line 2: Heterozygosity is missing a “t”, I would remove “overall gene diversity”.

Legend Table 1 Line 5: It should be mentioned that FIS are mean values calculated from the population estimates.

Legend Table 2: How many SSRs were analysed here? Please specify.

Legend Table 2 Line 3: To be consistent, you should also mention the method for calculating FIS.

Figure1: The distribution range of Q. suber indicated here would be very helpful.

Figure 2: I think the authors should use the abbreviations presented in Table 2 here.
In addition to the barplots, I would strongly recommend to plot genetic structure of one main result as pie charts on a map. The size of the pie charts could be scaled according to e.g. population-level heterozygosity. This Figure could then also replace Figure 1, which is not very informative on its own. Providing a spatial context would facilitate the interpretation of the results a lot.

Figure 3: How much variance is explained by each axis? What does the ellipse indicate? I think the ellipse could be removed.

Table S1: Which marker set was used for the calculation of FST? Please specify.

Figure S1: What does the black line indicate? This is not clear from the figure legend. Please specify.


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: POPULATION STRUCTURE IN QUERCUS SUBER L. REVEALED BY NUCLEAR MICROSATELLITE MARKERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: I read with interest the manuscript by Sousa et al. "Population structure in Quercus suber L. revealed by nuclear microsatellite markers". The manuscript presents a report on population genetic structure of a Mediterranean evergreen oak, Quercus suber, that has been extensively studied along with other hybridizing Quercus species in the last decades. I must say that the manuscript is well-written and is easy to follow. The authors apply standard analytical approaches for these types of studies, discuss the potential issues of the specific microsatellites that may affect further inference, and interpret their results in regard of the population structure they find. The authors propose that nuclear SSR markers chosen in this study “proved to be an effective means for detecting population structure in Quercus suber, despite being in much lower number than SNP markers”. The findings may shed light on still unclear issues on the way cork oak populations are phylogeographically structured as a product of past climatic events (e.g. glacial cycles) and human induced expansions/fragmentations.
I have however some comments that should be considered before acceptance of the manuscript for publication in PeerJ.
(A) There is ample literature in this topic, part of which was not even cited in the manuscript. Actually, some of the findings of the authors were already proposed earlier in other studies. Therefore, I consider the inclusion of these references, along with some suitable discussion in regard to previous findings would improve the quality of the present manuscript. In particular, Q. suber genetic structure has been largely studied using different types of molecular markers with varied inheritance:
-	A few relevant papers not cited in the manuscript and listed at the end of my report: plastidial RFLPs (Lumaret et al. 2005), PCR-RFLPs (López de Heredia et al. 2006, 2007) and sequence data (Costa et al. 2011), mtDNA (López de Heredia et al. 2018), isozymes (Elena-Rosselló y Cabrera, 1996; Jiménez et al. 1999; Toumi y Lumaret, 2001), nuclear ribosomal ITSs (López de Heredia et al. 2007) and AFLPs (Coelho et al. 2006; López de Heredia et al. 2007), nuclear microsatellites (Soto et al. 2003; Soto et al. 2007; López Aljorna et al. 2007; Burgarella et al. 2009; Lorenzo et al. 2009; Lumaret and Jabbour-Zahab 2009; Ramírez-Valiente et al. 2009), SNPs (López de Heredia et al. 2020).
-	Cited in the manuscript: cpDNA microsatellites (Magri et al. 2007), nuclear microsatellites (Ramírez-Valiente et al. 2010), SNPs from nuclear loci (Modesto et al. 2014), SNPs from GBS (Pina-Martins et al. 2019; Vanhove et al. 2021)

The areas explored in the aforementioned articles is variable, sometimes covering the whole distribution range of the species and many other times restricting the sampling to specific regions. To my knowledge, a large proportion of the battery of nSSRs analysed in the present manuscript has been already used before in Quercus suber. Nine of these SSRs were early transferred to cork oak by Soto et al. (2003, 2007), and then extensively used to study introgression with sister species (Burgarella et al. 2009), local scale-spatial structure (Soto et al. 2007) regional population structure (Lorenzo et al. 2009 or to find correlation with functional traits using samples from provenance trials (Ramírez-Valiente et al. 2009-2010).  Part of the information regarding the utility of these microsatellites included in these papers should be somehow included in the manuscript.
The interpretation for the population structure provided in the present manuscript should be also discussed in regard to the previous findings by Lumaret et al. (2005) using plastidial DNA and by López de Heredia et al. (20079 using plastidial DNA, nuclear ribosomal ITSs and AFLPs, not only to the more recent analysis with SNPs (Modesto et al. 2014, Pina-Martins et al. 2019, Vanhove et al. 2021), because both independent approaches already suggested three clusters for Q. suber in its whole distribution range (although not coincident with the clusters proposed here). While it is true that nuclear microsatellites here offer new insights on the genetic structure of cork oak populations (it is remarkable the behavior of the populations in Corsica, but see below), a more detailed discussion is required considering what other types of markers already showed.
REFERENCES
López-Aljorna A, Bueno MA, Aguinagalde I, Martín JP. (2007). Fingerprinting and genetic variability in cork oak (Quercus suber L.) elite tres using ISSR and SSR markers. Annals of Forest Science 64, 773–779. DOI: 10.1051/forest:2007057
López de Heredia U, Carrion JS, Jimenez P, Collada C, Gil L. (2007b) Molecular and palaeoecological evidence for multiple glacial refugia for evergreen oaks on the Iberian Peninsula. Journal of Biogeography 34(9):1505-1517 DOI: 10.1111/j.1365-2699.2007.01715.x
López de Heredia U, Jiménez P, Collada C, Simeone MC, Bellarosa R, Schirone B, Cervera MT, Gil L. (2007a). Multi-Marker Phylogeny of Three Evergreen Oaks Reveals Vicariant Patterns in the Western Mediterranean. Taxon 56(4):1209-1220 DOI: 10.2307/25065912
López de Heredia U, Vázquez F M, Soto Á. (2017). The role of hybridization on the adaptive potential of Mediterranean sclerophyllous oaks: the case of the Quercus ilex x Q. suber complex. En: Oaks Physiological Ecology. Exploring the Functional Diversity of Genus. Quercus, L. Eds. Gil-Pelegrín E, Peguero-Pina JJ, Sancho-Knapik D. Cham, Switzerland: Springer International Publishing, 239–260.
López de Heredia U, Mora-Márquez F, Goicoechea PG, Guillardín-Calvo L, Simeone MC, Soto Á. (2020). ddRAD Sequencing-Based Identification of Genomic Boundaries and Permeability in Quercus ilex and Q. suber Hybrids. Frontiers in Plant Science 11:564414. DOI: 10.3389/fpls.2020.564414
Lorenzo Z, Burgarella C, López de Heredia U, Lumaret R, Petit R J, Soto Á, Gil L. (2009). Relevance of genetics for conservation policies: the case of Minorcan cork oaks. Annals of Botany 104(6):1069-1076 DOI: 10.1093/aob/mcp200
Lumaret R, Tryphon-Dionnet M, Michaud H, Sanuy A, Ipotesi E, Born C, Mir C. (2005). Phylogeographical variation of chloroplast DNA in cork oak (Quercus suber). Annals of Botany 96(5):853-861. DOI: 10.1093/aob/mci237
Lumaret R, Jabbour-Zahab R. (2009). Ancient and current gene flow between two distantly related Mediterranean oak species, Quercus suber and Q. ilex. Annals of Botany 104(4):725-736. DOI: 10.1093/aob/mcp149
Ramírez-Valiente JA, Lorenzo Z, Soto A, Valladares F, Gil L, Aranda I. (2009). Elucidating the role of genetic drift and natural selection in cork oak differentiation regarding drought tolerance. Molecular Ecology 18:3803-3815. DOI: 10.1111/j.1365-294X.2009.04317.x
Ramírez-Valiente JA, Lorenzo Z, Soto A, Valladares F, Gil L, Aranda I. (2010). Natural selection on cork oak: allele frequency reveals divergent selection in cork oak populations along a temperature cline. Evolutionary Ecology 24:1031-1044. DOI: 10.1007/s10682-010-9365-6
Soto A, Lorenzo Z, Gil L. (2003). Nuclear microsatellite markers for the identification of Quercus ilex L. and Q. suber L. hybrids. Silvae Genetica 52:63-66.
Soto A, Lorenzo Z, Gil L. (2007). Differences in fine-scale genetic structure and dispersal in Quercus ilex L. and Q. suber L.: consequences for regeneration of mediterranean open woods. Heredity 99:601-607. DOI: 10.1038/sj.hdy.6801007
Toumi L, Lumaret R. (2001). Allozyme characterisation of four Mediterranean evergreen oak species. Biochemical Systematics and Ecology 29(8):799-817. DOI: 10.1016/S0305-1978(01)00024-2
Experimental design: (B) In relation to this latter point, the sampling scheme adopted in the manuscript gives me some doubts, both at global and local scales:
- Global sampling: I suggest including a layer with the distribution range for the species in Fig. 1 and then overlap the sampling sites. The authors can access the shapefile (or a jpg image) here: http://www.euforgen.org/species/quercus-suber/
According to this map, while there may be an over-representation of Portuguese populations (for obvious reasons), there is no representation of the populations from central Iberian Peninsula, the Landes nor from southeastern France.
This bias may be understandable providing the sampling was done in provenance trials rather in natural populations, but it must be considered in the discussion. For instance,  the adscription of Corsican samples to a different cluster, which is new to this manuscript, could be more easily explained if some samples form southeastern France (Provence) were included here. This would require further confirmation, but I would expect some kind of relationship between cork oak populations from these regions.
The lack of Central Iberian samples is more difficult to explain, as they form part of the international provenance trials for the species and in any case, they could have been sampled in natural populations. Some of the papers I referred above have analysed some of these populations with 9 out of the 13 microsatellites employed here.
- Local sampling:
Another issue related to the sampling strategy is that 22-30 samples might not be sufficient for certain analyses. For SNPs Pina-Martins et al. (2021) and Vanhove et al. (2021) used 6-7 samples for SNPs, and using 22-30 is an improvement, as the authors suggest, but it may still be short to be free from bias inferring many genetic diversity parameters, and interpretations should be cautious.

(C) Despite my comments above, I consider that, overall, the methodology employed in the manuscript is correct. It applies the standard analyses one would expect for the particular question the authors aim to answer. Actually, this is the type of analyses population genetics studies based on nSSRs use to perform. The analytical methods to explore the validity of the 13 nuclear microsatellites to infer population structure at a global scale are appropriate for this type of research (linkage between markers, null allele estimation, neutrality tests, H-W equilibrium estimation), and I appreciate the github link that includes genepop data and R scripts to build Fst based dendrograms and to the plots of principal component analysis, which is in line with the open research commitment of PeerJ.
Validity of the findings: I have also some comments regarding the methodology employed here that may compromise the validity of the findings:
- Several Structure clustering approaches are adopted including and excluding markers from the analysis. While there is evidence of some degree of linkage between markers (for instance between QpZag36 and QpZAG46), I’m not sure if this was considered when running all the Structure simulations for K=2 and K=3 with 13 or 11 “putatively neutral” markers.
- Regarding the optimal K number from Structure, I consider there is not enough support for a third cluster including Corsican populations. Indeed, the authors support this finding with the PCAs. This is correct, as pointed out by the authors, the number of genetic pools inferred by Structure (K) frequently requires independent support. The problem is that here, if the data from Fig. 3 are correct, the clusters inferred by plotting PC1 vs PC2 are inferred from two principal components that explain only 6.85 % of overall variation, and this is non-significant. Being strict, these results should not be presented, as they may induce to incorrect interpretations of the results. A clarification is needed here before acceptance.

(D) Quercus suber is a species with undoubted commercial values in rural areas from the Western Mediterranean. I am convinced that uncontrolled acorn transfer between areas has not been the rule in the past, even less between distant regions, but I expected at least some comments in the Discussion referred to the impact of human intervention in cork oak distribution. Some expansion of Q. suber woodlands after the last Glacial cycle might be favored by human action, possibly enabling local expansion of between adjacent areas. We cannot interpret the current population structure of species that have been handled extensively in the past without considering that they form part of highly anthropized ecosystems (even more in the Mediterranean region than in other areas of the world).

(E) There are only marginal references (Lines 462-466) to introgression of cork oak with other evergreen Quercus species (Q. ilex in the west and Q. cerris in the east of the distribution range). Both ancient and contemporary introgression levels have been extensively studied (even with 9 out of the 13 markers employed here), and the impact of this introgression in the population structure of cork oak could be better discussed. Some estimations of contemporary introgression in mixed Q. suber-Q. ilex stands were < 2% (average) (Burgarella et al. 2007), but this introgression could have been underestimated and, in some areas, introgression could affect the number of private alleles inferred with this set of microsatellites. At leas a reference in the Discussion is required.
Additional comments: (F) Minor comments:
As said before, the manuscript is well-written, the Figures (except for my comments above), tables, supplemental information and files in the github link are correct. I only found a typo in Line 95: "drivers" instead of "driver"


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: POPULATION STRUCTURE IN QUERCUS SUBER L. REVEALED BY NUCLEAR MICROSATELLITE MARKERS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have made satisfying efforts to revise the manuscript according to the suggestions raised by me and improved the manuscript substantially. However, in my view, some things still need to be adapted.

In my opinion, it is very difficult to interpret STRUCTURE results using non-neutral loci and the emphasis should be put on analyses performed with neutral markers. I think the Results section “Structure analyses” should be adapted accordingly by prominently presenting results for STRUCTURE and MavericK using 11 neutral markers. STRUCTURE results using nuSSR and EST-SSR markers only are, in my opinion, interesting additional information for the supplementary section and can be mentioned briefly but are not very relevant for the analysis of population structure.

I have some additional minor suggestions that might be worth to be addressed.

Titles and subtitles: In my opinion, the word “analyses” should be in singular in all the titles and subtitles.

Line 138: Are the authors sure about the higher error rate of SSRs compared to SNPs? Please consider to remove this statement or support with literature.

Line 150: “populations” should be singular.

Line 174: “and the distribution” could be changed to “together with the distribution”.

Line 267: “using, respectively,” should be changed to “, respectively, using”.

Line 315: “at populations” should be changed to “in populations”.

Lines 317-325: Please consider reformulating these sentences with emphasis on the neutral data set and consider trying to report Fst values more objectively. It is a bit difficult to me to understand what the authors mean with e.g. “using the “neutral” data set confirm the differentiation of the group formed by sites Lazio, Sicily, Sardinia and also of Corsica“. Is it the differentiation that formed the group or is this group formed by other factors?

Line 411: Line 331: “13 loci PCA scatterplots” should be replaced with something like “The PCA using 13 loci…”.

Line 546: “The existence of more than two clusters” should be changed to “The possible existence of more than two genetic clusters”.

Line 549: What do the authors mean with “population clusters”? Do you mean genetic clusters? Please specify.

Line 550: It is not very clear to me what is meant with “and the existence of several glacial refugia” in this context. Please consider to revise this sentence.

Line 907: There is a point after the question mark that should deleted.

Legend Table 2: “coefficient(Fis, Weir and Cockerham 1984)” should become “coefficient (Fis, Weir and Cockerham 1984)”

Table 2: In my opinion, genetic diversity estimates (observed &expected heterozygosity) should be presented per population here. This is a standard for reporting genetic diversity statistics and is interesting for the reader. Differences in genetic diversity between populations are not apparent in Table 1. Furthermore, I recommend to consider presenting the values using neutral markers instead of all 13.

Legend Figure 2: “Clustering plots from the analyses of the full data set and the neutral data set” is obsolete. Please adapt or remove.

Figure 3: In think an unrooted tree would be more appropriate instead of a phylogram.

Figure 4: The amount of variance explained per axis should be added to the figure or at least be mentioned in the figure legend. Knowing how much variation is contained in a single axis is very relevant for the interpretation of a PCA. The same is true for the supplementary Figure S3.

Figure S1: It is still not clear from the figure legend what the black line indicates. I think the answer of the authors to my initial comment on Figure S1 was related to Table S1.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: POPULATION STRUCTURE IN QUERCUS SUBER L. REVEALED BY NUCLEAR MICROSATELLITE MARKERS
Review round: 2
Reviewer: 2

Output:
Basic reporting: First of all, I’d like to apologize to the editor and the authors for the delay in submitting my report. I had a very close familiar hospitalized and I had to postpone all my academic duties to take care of him. I hope this delay will have no impact in a fast publication of an article that I consider now suitable for publication in PeerJ.
After reading carefully the revised version of the manuscript and the answer provided by the authors to my previous comments, I am quite satisfied and think that the manuscript has been considerably improved and that most of the weaknesses of the first version have been solved.
Experimental design: I agree with the authors when they consider the manuscript is not a review, but I think that now they offer the reader a complete view of the ample literature in the manuscript topic. Moreover, they have included relevant references that support and/or highlight their findings (Lines 409-422; Lines 530-533; Lines 557-570). I must say that I still disagree with the consideration of the PCA results as significant, and I must insist that a 6% of variance explained by the first two components using this type of markers running a PCA should not “be expected with a multidimensional data set such as this one”. In any case, the additional support for the presence of a cluster in Corsica might be sufficient to justify the authors’ hypothesis. I also appreciate the inclusion of a sentence justifying those “black regions” that could not be sampled because they were not present in the provenance trial.
Validity of the findings: Therefore, I consider now the manuscript is suitable for publication almost as it is. I have only three minor comments (one of them merely formal) that might be considered by the authors prior to definitive publication:
Lines 499-500: “Results from PCA and CA analyses also clearly distinguish the group formed by Lazio, Sicily and Sardinia, and separate Corsica from the remainder”. I must insist that the percentage of variance explained by the principal components is not significant. I suggest to remove “clearly”, regarding Corsica.

Figure 3: Please, consider using some bootstrapping or permutation procedure to estimate the significance of the UPGMA branches. Possibly, taking a look at the Fst matrix in the supplemental files, the Corsican branch would be robust (> 75).

Line 531: Q. suber in italics, and homogeneize the species nomenclature across the text (Q. suber or Quercus suber).
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: RELIABILITY AND ACCURACY OF ULTRASOUND IMAGE ANALYSES COMPLETED MANUALLY VERSUS AN AUTOMATED TOOL
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall the article is well written and able to be understood.  I would add one thing to help clarify the abstract slightly.  In the methods the low and high frequency images are explained (probe frequency) however, this is not explained in the abstract which can be confusing.  Perhaps just noting it is probe frequency is changing would help.
Experimental design: Overall, the methods are fine.  The one issue I had was with the repeated measures stats.  It was difficult to understand what measure was repeated.  The authors have a pre and a post measure in their data tables and they have two probe frequencies and two scans with a 10 minute break in between.  Were the repeated measures between the first and seconds scans (with the 10 minute break in between) or were they between measurements where the individual analyzing the data analyzed the same image twice?  If the pre post is between the two scans then I am very concerned about measuring the same fascicle each time.  Previous research (Infantolino and Challis, 2014) has shown pennation variability within one image so this variability alone could explain the pre-post differences.  If the scans change one would imagine this would become worse.  Perhaps measuring multiple pennation angles per scan and averaging would help to reduce this issue.
Validity of the findings: Overall the findings are valid based on the stats.  I would reiterate the issue I mentioned in the methods as a possible issue with the results in general.  The other thing I noticed while looking at the raw data is that the automatically measured pennation angles were in many cases well outside the realm of reported pennation angles in the literature (>60 degrees).  Is it possible to constrain the computer program to only look within a range of pennation angles (<60 degrees)?  If so, would this greatly influence the accuracy of the results?
Additional comments: I like the idea behind this study and agree it is very important work.  My two issues are the confusion with the repeated measures and the pennation angles that are well outside the realm of possibility.


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: RELIABILITY AND ACCURACY OF ULTRASOUND IMAGE ANALYSES COMPLETED MANUALLY VERSUS AN AUTOMATED TOOL
Review round: 1
Reviewer: 2

Output:
Basic reporting: General comments
Thank you for the opportunity to read and comment on the manuscript entitled “Reliability and validity of manual versus automatic ultrasound analyses”. The present study reports the reliability and validity of manual and automated methods to determine vastus lateralis architecture using B-mode ultrasonography. The manual analyses showed good reliability and the automatic analyses poor to moderate. However, absolute errors for both analyses were quite large, particularly in the automated analyses. The differences in outcome measures from the automated vs manual were very high and put into question the validity of the automated measurement program. These results are important for the field. Overall, the manuscript is well written, although there are sections requiring further improvement/clarification. I believe if they are met, it will form a much stronger manuscript. I also have some concerns regarding the methods used and more clarification on these are needed. Please refer to my specific comments below.

Specific comments
Tittle: It should be immediately clear that you’re testing the validity or previously published algorithms/automated analyses. I was under the impression that this was a newly developed coding/programming. This should be clear upfront.

Abstract: The authors should refer to fascicle angle instead of pennation angle. While pennation angle is generally used, it is conceptually wrong. Pennation angle is the angle between the fascicle and the muscle’s force line of action – tendon. Given that in pennate muscles, the aponeuroses are not necessarily in line with the tendon, we measure the angle of the fascicle instead of the angle of pennation. Please consider changing this throughout.
In the conclusion, I believe it’s better to provide a more general statement. What are the implications of these findings to the field? Why are they impactful? I also don’t see any inferences regarding the validity and the large errors in the automated analyses. Please consider including.

Introduction:
L91-92: This sentence is confusing. Please consider re-writing it

L94: this sentence is also unclear. Assessment of muscle architecture doesn’t directly imply number of sarcomeres in parallel and in series. It’s just that in pennate muscle we can fit more fascicles within the same space than a non-pennate muscle and that in lesser pennate muscles fascicles are often longer.

L94-97: Please revise the sentence.
L106-107: The authors suggest there is insufficient data regarding reliability and validity of automatic ultrasound analyses. I believe that published new automated analyses package/software are always validated at first glance. See Cronin et al 2020 (deep learning), Seynnes & Cronin 2020, Trackamte, UltraTrack (Farris & Litchwark 2016), etc. However, most of these manuscripts use single images with limited field of view, ie, they do not assess them using extended field of view.

L109: the authors suggest that only fascicle angle and length are part of the architectural measurements. However, muscle thickness is also very important and is strongly associated with maximal strength capacity and estimates of hypertrophy and its changes with training. I wonder why this has not been included in your analyses?

L110-11: there’s a problem with this statement. While it is generally accepted that automated analyses are compared against the manual ones to determine validity, this comparison will be highly dependent on the rater’s experience. If you have a sonographer that has no experience, the images will unlikely to be valid. But if the sonographer is highly experienced, it not only captures better, more valid, images but it also analyses them much better. However, one could also argue that the automated analyses from images obtained from both a non-experienced and experience sonographers and provides equally good/bad results, it will prove to be a valid coding/software. Haven said all of this, I what is the sonographer’s experience, how many images and analyses has the sonographer collected prior to this study, and why have you not included another rater to perform to capture the images and do the analyses? I also wonder if these analyses were blinded? All of that would have strengthen the methodological design of the paper.

In your hypothesis statement, can you provide an estimate of reliability beforehand? That, is, would you expect the reliability to be poor or good? And what sort of reliability are you referring to? This should be similarly applied to validity. These need further clarification….

Methods:
Why did the authors perform analyses in 23 participants only? Do you consider that enough for reliability and validity purposes? And why? Can you please justify your choice

Also, why was the study conducted on this specific population? If you’re determining the reliability and validity of a measurement, would you not want to test it in a population with very heterogenous characteristics? This would have allowed the results of the present study to be extrapolated to a broader range of population

The experimental design needs further information. I only understood that Trials 1 and 2 were used for reliability half-way through the methods. I think the sentence 124-125 is confusing: two independent data collection trials…. I think this could be improved to suggest you collected vastus lateralis images twice, 10 min apart, and that the images obtained from each time point were used to calculate fascicle angle and length and then estimate the within testing session reliability.

L126: Technically, ultrasound was used to capture images of the muscle of interest. The images allowed then for architecture to be calculated. Be clear.

L139: is this a secondary aim? Why was it done and why is it important? Please clarify that in the introduction. The reader won’t know why this was done.

L142-143: Two images per trial? So what did you do - took the average of fascicle angle and length for both? Sorry this is unclear

L143-144: What does “outer borders of the muscle” mean? The upper and lower apo? And what if you clearly see them but your probe is off angle and therefore fascicles aren't clearly delineated? I’d assume the main interest here is to see “fascicles” and therefore you wanted to guarantee fascicles were clearly visible.

L144: Please refer to the ultrasound images provided here.

L153-155: I'm concerned with this - vastus lateralis fascicles are not straight - they are curved and therefore you would be adding noise to your measurement if a straight line was considered. This will influence the validity of your measurement and affected the automated analyses. Please refer to Noorkoiv et al (2010, JAP).

L163: How is your manuscript different form the authors of this macro – ref 21? They showed validity of their automated programming in the ref 21

L171-172: If the automated program cannot identify fascicle angle and length from your "best" chosen image, it suggests that either (1) your image had poor quality or (2) the software doesn't do what it should be doing. Please clarify on this. It is also important to report how many times this occurred.

Statistical analyses:
L177: On what basis was this ICC chosen?

L178-179 & 181 & 185-189: Clarify how all the reliability measurements were calculated. The reader doesn't need to check Weir's paper to know how these were calculated. This also applies to the validity statistics.

L187: Why trial 1 only? Why didn't you repeat these to increase the number of comparisons to validate the automated analyses?

L193: ICC of 0.75 and SEM and MD of 14% and 7.4 degrees were reported. Does this suggest that, if an acute intervention was performed, you would need to change fascicle angle for at least 7.4 degrees for a change to be considered real? This is very impressive and practically impossible - changes in fascicle angle after fatiguing contractions or stretching are ~2.5 degrees. Do your data suggest they are possibly not real changes?
This should also be discussed further in the Discussion section.

L204: Somewhere it should be mentioned that the results show moderate reliability but very high errors!
L214-215: Do you consider these errors acceptable? They seem very high

L224: Can you speculate why the validity is worse for different frequencies?

Based on your figures, it seems to me that the only variable showing proportional bias is Figure A. Lower Frequency fascicle angle. All the others seem to increase the bias with increases in values.

Discussion:

L230: You need to make clear here that this is not your coding/program but that it has been published before. I suggest "...novel, previously published, automatic...."

L233-236: Please also refer to the SEM and other absolute reliability for this interpretation. ICCs only suggested that all subjects within the sample varied proportionally and therefore provides good between-subjects reliability; however, the absolute reliability is poor considering the expected changes in architecture with acute and chronic interventions.... this needs to be clarified.

L238: Tell the reader what TQ and SEE means.

L241-242: What do they add to? What's the implications of these findings? Please expand on this

L246-247: is this low? Which variables are these?

L249: The minimum provided here is significantly lower than the minimum SEM provided by the present study. Please discuss.

L251: Does this fit with the criterion adopted here? 0.63 does not seem an excellent score reliability.

L255-256:  These errors are large (19-31%). This is within the range of muscle architectural changes with training and detraining.
L2625-263: Why did you only analysed one fascicle? If this information was known prior to the study being performed, then you would expect to do something similar so you can discuss the findings to that specific study.

L264-266: Why didn't the authors expand further and analysed multiple fascicles? If this is indeed quick, it shouldn't have taken longer to analyse another fascicle of the same image. Please justify.

L269-271: This seems an unacceptable error and invalidates the automated program. On top of these differences between manual and automated analyses, you are also expecting a big between trial variation, further reassuring the inability of the automated program to accurately and reliability determine fascicle length and angle.

L274: What is a true validity calculation? Please be clear

L281-283: Why have you not performed those calculations to make it, at the least, comparable with the literature?

L289-293: Why were they not identifiable? If the images are performed under the same conditions, by the same experience investigator, would you not be able to place an image side by side and tell whether the region of the fascicles chosen are matched and therefore select the same fascicle? I also think analyses of a single fascicle is complicated. You may need to include that in your limitations. How can you guarantee the fascicle chosen was representative of the fascicles of the muscle?

L303-304: If the errors were large, would you consider this valid? The concept of validity needs to be clarified.

L306: Theres no mention of how these are applied to training, detraining, or acute interventions. A recent paper has shown that the changes in muscle architecture after disuse are method dependent (Sarto et al 2021 - 10.1249/MSS.0000000000002614). Considering the large errors showed here, this should be discussed!

Conclusion
L298-299: Can you please provide an explanation for this? Can you speculate as to why this is impacting on your measurements?

References:
Please check your reference throughout so that tittles are standardized. See ref 3 vs 2 - tittle has capital letters in each word whereas the other doesn’t.

Figures/Tables
Figure 1: If A and C are the same participant, there are clear differences in where the fascicle starts and ends and how the aponeuroses are determined – this is important because it significantly affects fascicle length and angle measurements.
It is unclear what the boxes in A and B are.

Figure 2: The x-axis of Figures A-D should be the average of the variable name, e.g., “average fascicle angle (degrees)”. Additionally, on the top of the figure, can you please write the variable name and in parentheses the US frequency?

Table 2: since it's unclear to me how you calculated any of these validity outcomes, it's hard to tell why this is in cm. The total error a can also be calculated as % value or arbitrary units, depending on how it is determined. Please clarify.
Experimental design: Full report is above.
Validity of the findings: N/A
Additional comments: No additional comments


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

Input:
Title: RELIABILITY AND ACCURACY OF ULTRASOUND IMAGE ANALYSES COMPLETED MANUALLY VERSUS AN AUTOMATED TOOL
Review round: 2
Reviewer: 1

Output:
Basic reporting: The reviewer thanks the authors for the responses to my comments.  The clarifications have helped with my understanding of the article.
Experimental design: I understand why you chose to not use an average fascicle value.  I am not thrilled with the idea but I can understand why you went in this direction.
Validity of the findings: I understand the constraints of the SMA macro produced results that were non-physiologic.  The additional wording you added helps to explain this.  While I wish that were not the case, I understand you are limited by the program you were testing.
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: RELIABILITY AND ACCURACY OF ULTRASOUND IMAGE ANALYSES COMPLETED MANUALLY VERSUS AN AUTOMATED TOOL
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors did a great job in addressing the questions and significantly improved the article. I have only provided minor comments/suggestions to further improve the manuscript. I have addressed them in blue throuhgout the word document attached.
Experimental design: N/A
Validity of the findings: N/A
Additional comments: No additional comments


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

Input:
Title: AGROCHEMICAL CONTROL OF GENE EXPRESSION USING EVOLVED SPLIT RNA POLYMERASE
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this manuscript, the authors use the traditional split T7 RNA polymerase in combination with the ABA receptor PYRABACTIN RESISTANCE 1 (PYR1),  a receptor that is known to interact with the chemical mandipropamid to reconstitute transcription. The manuscript is a standard manuscript in the field of split T7 RNA polymerases in which the authors have a ligand that binds to a protein and stabilizes T7 RNA polymerase and therefore transcription can be started.
The main concern that this reviewer has with this manuscript is the absence of crude data in the main figure, for instance, a sequencing denaturing acrylamide gel showing the accumulation of transcript of an SDS gel showing the accumulation of GFP (although this data is present in the supplementary figure).
In the opinion of this reviewer, the authors show that coupling of the agrochemical mandipropamid to a modified receptor can reconstitute T7RNAP and therefore the manuscript should be accepted.
Experimental design: The experimental design is standard for the field.
Validity of the findings: Data validation is standard for the field.
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: AGROCHEMICAL CONTROL OF GENE EXPRESSION USING EVOLVED SPLIT RNA POLYMERASE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Yuan and Miao describe the use of mandipropamid inducible T7 RNA polymerase system in E. coli which could also be utilized together with plant systems.  The study could be of interest to researchers, however, the overall description of experimental results are lacking details and clarity.  The authors need to improve their manuscript through additional experiments and clarifications.
Experimental design: The most important finding of the manuscript is mandipropamid-inducible T7 RNAP system.  The description is too brief and lacking details.  They need to provide additional experiments including in vitro assay to test association of the split T7 RNAP system.

They also discuss split-GFP based system, but the description is short and lacks clarity.

For figures, they use connecting lines without describing what they are.  The curve do not look like they were fit to data.  They should remove those lines unless they mean something.  For experiments, they do not report the number of replicates nor the statistical tests.
Validity of the findings: Overall, they need to provide detailed experimental setup with additional images.  For instance, it is difficult to understand the setup for E. coli grown in hollow stems of water spinach.  If the stems of water spinach were simply used as a container, how would it help them study plant-microbiome interaction as they proposed?  They could use microscope images of sections to better understand the plant and microbe interaction under the culture condition.
Additional comments: There are very few references and they seem to miss a number of relevant literature on the topic.


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: AGROCHEMICAL CONTROL OF GENE EXPRESSION USING EVOLVED SPLIT RNA POLYMERASE
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed all the concerns raised by this reviewer
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No comment


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

Input:
Title: AGROCHEMICAL CONTROL OF GENE EXPRESSION USING EVOLVED SPLIT RNA POLYMERASE
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors improved the manuscript with additional experiments and explanations in the main text.  They also included a number of relevant literature to put their work in the context of existing literature. Thus, I would like to recommend the work for publication.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: ARE ALL THERMOMETERS EQUAL? A STUDY OF THREE INFRARED THERMOMETERS TO DETECT FEVER IN AN AFRICAN OUTPATIENT CLINIC
Review round: 1
Reviewer: 1

Output:
Basic reporting: Excellent reporting--clear data and methods. Results and discussion are well-informed and articulated.

Minor typo on line 300-301, the word "Oral" has a space or a return between the "r" and the "a"
Experimental design: No comment
Validity of the findings: No comment.
Additional comments: The goal of this paper is to compare traditional oral thermometry with several newer methods adopted more globally during the COVID-19 pandemic. The article is written and organized in an approachable manner that will certainly serve the scientific and medical community, but also the lay person who reads this. Critically, the way this article is written and the data presented are in such a way that the average non-science reader can read and interpret the results.


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: ARE ALL THERMOMETERS EQUAL? A STUDY OF THREE INFRARED THERMOMETERS TO DETECT FEVER IN AN AFRICAN OUTPATIENT CLINIC
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.	The abstract should clearly state the oral thermometer is used as the gold standard.
2.	Line 101: Citations should appear in order (i.e., the first citation to appear in the text should be 1)
3.	Line 244: The classification target of fever vs no fever should be explained before describing the metrics used (sensitivity, specificity…)
4.	Line 265: Use of Bland Altman plots is described. This should be moved to the Methods section.
Experimental design: The research question is well defined and meaningful. The methods explain the experimental design well, with enough detail to replicate.
Validity of the findings: 1.	The authors should be commended for undertaking a rigorous comparison of various thermometers, which is a relevant issue given the global pandemic.
2.	The authors also do an excellent job of reviewing and summarizing the relevant literature. The authors comment on the literature for bias, but not for correlation and sensitivity. The discussion could be improved by similarly commenting on these metrics and comparing/contrasting with the authors’ own results.
Additional comments: no comment


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

Input:
Title: ARE ALL THERMOMETERS EQUAL? A STUDY OF THREE INFRARED THERMOMETERS TO DETECT FEVER IN AN AFRICAN OUTPATIENT CLINIC
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors provide an interesting research article. They use English throughout. But spelling mistakes in the result section ("Spearmann") and the abbreviation of body temperature measurement as BMT instead of BTM, which is used later in the article, should be corrected. The authors provide background information and relevant literature references. The structure of the article conforms to PeerJ standards.

Raw data is supplied and the findings and figures can be reproduced. The definition of fever as a body temperature greater than or equal to 38°C as stated in the statistical analysis section should be used throughout the other sections. The figures are relevant, well labeled and support the content.
Experimental design: The research question is well defined, relevant and meaningful and within the scope of the journal. The results help to narrow the identified knowledge gap. The investigations are performed according to technical and ethical standards. The methods are described with sufficient detail to allow replication of the findings.
Validity of the findings: Demographic data is not given on individual patient level. Apart from that the underlying data has been provided. The conclusions are well stated, linked to the research question and supported by the results.
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: A NEW GIGANTIC CARNIVORE (CARNIVORA, AMPHICYONIDAE) FROM THE LATE MIDDLE MIOCENE OF FRANCE
Review round: 1
Reviewer: 1

Output:
Basic reporting: During my reading I have made corrections to the English which, though perfectly comprehensible, could be improved for increased clarity and readability. My corrections are marked on the pdf.

All technical aspects as, as far as I can see, carried out in a professional manner.
Experimental design: This paper is descriptive and involves no experimental design. The paper ´clearly lays out what is new and why previous work on the topic needs to be updated.
Validity of the findings: This paper is composed of two distinct parts. The first is a description of a putative new Amphicyonidae taxon from the middle Miocene of southwest France. The second is a review of middle to early late Miocene amphicyonids from western Europe.

To begin with the second. This review is an update and broadening of that published by Viranta (1996). In the intervening years a number of new Amphicyonidae from the Miocene of Europe have been published. What makes the new review valuable is that the new taxa change a number of things related to amphicyonid ecology and extinction in the from the late middle Miocene onwards. I find this part of the paper well done and well worth publishing. However, Morales et al. (2021) recently published a new paper on amphicyonids of the late bearly Miocene of Czechia which should includes a rearrangement of taxa within Amphicyonini that should be added in revision to this manuscript regardless of agreement or disagreement over the generic assignments in that paper. Overall, however, this section of the manuscript requires only minor revision.

The other (first) part of the manuscript is the description of the new genus and species Tartarocyon cazanavei from the southwest France. This specimen (a mandibular ramus with a few teeth) is in my opinion interesting mainly because of its geographic provenance. I have no doubt that is a ‘local’ taxon, and it is certainly found on marine sediments. Nevertheless, I would like to see some evidence that the find site is not the result of substantial transport of a carcass down a river  and into the sea, where it came to rest. If that were the case the environment where the animal lived may have been well inland. The in-life illustration, though very skillfully done (I do like it), seems to me rather fanciful in two ways. First, I see it as unlikely that an amphicyonid of this size would be hunting along the shoreline in that fashion. Lions and brown hyenas do along the Namib coast, but they are after slow-moving marine mammals for which there is no evidence here and no presence in the illustration. Second, the by now classic paper by Carbone et al. (1999) based on fundamental energetic requirements, precludes carnivores as large as this amphicyonid having hunted prey as small as the moschids shown in the illustration. I find it far more likely, regardless of the geological provenance of the specimen, that the actual animal would have been hunting large prey and eating carrion well inland.

Regardless of this, which may be seen as quibbling, I find the taxonomic conclusion in the paper problematic. This only a single, incomplete specimen and the taxonomic distinction made is based essentially on a single character – the configuration of the distal part of the p4. In a group of taxa in which dental variability is high (cf. the illustrations of Paludocyon bohemicus in Morales et al., 2021 for an example) this is very poor evidence for generic distinction (which should in any case be based on phylogeny not phenetics). That this is a new species is certainly possible and I would not argue with making it a new species of some known genus, perhaps as (e.g.) aff. Amphicyon cazanavei. It just seems to me that the generic splitting introduced by Ginsburg (in Rössner & Heissig, 1999) is (was) overdone but is now being pursued by Morales and colleagues. My recommendation is to either act on my suggestion above or provide a much better argument for generic distinction of this specimen, specifically adding more direct comparison with related taxa. I do not expect this to be problematic.

Refs.
CARBONE, C., MACE, G. M., ROBERTS, S. C. & MACDONALD, D. W. 1999. — Energetic constraints on the diet of terrestrial carnivores. Nature 402: 286-288.
MORALES, J., FEJFAR, O., HEIZMANN, E., WAGNER, J., VALENCIANO, A. & ABELLA, J. 2021. — The Amphicyoninae (Amphicyonidae, Carnivora, Mammalia) of the early Miocene from Tuchořice, the Czech Republic. Fossil Imprint 77: 126-144.
RöSSNER, G. & HEISSIG, K. 1999. — The Land Mammals of Europe. Verlag Dr. F. Pfeil.
VIRANTA, S. 1996. — European Miocene Amphicyonidae — taxonomy, systematics and ecology. Acta Zoologica Fennica 204: 1-61.
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: A NEW GIGANTIC CARNIVORE (CARNIVORA, AMPHICYONIDAE) FROM THE LATE MIDDLE MIOCENE OF FRANCE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well structured, well figured, and does present the new fossil in its temporal and spatial context. I nevertheless suggest to shorten the chapter on amphicyonid evolution in the European Miocene to the very few points, the new taxon adds information to.
The language could be improved. I gave some hints in the commented pdf, but I suggest review by a native English speaker.
Experimental design: Given the restricted knowledge on Miocene amphicyonids of Europe and the very uncommon geological circumstances the fossil was found in, a publication is more than welcome and well within the scope of PeerJ.
The uncommon morphology of p4 adds importance to this publication. All methods are state of the art, and the references are adequate and complete.
Validity of the findings: The authors base their new taxon on a single character, the morphology of the distal accessory cuspid of p4. In my view, it would be necessary to discuss the variability of p4 in the closest relatives (Amphicyon) to strengthen this taxonomic decision. On the other hand, all points containing discussions on thaumastocyonines and haplocyonines could strongly be shortened, as relative molar sizes exclude the new mandible to belong to these subfamilies.
Additional comments: If the manuscript concentrates on the comparison to Amphicyon and is shortened by information the new taxon does not reveal new evidence, it will be highly welcome and will attract a lot of readers.
Several minor suggestions can be found in the attached pdf.


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: A NEW GIGANTIC CARNIVORE (CARNIVORA, AMPHICYONIDAE) FROM THE LATE MIDDLE MIOCENE OF FRANCE
Review round: 2
Reviewer: 1

Output:
Basic reporting: All points are fine, exeptions are given as a comment in the pdf.

 - A very new publication:     DOI: 10.1007/s12542-022-00610-0 should be included
 - Supplements with the references for the stratigraphical distributions would be helpful
Experimental design: no further comments
Validity of the findings: As in the first submitted version, the question to be debated is whether an uncommon p4 morphology is good enough to erect a new amphicyonid genus. In my view, one could probably separate a species.
Given the known variability within pseudarctine genera, I would avoid the erection of a new genus and follow the other reviewer in suggesting usage of aff. Amphicyon or Amphicyonini indet.
However, the added picture showing p4 morphologies among amphicyonids helps the reader to understand the position of the authors.
Everything else in this contribution is fine. Minor remarks can be found in the commented pdf.
Additional comments: No additional comments


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

Input:
Title: INDIVIDUAL CHARACTERISTICS, INCLUDING OLFACTORY EFFICIENCY, AGE, BODY MASS INDEX, SMOKING AND THE SEX HORMONES STATUS, AND FOOD PREFERENCES OF WOMEN IN POLAND
Review round: 1
Reviewer: 1

Output:
Basic reporting: Dear authors, I have read the manuscript with great interest. I found this study setting out to test an interesting research question: whether certain factors affect liking of different foods. The research question is interesting in that it could help understand what drives healthy and unhealthy food choice. The reporting is clear until the discussion, although the English is sometimes somewhat colloquial and may be improved to improve the clarity of the manuscript. The data was shared with the article, and the data structure and variables are clear. However, I have a number of questions with respect to the methods, a number of concerns regarding the data-analysis, and I believe the discussion section would need to be largely re-written and shortened before it is suitable for publication in PeerJ.
I nevertheless hope that my (many) comments find you in good spirit, and will allow improvement of the manuscript:

For the abstract, my suggestion would be to summarize instead of reporting results with statistics.

Paragraph in lines 101-108 would benefit from additional literature sources and more explanation on the causal effects drawn.

I would probably not call ‘chocolate’, ‘fruit’, ‘candies and jellybeans’ a ‘dish’, but a food type or food item instead.

The discussion is relatively long with respect to the other sections of the manuscript: it covers half of the main body of text. The discussion is speculative at places, and touches upon many topics not directly relevant for the study at hand. The discussion does not cover the same content and literature as the introduction.
Experimental design: The experimental design is correlational, which matches the research question and analyses proposed. The research fills a gap of knowledge.
With respect to the methods, I do have a few questions, specifically:

Lines 125-134: how was the sample size determined? Was there an a-priori expectation for the effect sizes?

Lines 132-134 are not immediately clear: How can people with high olfactory sensitivity be classified as anosmic?

Lines 146-155. On what criteria were these food types selected for the study? In addition: Having access to the pictures of the food types would help understand the methods and results of the study.

Line 155-156: the anchor labels do not make a lot of sense to use in English – perhaps good to also report the original Polish scale anchors.

The description of the threshold test is not very clear:
Line 162, 168, etc: In my understanding, a fragrance would mean a (complex) mixture of odorous molecules. It seems ‘fragrance’ is used here synonymous to ‘smell’ or ‘odor’.
Line 165: N-butanol is used, but presumably at different concentrations and not at a fixed concentration of 59.9 PPM. Or is the concentration of 59.9 PPM used as the first step (step 0) in the dilution series?
Line 166: air is not an inert gas.
Line 167: the sentence ‘providing a correct, positive answer twice in a row by all participants in the study ended the measurement cycle’ is not clear. Usually, in this type of test, a staircase procedure is used, in which the concentrations alternate between higher and lower dilution steps, but is not clear whether this was also done here? It is not clear what the task of the participants was; what constitutes a trial?

Lines 174-179: was the task to match odors to a name (i.e., multiple choice), or to free name the odors? In case of the latter, how are correct answers determined (in other words, would ‘lemon’, ‘citrus’, ‘7-up’, ‘limonene’ or ‘lemon scented cleaning product’ all count as a correct answers to the limonene odor?)

Table 1 & 2: It is surprising that fruit, and vegetables and salads are in the top 3 most liked food types. Additionally, I find it very surprising that the participants derived more pleasure from eating vegetables and salads than chocolate. This could be a translation issue (was it really ‘derived pleasure’ that was asked from the participants?), or, more damning for the conclusions of the article, it may be due to some type of response bias, e.g., demand characteristics or social desirability. Could the authors comment on this?

Table 1: Something seems odd about the n-butanol dilution step mean and standard deviation in Table 1. It does not make sense to calculate the average of a logarithmic scale. This leaves me to wonder: on what scale was olfactory sensitivity added to the analyses?
Validity of the findings: Regarding the validity of the findings, I have a number of concerns. It is sometimes not entirely clear how the analyses were performed, and whether the analyses are fit for the types of data used.

Specific comments are:
Lines 186-202: what was the nominal variable in the regression analysis? What was the dependent variable in each regression model? What is ‘ability to associate smells’? It might be good to spell out the levels of each predictor variable (e.g., how can participants differ on hormonal status in context of this study?).

In light of the research question, it may be informative to group food types into a few categories (e.g., ‘healthy’ and ‘unhealthy’, snacks vs meals, or by general food group) of which the means may be used in a few regression models, instead of running regression analyses for each food type separately.

It is not entirely clear what kind of regression analyses were done. What do t and F values denote in this context?
Were assumptions of the regression models checked beforehand? For example, the variable age is not normally distributed, with many more younger participants than older participants.

How were outliers handled, and missing data? I might imagine the person who has 60 pack years on a group average of .8 (sd = 4.7) is removed from the analyses?
Many of the significant p-values are close to p = .05, with relatively small effects. Was there some control for multiple testing?

Many of the points in the discussion are not directly supported by the data. In particular:
Lines 247-248: this relationship is speculatory, and the causal direction cannot be inferred from the data (e.g., vegetarians may rate meats as less pleasurable because they are vegetarians, instead of eating vegetarian because they don’t like meat). Also, the comparison to men can not be made with the current dataset. Was data on diet preferences collected in the present study? This conclusion is not supported by the data.
Lines 263-266: the only significant effects of BMI were on candies and on broth, and in both cases this relationship was negative, meaning that higher BMI resulted in lower liking of candies and broth, respectively. This conclusion is not supported by the data.
Lines 272-275: this relationship is plausible, but given that there is no data on consumption frequency of each of the food items, not directly supported by the data.
Lines 267-287: the history of Polish cuisine is interesting, but seems not directly relevant for the article.
Lines 288-297: please comment on whether this finding may be due to social desirability in answering the questions.
Lines 296-316: sense of smell is measured in two ways in the study, using olfactory sensitivity and naming ability. Only one of these (naming ability) showed significant relationships with food liking, but odor sensitivity is generally taken as a more basic measure of sense of smell. The general conclusions in these paragraphs are therefore not fully supported by the data.
Additional comments: additional specific comments:
line 56-57: it is not immediately clear what the percentages in this sentence mean
Line 112: I would maybe change this sentence to ‘…some considered less healthy with high energy value and some considered healthy, such as vegetables, fruit and fish.”
Line 155: a visual analogue scale?

Use of language – can be improved. A few examples (not exhaustive):
Line 21: ‘are’ or ‘mostly depend’
Line 46: remove ‘healthy’ and ‘unhealthy’
Line 48: remove ‘only’
Line 55: suggestion to remove ‘nowadays’
Line 161: remove ‘the’
Line 200: ‘predictrs’
My suggestion would be to change ‘women’ to ‘participants’ in the description of table 1.


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: INDIVIDUAL CHARACTERISTICS, INCLUDING OLFACTORY EFFICIENCY, AGE, BODY MASS INDEX, SMOKING AND THE SEX HORMONES STATUS, AND FOOD PREFERENCES OF WOMEN IN POLAND
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article must conform to professional standards of courtesy and expression, I would suggest you go through the report to check for incorrect expressions or technical terms.

The article should include sufficient introduction and background to demonstrate how the work fits into the broader field of knowledge and knowledge gap. Your introduction needs more detail - provide more justification for your study - hormonal levels and impact on smell which would potentially affect food preferences i.e expanding on the knowledge gap to be filled. Relevant prior literature should be appropriately referenced throughout the report.

All data has been made available in accordance with PeerJ Data Sharing policy.
Experimental design: The methods should be described with sufficient information to be reproducible by another investigator. It is not clear how the participants were recruited, what are the demographics of the group of women (transgender participants were they excluded or included), and how health interviews were conducted and data collected, and how anthropometric measurements taken and/ or data collected.
Validity of the findings: All underlying data have been provided; they are robust and statistically sound. Although the results are compelling, the data analysis could have consider factor analysis - principle component analysis and could have checked any correlations of some of the predictors which were not quite clear from your results. The discussion on correlation of predictors e.g sense of smell and greater pleasure should have been accompanied by results or reference to the tables with the results. The discussions should be focused on your results and backed up with literature and not vice versa. Conclusion are well stated and indicated were correlations could have occurred.
Additional comments: This is a very interesting research which would contribute to dissemination of science on impact of sense of smell on food preference and health. If they would need more detailed feedback on any of the sections, PeerJ should feel free to contact me.


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: INDIVIDUAL CHARACTERISTICS, INCLUDING OLFACTORY EFFICIENCY, AGE, BODY MASS INDEX, SMOKING AND THE SEX HORMONES STATUS, AND FOOD PREFERENCES OF WOMEN IN POLAND
Review round: 1
Reviewer: 3

Output:
Basic reporting: I regard the manuscript to fullfil the criteria of basic reporting set by the PeerJ journal.
Experimental design: Previously published literature on food preferences is large. The authors should make it clearer what is the knowledge gap they aim to fill. Is it food preferences of Polish women? I guess food preferences in many cultures have already studied.

For validation of the results from the olfactory test, it is great that the participants' nasal patency and symmetry of air flow through the nostrils were confirmed. The methods to measure olfacoty sensitivity (as detection threshold to n-butanol, a standard odorant) and odor identification test (althought employing only five odors) seem valid.

In contrast, the authors merely state (lines 143-144) "anthropometric data -- were collected." Were values for height and weight measured by the researchers or requested to self-report by the participants?

Regarding the assessment of food preferences it is good that the authors have explained the food items that are local (Polish) specialties. The authors mention that the food categories (total 24) for which the pleasantness were assessed included usually two or three dishes (shown with pictures to the participants). I suggest that you show the list of all items in a table. Please also describe why you used more than one picture for each food category and why only the most liked option from each category was rated for pleasantness.
Validity of the findings: The raw data have been provided as an Excel file.

The authors should discuss how well the set of two/three figures showed to the participants represented the food category in question and how the selection of the set of figures may influence the results.

The results tell about food preferences of Polish women, but their connection to overweight was not confirmed (although the Introduction discusses overweight). It could be that pleasantness of food do not explain much of their use. Furthermore, self-reported responses are prone to bias from wish to provide responses that are socially acceptable. The authors could consider and discuss these challeges.

The Conclusions is a mere summary of the results; it does not really make conclusions based on the results.
Additional comments: Abstract includes too much details on results of statistical analysis, which makes it difficult for a reader to follow.


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: INDIVIDUAL CHARACTERISTICS, INCLUDING OLFACTORY EFFICIENCY, AGE, BODY MASS INDEX, SMOKING AND THE SEX HORMONES STATUS, AND FOOD PREFERENCES OF WOMEN IN POLAND
Review round: 2
Reviewer: 1

Output:
Basic reporting: Thank you for giving me the opportunity to review this revised version of the manuscript. It has improved a lot, especially textually. The additions are very useful, especially the pictures of the different food types, and the comment on social desirability in the Discussion section, for example.

With respect to the basic reporting, I still feel the discussion section is very long, and could be compressed more. Findings could be discussed in more general terms, reflecting the literature from the introduction, instead of separately for each predictor and food type. I leave it up to the editor to decide if the current length is acceptable for the journal.
Experimental design: As before, the design is correlational and correct.
Validity of the findings: Given the information in the manuscript, and the underlying data provided, I was able to re-analyse the data with the same results. That is very nice!

Regarding the validity of the findings, I still have a number of concerns. The authors have clarified many of the questions I had regarding the analyses (e.g., it is made clear that the analysis was a GLM, and hormonal status is clarified), but it remains questionable if the data supports the conclusions.

For example, the decision to keep strong outliers (i.e., someone with 60 pack years on a sample average of .8, SD = 4.7, so this person is approx. 12 standard deviations from the mean) in, raises doubt about the conclusions regarding smoking and food preference. It is possible, for example, that the reported relationship between smoking and disliking of bread is entirely due to this single person’s disliking of bread (nb. actually, when looking at the data, it is more likely removal of this individual makes the relationship between smoking and bread liking stronger).

Additionally, regarding the assumptions, the main assumption for regression is that there is a linear relationship between the predictor and dependent. If olfactory sensitivity entered the analysis in the format reported in Table 1 (or the data-file), i.e., the number of times the original concentration is diluted, this assumption is not met – in this form, olfactory sensitivity is neither normally distributed nor linearly associated with any of the dependent variables. If some transformation will take place (e.g., taking the natural log, or the dilution step), the analysis is probably valid.

I would find the manuscript publishable if the authors do the analyses again with the dilution steps as predictor instead of the raw value for number of times 59.9 PPM n-butanol was diluted as the operationalization for olfactory sensitivity, and take an approach to outliers (for example, replace using the Median Absolute Deviation method (Leys et al., 2013), or remove). This will affect the conclusions.

Leys, C., Ley, C., Klein, O., Bernard, P., & Licata, L. (2013). Detecting outliers: Do not use standard deviation around the mean, use absolute deviation around the median. Journal of experimental social psychology, 49(4), 764-766.
Additional comments: I don't have any general comments at this point, but a few additional specific comments, often in response to the rebuttal:
Lines 39-41: use M as abbreviation for averages (not x) – would further suggest to report SDs with to any means reported.
lines 199-201: I think my previous comment on people with obstructed nasal airflow, increased olfactory sensitivity and anosmia remains valid. My suggestion would be to rephrase this sentence as follows: "A transient nasal obstruction could excessively affect the results of the olfactory efficiency test, so the subjects with obstructed airflow could be classified as anosmic on the test day."
line 236: my suggestion would be to translate 'przyjemny' with 'pleasantness' and not with 'pleasure', for the purpose of the manuscript.
lines 250-252: it is not clear what these numbers are, there are no units. I think the numbers represent the times the n-butanol in the original concentration (59.9 ppm) is diluted, but this is not clear from the text.
Lines 285-289: the dependent variables, I think, are the rated pleasantness for each of these food types?
Line 352: olfactory memory?
Line 454: detection threshold.
Line 456: the standard deviation was, according to table 1, 15550 on a mean of 7456.
Table 1: a N-butanol dilution step mean of 7456 (SD = 15550) is not sensible and does not provide direct meaningful information about how sensitive the participants were for smells. This is the number of times the odor is diluted, not the dilution step. The median is step 10. There were 16 dilution steps, and the mean of these 16 should be used and reported.

N.B. these line numbers refer to the line numbers in the revised manuscript with tracked changes.


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: INDIVIDUAL CHARACTERISTICS, INCLUDING OLFACTORY EFFICIENCY, AGE, BODY MASS INDEX, SMOKING AND THE SEX HORMONES STATUS, AND FOOD PREFERENCES OF WOMEN IN POLAND
Review round: 2
Reviewer: 2

Output:
Basic reporting: The document has been improved in using clear and unambiguous professional English. The literature is still lacking on the interaction or association of hormone levels, taste and smell - the editions in lines 107 to 109 refers to menstrual cycle and calorie intake and body weight which is not the same as the link of hormone levels  with taste and olfactory sensitivity. I suggest this is made clear the possible link between these three as this justifies the inclusion in the study whilst backing up the discussion which linked olfactory sensitivity to hormone levels.
Experimental design: A lot of improvements made to the methodology, and methods describe with sufficient detail and information to replicate.
Validity of the findings: No comment
Additional comments: No additional comments


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

Input:
Title: INDIVIDUAL CHARACTERISTICS, INCLUDING OLFACTORY EFFICIENCY, AGE, BODY MASS INDEX, SMOKING AND THE SEX HORMONES STATUS, AND FOOD PREFERENCES OF WOMEN IN POLAND
Review round: 2
Reviewer: 3

Output:
Basic reporting: This is OK. However, fluency of text could still be improved (see section 4).
Experimental design: The Authors have considered my comments and revised the manuscript accordingly.
Validity of the findings: The Authors have considered my comments and revised the manuscript accordingly.
Additional comments: The Authors have carefully considered my comments and revised the manuscript accordingly. I regard the manuscript much improved and have no further major concerns. However, fluency of the Abstract could still be improved. It is not clear what the values after the "x" are. Adding a note to Methods secotion of the Abstract that food preferences were measured using 9-point hedonic scale could help. In addition, "better sense of smell" is now ambiguous in "Women with a better sense of smell (shown in olfactometric measurements) preferred sour products, women with better smell (manifested as an ability to name scents) preferred sausages and ham." and only the extra info in the parentheses reveal the difference. I suggest to describe the differences directly, for example "Women performing better in olfactometric measurements preferred sour products, whereas women with better ability to name scents preferred sausages and ham."


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: INDIVIDUAL CHARACTERISTICS, INCLUDING OLFACTORY EFFICIENCY, AGE, BODY MASS INDEX, SMOKING AND THE SEX HORMONES STATUS, AND FOOD PREFERENCES OF WOMEN IN POLAND
Review round: 3
Reviewer: 1

Output:
Basic reporting: The manuscript has seen 3 rounds of revisions, and it has improved greatly. It is an interesting paper with interesting findings.
Experimental design: no further comments
Validity of the findings: I believe these findings presented are valid. The statistical analyses in the revised version seem sound.
Additional comments: The only suggestion I would like to add at this point is to rephrase the following type of sentence:
'Variances in broth preferences were explained in 13%, ... ' (e.g., in line 288, and the following lines). My suggestion would be to change it into 'The model for broth preferences explained 13% of the variance, ...'.


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: LEPTIN ADMINISTRATION DOES NOT INFLUENCE MIGRATORY BEHAVIOUR IN WHITE-THROATED SPARROWS (ZONOTRICHIA ALBICOLLIS)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is well written, with sufficient, relevant references, and the background information sets up the question sufficiently. Apart from a few editing mistakes (an extra "used" in line 185 and a typo "recordins" in line 168), I could find no other grammatical or spelling errors. The figures are clear, although the figure legend for Figure 3 has text that is clearly copied and pasted from Figure 2, and does not belong in the Figure 3 legend.

The study clearly states the hypothesis and predictions, and the data attempt to address those predictions.
Experimental design: The study is original primary research, the research question is well defined, interesting and meaningful. However, I am not convinced that the way the study was carried out is a decisive test of the stated hypotheses. There are, in fact, two aspects of how the study was set up that might be responsible for the negative (non-significant) results found by the authors, which then in turn does not allow them to draw the strong conclusions they currently draw from the study. These two aspects are:

1) the birds were injected with (murine) leptin twice per day, 6 hours apart, in the light phase. However, the behaviour that was being quantified happens during the dark phase, 8h after the second injection of the day. The authors do not mention anything about the biological half-life of the injected leptin, but I would be surprised if it is still present by the time the behaviour is measured. All the other studies they cite that have done similar things (Henderson et al 2018, Cerasale et al 2011, Zajac et al 2011) quantified behaviours that happened shortly after the injection, not 8-18h later. It is therefor possible that migratory restlessness does respond to leptin, but only acutely.

2) the other prediction was that the birds would gain less fat if injected with leptin. This may suffer from the same problem as above, if the biological half-life is short, as they could easily compensate for the suppressing effect of leptin in the hours after it has worn off, or indeed in the week after the treatments were stopped. There is, however, an even more fundamental problem with the experimental design to test this prediction, and that is that the birds were food restricted for the entire duration of the experiment. I do not understand how the authors expected the birds to gain fat (and indeed, they did not, even though they should when building up to migration), if their access to food was not ad libitum during this period. If your prediction is that the treatment will decrease the fat gain, then setting up the foraging conditions such that fat gain is less likely is not a good test of your hypothesis.
Validity of the findings: At face value, the analyses carried out seem valid and appropriate, and the data collected seem valid as well, although it would have been nice to quantify a little bit how the validation of the EthoVision data came out (e.g. correlation between EthoVision and manual scores for the sample used in the validation). The data contained many zeros and were square root transformed. It is not clear whether after transformation, the data were acceptable for the models. If many zeros were present, there would still be a lower cut-off on the data that is not ideal for linear models.

The raw data are provided for the migratory restlessness, but not for the fat data. These should also be provided (e.g. in a separate tab of the spreadsheet).

The statistical analyses are good, although I would have liked a bit more explanation about how models are averaged. I know this is a common procedure, I just don't know how it's done. Also, I do not understand how Phase and Trial can be in the same analysis together, as they must be highly co-linear. I may not quite understand the nature of the models being fitted, though.

The direct conclusions from the analysis are fine (e.g. there are no significant changes in fat reserves), but the more general conclusions drawn from those findings (e.g. "leptin has not direct influence on avian migratory motivation or behaviour"), are not, due to the design issues outlined earlier. Conclusions about birds being insensitive to leptin are also too far, as even if the experiment had worked as planned, it is testing the effects of EXTRA leptin, on top of what may already be circulating in the bird. So at best they are insensitive to the increases in leptin administered in the experiment.
Additional comments: In the introduction, the authors lay out the history of avian leptin, and the fact that for a long time, the leptin used as "avian" as in fact murine leptin. I think it would be more informative if the authors used "murine leptin" and "avian leptin" throughout the paper, so the reader can follow better. In addition, I would like the authors to justify why they used murine leptin, after setting out in the introduction that "using an erroneous sequence has resulted in a distinct gap in the knowledge on the function on leptin in birds".

Also, if the authors could clearly state which species each study that they cite in the introduction was conducted, that would be very helpful, as "bird" covers 10,000 species.

The statement about the effect of leptin on migratory behaviour (line 84-85) seems to come out of nowhere. Later on the same page, evidence is reviewed that justifies looking for such an effect. I suggest moving that sentence to after the justifying evidence has been reviewed.

The methods could be explained a bit better, especially the "natural" photoperiod the birds were on when being moved to 8L:16D is never stated. Figure 1 is very helpful, because many of those details were unclear from the text. E.g. the text (line 138-140) suggested to me the food restriction was for only 2 weeks, but the figure makes clear it lasted 5 weeks, and covered the entire treatment period and beyond. It is never made clear what the parameters of the food restriction were, and whether they were individual to get a certain fat level or body mass.

I have two minor questions about the tables: in both Table 1.1 and table 1.3, the SE and the 95%CI don't seem to correspond very well for two variables: in Table 1, Treatment has an SE of 0.34 and a 95%CI of -22.07 to 13.28, while in Table 3, Morph has an SE of 0.17, and a 95%CI of -10.35 to 30.09. That seems like quite a mismatch...