Head Sexual Characterization of Sanmartinero Creole Bovine Breed Assessed by Geometric Morphometric Methods

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors Dear authors, The article entitled "Head sexual characterization of Sanmartinero Creole bovine breed assessed by geometric morphometric methods" has been revised. The use of geometric morphometric methods to characterize sexual domorphism in the breed in question allows for the establishment of greater criteria for sexual differentiation and characterization. In addition, the findings in your research corroborate studies that suggest that certain parameters for males are related to sexual selection, hormonal factors, and territorial dominance. However, the information in the text needs to be better organized. Some information in the materials and methods requires attention, as well as figures and graphs throughout the text. The PDF version for review of the manuscript had formatting errors (I do not know if this was due to the manuscript submitted or after the file was transformed into the version to be evaluated). If this was due to the formatting of the PDF file, please disregard them. Finally, the article requires corrections. All comments are in balloons in the PDF file.        

Comments for author File: Comments.pdf

Author Response

Thank you very much for reviewing our manuscript. We appreciate your valuable complementary comments and suggestions. We have made suggested changes and have revised the manuscript accordingly. We hope that you find our answers satisfactory

First reviewer’s responses

Comment

Dear authors. The article entitled "Head sexual characterization of Sanmartinero Creole bovine breed assessed by geometric morphometric methods" has been revised. The use of geometric morphometric methods to characterize sexual dimorphism in the breed in question allows for the establishment of greater criteria for sexual differentiation and characterization. In addition, the findings in your research corroborate studies that suggest that certain parameters for males are related to sexual selection, hormonal factors, and territorial dominance. However, the information in the text needs to be better organized. Some information in the materials and methods requires attention, as well as figures and graphs throughout the text. The PDF version for review of the manuscript had formatting errors (I do not know if this was due to the manuscript submitted or after the file was transformed into the version to be evaluated). If this was due to the formatting of the PDF file, please disregard them. Finally, the article requires corrections. All comments are in balloons in the PDF file.   

Geometric morphometrics are performed on different species in different contexts. I believe that instead of explaining the method (which is relatively well-known), it would be more useful to justify why this is done on the breed in question. Highlighting its importance and correlating it with the bioclimatic aspects of Colombia, as done at the beginning of the "Introduction" section.

Response.  Corrected in the text

This paragraph could be removed from the introduction without prejudice. It would be more pertinent to use it in the discussion, in order to justify the use of geometric morphometrics and not linear morphometrics. The paragraph before this one already explains the two methods well, and the paragraph after this one justifies the use of geometric morphometrics to characterize shape and sexual dimorphism. Therefore, it would be more useful to address the justification of what is best for the proposed work in its "discussion" section.

Response.  Corrected in the text

 

This information is important for your introduction. However, it is out of context. You could move it to after the paragraph that ends on line 107. This would make the text more organized, addressing the general characteristics of sexual dimorphism, as well as its applications, and then moving on to a more specific part that is linked to your research on "cattle skulls". Then continue in the last paragraph where you present the objective of the research.

Response. Corrected in the text according to your comment

These images (Figure 2) could be oriented vertically to make it easier to see and understand the reference points. Furthermore, the numbers used in the grid are too small. If the article is accepted, it will probably be impossible to see them when it is laid out for publication.

Response. Both images have been flipped vertically. Used program (MorphoJ) does not allow us to change the size of the numbers. If necessary, we will do it manually.

Figure 3. I don't understand this graph. It could be more informative. Use captions to make it more self-explanatory.

Response. It is a mere histogram plot comparing distribution of shapes between sexes. It is quite used and known in GM researches.

 English corrected.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear author, congratulation for your work. In order to improve the quality of the manuscript I would like to suggest some points as below:

- The abstract does not present clearly the sexual dimorphism with some morphological charachteristcs, but just “males present more massive heads. Sexes appeared to be different for size, but also for shape”. To my opinion this is not sufficient and show almost nothing how male differently form the female. Maybe, in the abstract, better to add this sentence; “male had significantly larger heads based on centroid size (U = 714, p = 0.0004), confirming true sexual size differences, and PCA revealed overlapping head shapes with sexual dimorphism concentrated at midline sagittal landmarks (between the most rostral and caudal orbit points) and paired lateral points, indicating males have broader and longer heads”.

-In the Introduction part from line 64 – 85, to my opinion are irrelevant information.

-References in the line 103 should be (22-24).

-A typo error is found in the line 112  …“breed. ”  and “….. sex This.. ”

-In some parts of the manuscript there is used skull, in fact here the contexts should be only head and not skull, like in the sentence: “Males have wider heads and longer skulls” lines (226 and284). To my opinion should use only the word “head” and to give exact anatomotopografical references where is more wide and where is more longer.

 

Author Response

Thank you very much for reviewing our manuscript. We appreciate your valuable complementary comments and suggestions. We have made suggested changes and have revised the manuscript accordingly. We hope that you find our answers satisfactory

Second reviewer’s responses

Comment

Dear author, congratulation for your work. In order to improve the quality of the manuscript I would like to suggest some points as below:

- The abstract does not present clearly the sexual dimorphism with some morphological charachteristcs, but just “males present more massive heads. Sexes appeared to be different for size, but also for shape”. To my opinion this is not sufficient and show almost nothing how male differently form the female. Maybe, in the abstract, better to add this sentence; “male had significantly larger heads based on centroid size (U = 714, p = 0.0004), confirming true sexual size differences, and PCA revealed overlapping head shapes with sexual dimorphism concentrated at midline sagittal landmarks (between the most rostral and caudal orbit points) and paired lateral points, indicating males have broader and longer heads”.

-In the Introduction part from line 64 – 85, to my opinion are irrelevant information.

-References in the line 103 should be (22-24).

-A typo error is found in the line 112  …“breed. ”  and “….. sex This.. ”

-In some parts of the manuscript there is used skull, in fact here the contexts should be only head and not skull, like in the sentence: “Males have wider heads and longer skulls” lines (226 and284). To my opinion should use only the word “head” and to give exact anatomotopografical references where is more wide and where is more longer.

Response. We appreciate your valuable complementary comments and suggestions

All suggestions and comments were corrected in the text.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This is a relatively straight-forward study investigating sexual dimorphism in Sanmartinero Creole cow head morphology using geometric morphometrics to identify new character information of the breed to preserve its uniqueness.

I provide my editorial comments in a Page:Line number format below:

1:23-24: This is not really the definition of GMM, so please refine definition.

1:25-26: This sentence is repetition of other ideas in the abstract and can be removed.

1:27-28: Unnecessary sentence- remove or be specific.

1:32: Not really ensuring a homogeneous sample but rather eliminating additional variables.

1:32-33: Combine this sentence with the previous sentence for clarification.

1:34: Change to "Sexual dimorphism was found in form..."

1:35-36: Remove this sentence and summarize shape differences in previous sentence.

1: 36: Remove “all these evaluated characters” and just state the two listed characters.

2:45: If the cow was introduced, then the breed wasn’t formed through natural selection, but it could have adapted since introduction.

2:45: Add “century” after XVth.

2:48: Remove “s” from forages.

2:51: Provide parenthetical definitions for mesolinean and eumetric.

2:55: These two sentences need verbs. Also, I am going to stop copy-editing here and just focus on scientific suggestions unless a suggestion improves understanding for the reader.

2:60: I am not sure what “stable free representation” means- please expand.

2:60-62: This is not necessarily part of the definition of morphometry, as that may be a factor one is trying to assess using morphometry. Truly, morphometry is just the measurement of shape.

2:65: Traditional morphometry can include more complex measures than distances, such as volume, area, etc.

2:66: I don’t know if efficient is the correct term here, since it is decidedly more time consuming, so one might argue less efficient, though it can capture more subtle variation than traditional measures.

2:69: Do you mean “configuration” rather than conformation?

2:72-73: People have been doing that well before GMM was invented (be careful not to oversell GMM).

2:74: Change “Shape analysis” to GMM, since traditional methods are still shape analysis.

2:81-83: This paragraph is about research done but really should provide brief summaries of the results, otherwise it is just repeating previous information.

3:100-101: Sexual dimorphism is far more than just body size differences and can include a variety of other things (shape, coloration, calls, displays, etc.).

3:101-103: I think you are trying to say that just size is not enough to ascertain sexual dimorphism, but shape is also required, but you don't say that expectedly. I could be wrong, though, in which case, I am not sure of the point being made in this section.

3:110: “Few studies”- Have any studies done this? The citations for this sentence are general GMM references, so I am not sure.

3:112: Geometric morphometrics is in the reverse order (“morphometric geometrics”) here and on 1:19 and 5:175, so be sure to edit those.

3:126: This is a relatively uneven sample size, especially since you have 14 landmarks (= 28 variables). The general rule is n+2 specimens where n= the number of variables in sample, though DFA performs best when specimen n = 4x the number of variables.

5:165-166: Why are only the first two PCs considered?

5:167: Change “show” to “test for”

5:171-172: This paragraph seems out of place in the Methods section and can be moved to the introduction.

5:178: The ANOVA is testing for consistency, not correctness.

5:179: Similarly, the ANOVA is testing for intra-observer error, not to “verify the landmarks digitization”.

5:185-6: What method is used to determine the allometric effect percentage? I don't see it specified in the methods. You also didn’t discuss the allometric slope test in your methods, so be sure to add both of those.

5:189: Similarly, discuss the regression in your methods.

5:193: It is standard practice to present the PCA (PC1 versus PC2), so please add that.

5:194-195: Given your 100% correct reclassification of the specimens into their known sexes, I would expect some separation to be evident in the PCA, since a DFA then minimizes the variation within a group and maximizes the variation between groups. Why do you think the DFA is so successful if the PCA doesn't show any separation?

6:205-206: What is happening to the TPS in the middle region? It almost appears to be folded over itself into three-dimensional space, which shouldn't be happening since this is a 2D analysis. Also, rotate Figure 2 90 degrees counterclockwise.

7:214: Please explain what Frequency means here and is the X-axis the discriminant function score for each individual?

7:214: I am unfamiliar with the term “carapace” usage in bovids but see it in reference to shells. Please define what it means here.

7:220: Why did you include these two citations (31,32) here? There are so many more on sexual dimorphism, including review articles (especially in the 1990s), that I suggest citing one of those, as they are more general.

7:233-243: I suggest replacing the references to non-mammals (since they have selection parameters with that apply less to mammals, such as reproduction physiology and temperature regulation) with mammalian examples, as sexual dimorphism is widely studied using GMM, especially in primates, but also in other artiodactyls.

7:247-248: Fighting may take a lot of energy, but so does pregnancy and lactation. It is likely that energetic requires are similar between males and females, holding body size constant.

8:254-255: This point is not clear. Why would natural selection operate differently on males and females if they are in the same group? Please expand on this or remove natural selection as sexual selection is sufficient to explain the differences we see here.

In the end, you are confirming that there is a form difference in these bovids, and that it is following the expected pattern found in mammals (8:270-274). Since the study is to define uniqueness in the breed in regard to conservation, you need to expand that part of the discussion. In doing so, your article will be more impactful.

Comments on the Quality of English Language

There needs to be some further copy-editing for language clarity. I did some, but stopped at the top of page two.

Author Response

Thank you very much for reviewing our manuscript. We appreciate your valuable complementary comments and suggestions. We have made suggested changes and have revised the manuscript accordingly. We hope that you find our answers satisfactory

Third reviewer’s responses

Comment

This is a relatively straight-forward study investigating sexual dimorphism in Sanmartinero Creole cow head morphology using geometric morphometrics to identify new character information of the breed to preserve its uniqueness.

I provide my editorial comments in a Page:Line number format below:

1:23-24: This is not really the definition of GMM, so please refine definition.

1:25-26: This sentence is repetition of other ideas in the abstract and can be removed.

1:27-28: Unnecessary sentence- remove or be specific.

1:32: Not really ensuring a homogeneous sample but rather eliminating additional variables.

Response. It was corrected in the text

1:32-33: Combine this sentence with the previous sentence for clarification.

1:34: Change to "Sexual dimorphism was found in form..."

Response. It was corrected in the text

1:35-36: Remove this sentence and summarize shape differences in previous sentence.

1: 36: Remove “all these evaluated characters” and just state the two listed characters.

Response It was corrected in the text

2:45: If the cow was introduced, then the breed wasn’t formed through natural selection, but it could have adapted since introduction.

Response. Through natural selection over more than 500 years, it adapted to the region's climatic conditions. It was corrected in the text

2:45: Add “century” after XVth.

Response. It was corrected in the text

2:48: Remove “s” from forages.

Response. Removed

2:51: Provide parenthetical definitions for mesolinean and eumetric.

Response. Added to the text

2:55: These two sentences need verbs. Also, I am going to stop copy-editing here and just focus on scientific suggestions unless a suggestion improves understanding for the reader.

2:60: I am not sure what “stable free representation” means- please expand.

Response. It was corrected in the text

2:60-62: This is not necessarily part of the definition of morphometry, as that may be a factor one is trying to assess using morphometry. Truly, morphometry is just the measurement of shape.

2:65: Traditional morphometry can include more complex measures than distances, such as volume, area, etc.

2:66: I don’t know if efficient is the correct term here, since it is decidedly more time consuming, so one might argue less efficient, though it can capture more subtle variation than traditional measures.

2:69: Do you mean “configuration” rather than conformation?

Response. It was corrected in the text

2:72-73: People have been doing that well before GMM was invented (be careful not to oversell GMM).

2:74: Change “Shape analysis” to GMM, since traditional methods are still shape analysis. Response. It was corrected in the text

2:81-83: This paragraph is about research done but really should provide brief summaries of the results, otherwise it is just repeating previous information.

3:100-101: Sexual dimorphism is far more than just body size differences and can include a variety of other things (shape, coloration, calls, displays, etc.).

Response In the text say: “Significant differences are manifested in body size, color and morphology” (see Ref. 16 and 17)

3:101-103: I think you are trying to say that just size is not enough to ascertain sexual dimorphism, but shape is also required, but you don't say that expectedly. I could be wrong, though, in which case, I am not sure of the point being made in this section.

3:110: “Few studies”- Have any studies done this? The citations for this sentence are general GMM references, so I am not sure.

Response. Few studies on Sanmartinero Creole cattle. Corrected

3:112: Geometric morphometrics is in the reverse order (“morphometric geometrics”) here and on 1:19 and 5:175, so be sure to edit those.

3:126: This is a relatively uneven sample size, especially since you have 14 landmarks (= 28 variables). The general rule is n+2 specimens where n= the number of variables in sample, though DFA performs best when specimen n = 4x the number of variables.

Response. In GM, sample size recommendations generally suggest having at least 15-20 specimens per group to achieve reliable estimates of mean shape and variance. However, the ideal sample size can vary depending on the specific research question, the complexity of the shape variation and, effectively, the number of landmarks used. Out sample was of 42 animals. The complexity of shape variation being studied was considered low. However, the results are relevant to this study in the Sanmartinero Creole bovine.

5:165-166: Why are only the first two PCs considered?

Response. First two PCs explained a 58.89%, and sufficient to explain landmarks which contribute more to the differences between genders.

5:167: Change “show” to “test for”

Response. Corrected

5:171-172: This paragraph seems out of place in the Methods section and can be moved to the introduction.

Response. Deleted

5:178: The ANOVA is testing for consistency, not correctness.

Response. In this case, the test was used to test goodness between replicas.

5:179: Similarly, the ANOVA is testing for intra-observer error, not to “verify the landmarks digitization”.

Response. Corrected

5:185-6: What method is used to determine the allometric effect percentage? I don't see it specified in the methods. You also didn’t discuss the allometric slope test in your methods, so be sure to add both of those.

Response. This represents the proportion of variation for which the regression accounts as a percentage of the total variation. It has been added to the text (ln. 185-187)

5:189: Similarly, discuss the regression in your methods.

Response. A short explanation has been introduced in the methods section (ln. 161-162)

5:193: It is standard practice to present the PCA (PC1 versus PC2), so please add that.

Response. This new figure (figure 2) has been added.

5:194-195: Given your 100% correct reclassification of the specimens into their known sexes, I would expect some separation to be evident in the PCA, since a DFA then minimizes the variation within a group and maximizes the variation between groups. Why do you think the DFA is so successful if the PCA doesn't show any separation?

Response. As it is stated, “PCA does not assume a priori division and concentrates on the relation between variables and individuals of the same sample, reducing the dimension of original data to a new group of variables with axis or orthogonal components” (ln. 199-202)

6:205-206: What is happening to the TPS in the middle region? It almost appears to be folded over itself into three-dimensional space, which shouldn't be happening since this is a 2D analysis. Also, rotate Figure 2 90 degrees counterclockwise.

Response.  Figure has been rotated. Transfprmation grid is merely to better visualize the nature of the geometric changes

7:214: Please explain what Frequency means here and is the X-axis the discriminant function score for each individual?

Response.  Frequencies correspond to number of individuals (duplicated)

7:214: I am unfamiliar with the term “carapace” usage in bovids but see it in reference to shells. Please define what it means here.

Response.  Corrected (an erroneous translation)

7:220: Why did you include these two citations (31,32) here? There are so many more on sexual dimorphism, including review articles (especially in the 1990s), that I suggest citing one of those, as they are more general.

Response. Aggregated

7:233-243: I suggest replacing the references to non-mammals (since they have selection parameters with that apply less to mammals, such as reproduction physiology and temperature regulation) with mammalian examples, as sexual dimorphism is widely studied using GMM, especially in primates, but also in other artiodactyls.

7:247-248: Fighting may take a lot of energy, but so does pregnancy and lactation. It is likely that energetic requires are similar between males and females, holding body size constant.

8:254-255: This point is not clear. Why would natural selection operate differently on males and females if they are in the same group? Please expand on this or remove natural selection as sexual selection is sufficient to explain the differences we see here.

Response. The sentence is supported by a bibliographic reference

In the end, you are confirming that there is a form difference in these bovids, and that it is following the expected pattern found in mammals (8:270-274). Since the study is to define uniqueness in the breed in regard to conservation, you need to expand that part of the discussion. In doing so, your article will be more impactful.

Response. Paragraph has been extended (ln. 298-300)

English corrected

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The study presents a relevant exploration of sexual dimorphism in Sanmartinero Creole cattle using geometric morphometrics, yet it has several limitations. The sample size is relatively small and unevenly distributed between sexes (13 males ,30 females). Additionally, while the study claims to eliminate dietary effects through uniform feeding, other potential confounders such as age, genetic variability, or environmental exposure are not addressed. The methodology lacks detailed justification for the selection of 14 landmarks and does not specify the type of multivariate analyses used, limiting reproducibility. The discussion is descriptive rather than analytical and does not sufficiently compare findings to existing literature on sexual dimorphism. The study also lacks a detailed explanation of potential age-related variation, especially given the broader age range among females (2–4 years), which could influence head morphology due to ongoing growth. Moreover, while the morphometric analysis is technically sound, the absence of visual representations for key results (e.g., PCA plots) limits interpretability. The discussion reiterates existing literature without critically integrating how findings from Sanmartinero cattle compare with other breeds in a meaningful evolutionary or ecological context. Additionally, potential environmental, genetic, or management-related confounders are acknowledged but not systematically explored. Finally, the conclusion highlights the importance of morphometrics for breed conservation, but it stops short of proposing practical applications or future directions for breeding strategies or conservation policies.

Comments on the Quality of English Language

 The English could be improved to more clearly express the research.

Author Response

Thank you very much for reviewing our manuscript. We appreciate your valuable complementary comments and suggestions. We have made suggested changes and have revised the manuscript accordingly. We hope that you find our answers satisfactory

Fourth reviewer’s responses

 Comment

The study presents a relevant exploration of sexual dimorphism in Sanmartinero Creole cattle using geometric morphometrics, yet it has several limitations. The sample size is relatively small and unevenly distributed between sexes (13 males ,30 females). Additionally, while the study claims to eliminate dietary effects through uniform feeding, other potential confounders such as age, genetic variability, or environmental exposure are not addressed. The methodology lacks detailed justification for the selection of 14 landmarks and does not specify the type of multivariate analyses used, limiting reproducibility. The discussion is descriptive rather than analytical and does not sufficiently compare findings to existing literature on sexual dimorphism. The study also lacks a detailed explanation of potential age-related variation, especially given the broader age range among females (2–4 years), which could influence head morphology due to ongoing growth. Moreover, while the morphometric analysis is technically sound, the absence of visual representations for key results (e.g., PCA plots) limits interpretability. The discussion reiterates existing literature without critically integrating how findings from Sanmartinero cattle compare with other breeds in a meaningful evolutionary or ecological context. Additionally, potential environmental, genetic, or management-related confounders are acknowledged but not systematically explored. Finally, the conclusion highlights the importance of morphometrics for breed conservation, but it stops short of proposing practical applications or future directions for breeding strategies or conservation policies.

Response. We appreciate your valuable complementary comments and suggestions

Comparisons with other findings in existing literature are not made, as studies in bovine, and even more so in studies of the head, are limited. References 21, 35, and 36 are included. This is a first study of the shape and size of the head of the Sanmartinero Creole cattle.

English corrected

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors,

After the corrections and justifications, I agree with the new version of the manuscript. Therefore, my opinion is that the research is accepted.

Author Response

Comment

Dear authors,

After the corrections and justifications, I agree with the new version of the manuscript. Therefore, my opinion is that the research is accepted.

Response. We appreciate your valuable complementary comments and suggestions.

Reviewer 3 Report

Comments and Suggestions for Authors

A number of my concerns were addressed, but several were not, so I include those again and a new one regarding the distribution of specimens in PCA space below. I provide my editorial comments in a Page:Line number format below:

1:23-24: This is not really the definition of GMM, so please provide standard definition.

1:25-26: This sentence is repetition of other ideas in the abstract and can be removed.

1:32-33: Combine this sentence with the previous sentence for clarification.

2:55: These two sentences need verbs. Also, I am going to stop copy-editing here and just focus on scientific suggestions unless a suggestion improves understanding for the reader.

(still has the same language, though text is highlighted) 2:60-62: This is not necessarily part of the definition of morphometry, as that may be a factor one is trying to assess using morphometry. Truly, morphometry is just the measurement of shape.

(volume is not a distance) 2:65: Traditional morphometry can include more complex measures than distances, such as volume, area, etc.

2:66: I don’t know if efficient is the correct term here, since it is decidedly more time consuming, so one might argue less efficient, though it can capture more subtle variation than traditional measures.

(text is highlighted but unchanged) 2:72-73: People have been doing that well before GMM was invented (be careful not to oversell GMM).

3:101-103: I think you are trying to say that just size is not enough to ascertain sexual dimorphism, but shape is also required, but you don't say that expectedly. I could be wrong, though, in which case, I am not sure of the point being made in this section.

3:110: “Few studies”- Have any studies done this? The citations for this sentence are general GMM references, so I am not sure. (You can say “no studies” if none have been done)

3:126: This is a relatively uneven sample size, especially since you have 14 landmarks (= 28 variables). The general rule is n+2 specimens where n= the number of variables in sample, though DFA performs best when specimen n = 4x the number of variables. Regarding the authors’ response: sample size can impact the results regardless of research question. Considering some of the issues I bring up below, do you think the sample size could be an issue? If so, I suggest acknowledging that in the discussion. This happens frequently especially in paleontological studies where samples size tend to be sample, but it should be mentioned.

5:165-166: Why are only the first two PCs considered? Regarding the authors’ response: That then leaves 41% of the shape variance out of the interpretation. That also raises the question: What data did you submit to the DFA? Was it all landmark coordinates or the PC scores for the first two PCs?

5:178: The ANOVA is testing for consistency, not correctness. Regarding the authors’ response: The goodness of fit between replicas is still testing for consistency in digitization, not correctness.

New comment: There is something odd going on in these data. What does the distribution on PC3 look like? What is going on with that outlier (labeled H)? If that is removed, then I predict the new PC 1 would go right through both of these clusters, but the clusters include males and females. I think the authors do need to spend more time thinking and then writing about what is happening in this PCA. I understand that PCA has no a priori assumption about group membership, but I admit that I was surprised to see so little distinction between the sexes, yet the presence of two clusters. What do you think is happening?

5:194-195: Given your 100% correct reclassification of the specimens into their known sexes, I would expect some separation to be evident in the PCA, since a DFA then minimizes the variation within a group and maximizes the variation between groups. Why do you think the DFA is so successful if the PCA doesn't show any separation?

6:205-206: What is happening to the TPS in the middle region? It almost appears to be folded over itself into three-dimensional space, which shouldn't be happening since this is a 2D analysis. Regarding the authors’ response: Indeed- the visualization is a tool to help understand the results. That is why I ask about the oddness of that region- what do you think that result tells you (and the reader)?

Figure 2: please make landmark numbers larger

7:214: Please explain what Frequency means here and is the X-axis the discriminant function score for each individual? Regarding the authors’ response: Can you expand on that method more in your Methods section?

7:233-243: I suggest replacing the references to non-mammals (since they have selection parameters with that apply less to mammals, such as reproduction physiology and temperature regulation) with mammalian examples, as sexual dimorphism is widely studied using GMM, especially in primates, but also in other artiodactyls.

7:247-248: Fighting may take a lot of energy, but so does pregnancy and lactation. It is likely that energetic requires are similar between males and females, holding body size constant.

8:254-255: This point is not clear. Why would natural selection operate differently on males and females if they are in the same group? Please expand on this or remove natural selection as sexual selection is sufficient to explain the differences we see here. Regarding the authors response: Since it caught my attention, it will certainly catch other readers’ attention. Please provide a brief summary/expansion.

Author Response

Third reviewer’s responses Round 2

Response. We appreciate your valuable complementary comments and suggestions; But we do not agree with some comments. In round 1 it was answered and corrected.

We request the editor to judge the convenience of publishing the manuscript.

Comment

A number of my concerns were addressed, but several were not, so I include those again and a new one regarding the distribution of specimens in PCA space below. I provide my editorial comments in a Page:Line number format below:

1:23-24: This is not really the definition of GMM, so please provide standard definition.

Response. In round 1 it was corrected

1:25-26: This sentence is repetition of other ideas in the abstract and can be removed.

Response. In round 1 it was corrected

1:32-33: Combine this sentence with the previous sentence for clarification.

Response. In round 1 it was corrected

2:55: These two sentences need verbs. Also, I am going to stop copy-editing here and just focus on scientific suggestions unless a suggestion improves understanding for the reader.

Response. In round 1 it was corrected

(still has the same language, though text is highlighted) 2:60-62: This is not necessarily part of the definition of morphometry, as that may be a factor one is trying to assess using morphometry. Truly, morphometry is just the measurement of shape.

Response. In round 1 it was corrected. Also, it has a reference that supports the sentence.

(volume is not a distance) 2:65: Traditional morphometry can include more complex measures than distances, such as volume, area, etc.

Response. In round 1 it was corrected

2:66: I don’t know if efficient is the correct term here, since it is decidedly more time consuming, so one might argue less efficient, though it can capture more subtle variation than traditional measures.

Response. In round 1 it was corrected. Efficient refers to the ability to analyze shape and size simultaneously. This phrase is supported by reference 4 and 5.

(text is highlighted but unchanged) 2:72-73: People have been doing that well before GMM was invented (be careful not to oversell GMM).

Response. In round 1 it was corrected

3:101-103: I think you are trying to say that just size is not enough to ascertain sexual dimorphism, but shape is also required, but you don't say that expectedly. I could be wrong, though, in which case, I am not sure of the point being made in this section.

Response. In round 1 it was corrected.

3:110: “Few studies”- Have any studies done this? The citations for this sentence are general GMM references, so I am not sure. (You can say “no studies” if none have been done)

Response. In round 1 it was corrected. In the text it says: “in Sanmartinero Creole bovine. Corrected

3:126: This is a relatively uneven sample size, especially since you have 14 landmarks (= 28 variables). The general rule is n+2 specimens where n= the number of variables in sample, though DFA performs best when specimen n = 4x the number of variables. Regarding the authors’ response: sample size can impact the results regardless of research question. Considering some of the issues I bring up below, do you think the sample size could be an issue? If so, I suggest acknowledging that in the discussion. This happens frequently especially in paleontological studies where samples size tend to be sample, but it should be mentioned.

Response: In round 1 it appears the response. In GM, sample size recommendations generally suggest having at least 15-20 specimens per group to achieve reliable estimates of mean shape and variance. However, the ideal sample size can vary depending on the specific research question, the complexity of the shape variation and, effectively, the number of landmarks used. Our sample was of 42 animals. We know that the complexity of shape variation being studied was considered low. However, the results are relevant to this study in the Sanmartinero Creole bovine. In any case, a new paragraph has been added (lines 136-140).

5:165-166: Why are only the first two PCs considered? Regarding the authors’ response: That then leaves 41% of the shape variance out of the interpretation. That also raises the question: What data did you submit to the DFA? Was it all landmark coordinates or the PC scores for the first two PCs?

Response: In round 1 the answer was. First two PCs explained a 58.89%, and sufficient to explain landmarks which contribute more to the differences between genders.

5:178: The ANOVA is testing for consistency, not correctness. Regarding the authors’ response: The goodness of fit between replicas is still testing for consistency in digitization, not correctness.

Response: In round 1 the answer was. “In this case, the test was used to test goodness between replicas

New comment: There is something odd going on in these data. What does the distribution on PC3 look like? What is going on with that outlier (labeled H)? If that is removed, then I predict the new PC 1 would go right through both of these clusters, but the clusters include males and females. I think the authors do need to spend more time thinking and then writing about what is happening in this PCA. I understand that PCA has no a priori assumption about group membership, but I admit that I was surprised to see so little distinction between the sexes, yet the presence of two clusters. What do you think is happening?

Response: “H” and “M” were acronyms for females and males, respectively. These letters have been removed from the new figure. On the other hand, as it is stated (ln. 199-200), the first two components explained a 58.89% of the total variation, so the information offered by PC3 seems irrelevant. Moreover, the lack of plotted differentiation between groups in PC1/PC2 space does not mean that the groups are undifferentiated, as, PCA does not assume a priori division and concentrates on the relation between variables and individuals of the same sample (lines 201-205).

5:194-195: Given your 100% correct reclassification of the specimens into their known sexes, I would expect some separation to be evident in the PCA, since a DFA then minimizes the variation within a group and maximizes the variation between groups. Why do you think the DFA is so successful if the PCA doesn't show any separation?

Response: In round 1 the answer was. “As it is stated, “PCA does not assume a priori division and concentrates on the relation between variables and individuals of the same sample, reducing the dimension of original data to a new group of variables with axis or orthogonal components” (ln. 199-202)

6:205-206: What is happening to the TPS in the middle region? It almost appears to be folded over itself into three-dimensional space, which shouldn't be happening since this is a 2D analysis. Regarding the authors’ response: Indeed- the visualization is a tool to help understand the results. That is why I ask about the oddness of that region- what do you think that result tells you (and the reader)?

Response: In round 1 the answer was. Figure has been rotated. Transformation grid is merely to better visualize the nature of the geometric changes

Figure 2: please make landmark numbers larger

Response: the numbers in figure 3 are difficult to be modified, as they are directly generated with the software. In any case, the discussion includes clearly these anatomical reference points, which are more informative when interpreted on their bony support

7:214: Please explain what Frequency means here and is the X-axis the discriminant function score for each individual? Regarding the authors’ response: Can you expand on that method more in your Methods section?

Response. In round 1 there is the response. Frequencies correspond to number of individuals (duplicated). In any case, a new figure with no X-axis values (which are irrelevant for the interpretation) has been added.

7:233-243: I suggest replacing the references to non-mammals (since they have selection parameters with that apply less to mammals, such as reproduction physiology and temperature regulation) with mammalian examples, as sexual dimorphism is widely studied using GMM, especially in primates, but also in other artiodactyls.

Response: In round 1 the answer was. The sentence is supported by a bibliographic reference

7:247-248: Fighting may take a lot of energy, but so does pregnancy and lactation. It is likely that energetic requires are similar between males and females, holding body size constant.

Response: interesting question, but it seems irrelevant on head, which is supported mainly on a bony basis and muscle masses are mainly those related to masticatory function

8:254-255: This point is not clear. Why would natural selection operate differently on males and females if they are in the same group? Please expand on this or remove natural selection as sexual selection is sufficient to explain the differences we see here. Regarding the authors response: Since it caught my attention, it will certainly catch other readers’ attention. Please provide a brief summary/expansion.

Response. They are from the same group, in this case Sanmartinero Creole bovine, but different sexes. The sentence is supported by a bibliographic reference (see ref. 37,38).

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The sample size especially for males was relatively small. The article needs clearer language and more explanation of the practical implications for breeders and conservation efforts. Still, the study provide useful data for understanding morphological traits

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Fourth reviewer’s responses Round 2

 Comment

The sample size especially for males was relatively small. The article needs clearer language and more explanation of the practical implications for breeders and conservation efforts. Still, the study provide useful data for understanding morphological traits

Response. We appreciate your valuable complementary comments and suggestions

English corrected