PONE-D-25-13659Increased brain size of the dwarf Channel Island fox (Urocyon littoralis ) challenges “Island Syndrome” and suggests little evidence of domesticationPLOS ONE

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First clarify the taxonomic scale of study. If subspecies is available for grey fox this should be considered and implemented. Sexual dimorphism should also be tested more explicitly together with the implementation of stats tests for slope between grey and island fox. If available, report the area of each island and run a simple correlation test between average skull length and island area.Provide raw data as appendix or in a repository and implemented literature and other concepts as advised by reviewers.  

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Additional Editor Comments:

The paper reads well and it is nice however my main point of concern is your way of considering U. cinereoargenteus as a single species vs 6 subspecies. In theory, your paper will be much stronger if you could account for subspecies differences also within your grey fox sample. Also you should introduce sexual dimorphism earlier in the paper and test for it. One consequence of island syndrome might also be increase in sexual size dimorphism (see: Raia, P., Barbera, C., & Conte, M. (2003). The fast life of a dwarfed giant. Evolutionary Ecology, 17, 293-312). You have an excellent opportunity to test for it by using simple SSD ratios for each of your island subspecies. As an example see on how to compute this see: Cardini, A., & Elton, S. (2008). Variation in guenon skulls (II): sexual dimorphism. Journal of Human Evolution, 54(5), 638-647.

Below I provide more specific points of concerns for each section that I hope you can find useful to increase clarity in your paper. I think the paper include quite valuable data and information. If you could make raw data available [eg. measurements at least] that will also be good.

Introduction

Line 58-59: add scientific names since this is first time you mention these species

Line 66: perhaps refer to Figure 1 and add some arrows on the map showing the potential route of island colonisation with dates. Add in the map also a mini-map outlining the area within the broader context of North American continent

Mat & Method:

Line 92: since you refer to the island subspecies it is worth providing more details on your sample distribution for U. cinereoargenteus for which there have been recorded about 16 subspecies....did you use only skulls of one population or did you mix specimens from different locations / subspecies....in that case it might be important to outline this. You want to produce a fair comparison across subspecies (variation of subspecies of A vs variation of subspecies of B) otherwise the comparison will be unfair (A might vary more geographically making the false impression of bigger variation compared to subspecies of B).

Line 126: do you mean cranial morphometry? You do not present any geometric morphometric analysis that is also based upon anatomical landmarks but in geometric morphometrics the raw data are coordinates and NOT linear distances between landmarks.

Line 138: nice one....I wonder how does this compare to the Van Valkenburgh equations on Canidae only. It would make more sense to use Canidae specific equation for a more accurate body mass reconstruction

Results

Line 161: because you are talking about scaling I was expecting a test of slope differences and not a test of variance. It might be worth exploring the regression of Skull length (X) vs Brain Size between the two subspecies or different subspecies. This test will address the question: does grey fox and island fox brain size scale in the same way? Also I can see Sex in Figure S1 -this should be in the paper since it is your very first result- but Sex is not mentioned in the Introduction / Mat-Methods...if you got sexual dimorphism you should first test for differences in relative brain size between species and sex using a model "BrainSize~Species+Sex+Species*Sex"....if Species*Sex is significant you might need to do the analyses separated by sex (e.g. compare males with males only). IF you have sufficient subspecies sample size for U. cinereoargenteus than your unit of analysis [also for sex] should be "subspecies" and not species.

Line 162: why did you use Wilcoxon [generally it applies to dependent data that have a time structure [e.g. before and after]...you should use simple t-test or non-parametric Mann-Whitney if your groups are species.

Line 165: Report slopes and intercepts (and 95% CI if necessary) for your models in a table....slopes of the two Urocyon seem parallel to me but without any stats test we will never know that for sure...try and test for difference using ANCOVA and eventually check if slope differ between subspecies (if n within subspecies is big enough).

Line 177: delete "with"

Line 179-181: leave it out for the discussion

Line 191: you cannot talk of shape for plot 5B -it is mainly size, right?-. Different thing is plot 5C. A log transformation of the data might make the plot also a bit better. You can clearly see the issue of species (big yellow convex hull) vs subspecies (convex hulls there are much much smaller)

Line 192-197: you should test for differences using MANOVA/CVA...if test is significant then with post-hoc you can identify the pair of taxa that are more disparate between each other...based on the plot there seems to be no difference between subspecies of island fox so statistically it is more correct to not to consider the populations as separate analytical entities [if they do not differ and you have no subspecies of U.cinereoargenteus it is more correct to merge them into a single group...which is obviously not what you want to do!]

Line 225: if you manage to find subspecies for U. cinereoargenteus perhaps your differences might be even larger...at the moment the only true difference I feel to trust if the one between grey and island fox.

Discussion

Line 269: nice explanation perhaps it is worth talking also about the competitors...do they have many on the island or reduced competition (check for theoretical background: Raia, P., & Meiri, S. (2006). The island rule in large mammals: paleontology meets ecology. Evolution, 60(8), 1731-1742)? does diet change between gray and island fox? any refs on diet?

Line 290: you can check for this in your data testing the association between average skull size and island area for the six island subspecies (van der Geer, A. A., van den Bergh, G. D., Lyras, G. A., Prasetyo, U. W., Due, R. A., Setiyabudi, E., & Drinia, H. (2016). The effect of area and isolation on insular dwarf proboscideans. Journal of Biogeography, 43(8), 1656-1666)

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

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Reviewer #1: General remark:

One of the most significant aspects of this paper is the size of its sample. Most studies on the brain size of insular mammals rely on fossil specimens, often with very limited sample sizes (sometimes just a single specimen). In contrast, this paper examines 297 specimens, which is a considerable strength that could be emphasized more.

I also have one suggestion that the authors may find interesting. It is related to the brain anatomy.

The authors noticed that the island fox has a reduced length in the prefrontal area. I believe this is a consequence of its shorter rostrum. In general, canids that have relatively short faces have relatively high and massive frontal brain lobes. A somewhat similar case to Urocyon is the living raccoon dog Nyctereutes procyonoides. That species has a shorter muzzle than its Pliocene relatives. The muzzle shortening led to a shortening of the proreal gyrus length (Lyras 2009).

On Table 2, I have three minor comments:

1. Csiki-Sava et al. (2018) compare Homo floresienceis with an early form of Homo erectus ‘Homo erectus (early form)’. I suggest they add a similar parenthesis in their table, as brain sizes differ significantly between early and late forms of Homo erectus.

2. They list Gallogoral meneghinii as the ancestor of Myotragus balearicus. Actually that is not true, and neither the reference they cite (Köhler et al., 2004), says so. Gallogoral meneghinii is a Pleistocene relative of Myotragus. Myotragus is phylogenetically related to the Miocene archaic goats Aragoral and Norbertia (Rozzi, 2013). Unfortunately, their brain sizes are unknown. However, we do know that the brains Late Miocene bovids are in general smaller than those of modern bovids (Liakopoulou et al., 2024). That makes the reduction of brain size of Myotragus less dramatic than that reported by Köhler et al. (2004). To keep things simple, I suggest keeping Gallogoral meneghinii in the table but adding a footnote clarifying that it is a Pleistocene relative of Myotragus. Additionally, the footnote could mention that the reduction in encephalization appears less dramatic when compared to Late Miocene taxa (Liakopoulou et al., 2024).

3. They list Cervus elaphus as the ancestor of Candiacervus ropalophorus. It has been suggested that the fallow deer is the living relative of Candiacervus (van der Geer, 2018). I am sure that the replacement of Cervus elaphus with Dama dama will not significantly alter the values in the table.

Citations:

Csiki-Sava Z, Vremir M, Meng J, Brusatte SL, Norell MA. 2018. Dome-headed, small-brained island mammal from the Late Cretaceous of Romania. Proc Natl Acad Sci U S A. 115(19):4857–4862.

Köhler M, Moyà-Solà S. 2004. Reduction of brain and sense organs in the fossil insular bovid Myotragus. Brain Behav Evol. 63(3):125–140.

Liakopoulou D, Roussiakis S, Lyras G. 2024. The brain of Myotragus balearicus, an insular bovid from the Balearics. Hist. Biol. 1–8.

Lyras G.A. 2009. The evolution of the brain in Canidae (Mammalia: Carnivora). Scripta Geologica 139: 1-93.

Rozzi R. 2013. Palaeobiogeography and evolution of insular bovids: ecogeographic patterns of body mass variation and morphological changes. Unpublished Ph.D. Thesis. Sapinza Università di Roma, Dipartimento di Scienze sella Terra.

van der Geer A.A.E. 2018. Uniformity in variety: Antler morphology and evolution in a predator-free environment. Palaeontol Electron. 25.2.a23.

Reviewer #2: This is a thorough study addressing brain size and some external anatomical features of the brain of the Channel Island foxes of several islands respective to their mainland congener, the gray fox. It is an excellent study, well structured and easy to read. I have no suggestions for improvement, but have a very few remarks. First of all, I selected no for data availability, because this is not clear to me: are the CT-scans available for external brain morphology? Then, in the Introduction, line 51, what means 'simplified locomotion'? I'm not aware of such a feature in island endemic mammals, but my guess is that here reduced dispersal abilities are meant, such as loss of running in vertebrates, of flight in birds. In any case, 'simplified' needs to be explained here. Same with coloration, it's rather dull than simplified, and applies only (?) birds (mammals can e.g. develop spotted patterns, white-tipped tail etc., which is not 'simplified coloration'. Further, in line 60 no reference is given for the body size reduction in the island foxes (could be Lyras et al. 2010 JoB; but perhaps there is a more recent reference). Then, page 4 lines 82-83 the definition of endemic includes also mainlands, so the addition 'island' or 'insular' is needed here. There are many more living endemic canids, but if you excluse the mainlands, then the statement is almost true (there is also the Cozumel island fox). Discussion page 13 line 239 reduced predatory pressure does not apply mostly to small vertebrates; I'd argue the opposite. Elephants evolve dwarfism when no predators are around, and they are not small. Also, for rodents, predatory pressure is not lower on islands, but shifted to birds only (which can grow gigantic). Page 15 last paragraph, this link with increased arboreality is very interesting, and looks like a strong point. So I was wondering, how is the situation on San Nicolas? Page 16 line 316 I don't agree that the first domestic dogs in the late Pleistocene were mostly hunting assistants; archaeological evidence suggests rather that they were primarily food items (as with horses). Lastly, in the bibliography, a few titles are erroneously given in caps (e.g. 11, 27, 36

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PONE-D-25-13659R1Increased brain size of the dwarf Channel Island fox (Urocyon littoralis ) challenges “Island Syndrome” and suggests little evidence of domesticationPLOS ONE

Dear Dr. Schoenberger,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Double check the use of ANOVA and MANOVA providing more appropriate results. Use covariance matrix for the PCA and make sure you exclude the juveniles from the sample.See the analytical report for advise on how to perform MANOVA and pairwise testing.  

Please submit your revised manuscript by Jul 19 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Carlo Meloro

Academic Editor

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Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

The paper is almost ready, I just found few minor issues that you can hopefully be implemented very quickly before official acceptance.

Line 193: "Carnivora" upper case

Line 210: It is ANCOVA and NOT ANOVA. Modify as: "We then used ANCOVA, including interaction terms, to test whether slopes and intercepts of these scaling relationships differed significantly between species".

Line 216: I do not think you used "Welch's ANOVA". This is used when variances are not equal, in that case the post doc should be Dunnett's T3.

For an example on how to report this correctly see Meloro (2011, JVP: https://doi.org/10.1080/02724634.2011.550357). You first do a one-way ANOVA followed by a LEvene's test (LEvene's will tell you if variance is homogenous -p > 0.05- or not). If Levene's is NOT significant, you can use Tukey post doc -that will satisfy the assumption of equal variances.

Line 223-224: it is ok to perform MANOVA only on PC1 and PC2 although you might try to cover at least 95% of total variance (e.g. first 5 PCs) and provide results as expressed by Wilk's lambda and Pillai Trace.

Your PCA was probably based on Correlation matrix...you should use Covariance matrix if your input variables are log transformed measurements. See the attach doc "Urocyon_tests". I selected only Adult specimens and provided some examples on analyses run using PAST and SPSS. It is fine to combine all U. cinereoargenteus subspecies but still you need to implement MANOVA more appropriately. ANOVA that follows MANOVA is not appropriate, so please use PAST or try to use simple script in R to run Hotelling's pairwise test after MANOVA. An example is below:

manova_model <- manova(cbind(PC1, PC2, PC3) ~ species

Assuming that your selected PCs are in PC_data

For pairwise comparisons use the library (pairwiseAdonis)

result <- pairwise.adonis(PC_data, factors = species, sim.method = "euclidean", p.adjust.m = "bonferroni")

Report pairwise differences after MANOVA not based on each single PC.

Please clarify if you removed the Juveniles from the sample, I think they should be removed.

Figure 4: It is not clear to what line the R2 and p value refers to. Report eventually the general equation and the R2 next to the general regression line it refers to or use a legend.

You can check the example below:

https://link.springer.com/article/10.1007/s10914-020-09513-w

Only the general equation and the R2 which will be relevant

Figure 5 A and B are identical...perhaps put one or the other in the Appendix

Figure 6: you got one outlier for Fig 6A and 6C. Check carefully, if you remove this individual and PC loadings change that must be removed from the analyses, if not you can keep it. Maybe this occurs when using correlation matrix. See the attached example using the covariance matrix.

I noted from your TAble that you also measured Juveniles...they should be excluded.

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

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Reviewer #2: Yes

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Reviewer #1: (No Response)

Reviewer #2: (No Response)

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Submitted filename: Urocyon_Tests.pdf

Attachment

Submitted filename: Urocyon.dat

Increased brain size of the dwarf Channel Island fox (Urocyon littoralis ) challenges “Island Syndrome” and suggests little evidence of domestication

PONE-D-25-13659R2

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Additional Editor Comments (optional):

You did a good job, I just spotted few things you can adjust during production process:

line 274: representing 87.78% of variance for log-raw measurements, and 69.40% of variance for log-normalized measurements - something not right here because if one PC is 87.78% the second one cannot be 69.40%. You should write PC1 explains 82% of variance and PC2 6%

line 286: "most substantial differences by tens of orders of magnitude greater were found between" remember the P values does not give you the idea of magnitude differences so remove this...you just got highly significant differences.....be aware that this using all variables...if you re-try using only the PCs that explain 95% of variance (e.g. the first five...) you will see few non-significant differences. That might be more worth reporting, this is because MANOVA sometimes overfit differences. Your smallest group should have at least double the number of your variables. In your case if you have 11 variables the smallest group should have at least 22 cases for solid interpretation of MANOVA.

From this perspective the table in the paper is probably not that important -everything is significant- and it can go in the Appendix if you want.

Line 499: "DOIs: 10.17602/M2/M740219 (island fox 500 endocast), 10.17602/M2/M740195 (gray fox endocast), 10.17602/M2/M740169 (island fox raw

501 CT data), 10.17602/M2/M740136 (gray fox raw CT data)". I think these are ID used by morphosource ...if you copy and paste in a browser it does not open your scan so just double check.

Reviewers' comments: