DNA Metabarcoding Reveals Seasonal Variations in Crop-Foraging Behavior of Wild Rhesus Macaques (Macaca mulatta)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors present an interesting study, surely worth publishing. That said, there are several major issues to fix before acceptance:

1. In the introduction (around line 76), missing more details on how DNA metabarcoding is currently used to answer similar questions. The literature used is very limited (only 28 references) considering the topic of investigation (well studied, although the authors try to use a novel approach, or at least on a novel species, not clear).
2. The sampling method is not so clear. Yes you took probably independent samples, but maybe from the same group (if the threshold is just 1 m), so with similar feeding habits. Did you check if some of the samples that were nearby were related in terms of diet? From table S1 is it clear that many samples were taken from nearby spots. So maybe need to somehow average them (if very similar, meaning they come from the same cohesive group), otherwise the analyses will be biased
3. In results and discussion, some results are threated as significant while they are not, just need to say that the differences were not significant, not saying one is higher but not significant, simply they were not different. 
4. More emphasis throughout key parts of the paper (title, abstract, conclusion) should be placed on the novelty of the method (but still need to introduce why it is novel). 

 

Other minor points:

- Line 136 Macaca

- Line 143, PERMANOVA is listed as method but not presented

- Fig 1A is not visible, need to make it bigger

- Fig 3B From the graph it is not so clear the separation, just in winter the niche is larger. Also PCoA seems to perform very bad (only 47% explained by the 2 axes, so I suggest to try NMDS as it is more flexible).

- Line 294. The link with the SDGs is not well explained

Author Response

Comments 1: [In the introduction (around line 76), missing more details on how DNA metabarcoding is currently used to answer similar questions. The literature used is very limited (only 28 references) considering the topic of investigation (well studied, although the authors try to use a novel approach, or at least on a novel species, not clear)]

Response 1: [Thank you for pointing this out. We agree with this comment. Therefore, we have revised the introduction to more comprehensively demonstrate the application of DNA metabarcoding in similar studies. We have supplemented the introduction with relevant content that highlights the advantages of this technology and its latest advancements in the study of wildlife diets. These changes can be found on page 2, lines 71-91 of the revised manuscript. Additionally, to enhance the academic and referential value of the study, we have further expanded the list of references.]

 

Comments 2: [The sampling method is not so clear. Yes you took probably independent samples, but maybe from the same group (if the threshold is just 1 m), so with similar feeding habits. Did you check if some of the samples that were nearby were related in terms of diet? From table S1 is it clear that many samples were taken from nearby spots. So maybe need to somehow average them (if very similar, meaning they come from the same cohesive group), otherwise the analyses will be biased]

Response 2: [Thank you for your valuable comments. We have revised the description of the sampling methods, as we recognize that the original description was not clear enough. Before formal sampling, we conducted a detailed preliminary survey of the macaque population distribution in the study area. Based on the results of this survey, we systematically sampled multiple wild macaque populations across various sites in Qiannan Buyi and Miao Autonomous Prefecture, Guizhou Province, ensuring the diversity and representativeness of our samples by covering different independent populations. For each identified macaque population, we collected multiple samples to maximize their representativeness. The "1-meter sampling distance" mentioned in the text refers to the spatial interval between repeated samples collected within each population, not the separation distance between different populations. In fact, the distance between different populations is much greater than 1 meter, ensuring their ecological and social independence. These revisions can be found on page 3, lines 106-120 of the revised manuscript.]

 

Comments 3: [In results and discussion, some results are threated as significant while they are not, just need to say that the differences were not significant, not saying one is higher but not significant, simply they were not different.]

Response 3: Thank you for pointing this out. We agree with this comment. Therefore, We have revised the Results and Discussion sections to accurately reflect that the differences were not significant, without implying any directionality. This ensures our conclusions are based on the actual statistical outcomes. These changes can be found on pages 7, 8, and 9, lines 246-336 of the revised manuscript.]

 

Comments 4: [More emphasis throughout key parts of the paper (title, abstract, conclusion) should be placed on the novelty of the method (but still need to introduce why it is novel).]

Response 4: [Thank you for pointing this out. We agree with this comment. Therefore, We have now placed greater emphasis on the novelty of the method throughout key parts of the paper (title, abstract, conclusion) and briefly introduced why it is novel. These changes can be found on pages 1 and 9 of the revised manuscript.]

 

Comments 5: [Line 136 Macaca]

Response 5 [Thank you for pointing this out. We agree with this comment. Therefore, We have revised this part of the content as suggested. This change can be found on page 4, lines 155-156 of the revised manuscript.]

 

Comments 6: [Line 143, PERMANOVA is listed as method but not presented]

Response 6: [Thank you for pointing this out. We agree with this comment. As I later changed the analysis method, this part has been removed. This change can be found on page 4, lines 143-165 of the revised manuscript.]

 

Comments 7: [Fig 1A is not visible, need to make it bigger]

Response 7: [Thank you for pointing this out. We agree with this comment. Therefore, We have adjusted the size of Fig 1A to make it more visible and clear in the manuscript. This change can be found on page 5 of the revised manuscript.]

 

Comments 8: [Fig 3B From the graph it is not so clear the separation, just in winter the niche is larger. Also PCoA seems to perform very bad (only 47% explained by the 2 axes, so I suggest to try NMDS as it is more flexible).]

Response 8: [Thank you for pointing this out. We agree with this comment. Therefore, We have replaced the PCoA plot with an NMDS plot in Figure 3B. The NMDS plot provides a clearer visualization of the community structure and better represents the differences between the samples. These changes can be found on pages 4, 6, and 7 of the revised manuscript.]

 

Comments 9: [Line 294. The link with the SDGs is not well explained]

Response 9: [Thank you for pointing this out. We agree with this comment. Therefore, We have supplemented the connection with the Sustainable Development Goals (SDGs) in the discussion section as suggested, to more clearly demonstrate the relevance and significance of our research. This change can be found on page 9, lines 319-325 of the revised manuscript.]

Reviewer 2 Report

Comments and Suggestions for Authors

The authors presented the diet of rhesus macaques in China through fecal analyses. The findings are important, suggesting certain trends based on which hypotheses could be confirmed. Furthermore, these findings have significant management implications for the welfare of both the macaque and humans.

The research design, field and laboratory work, and analyses were appropriate. Results and discussion have been presented in a clear and informative manner, making them accessible to the reader. Some amendments would further improve the manuscript.

Comments

Lines 159, 161 (Fig. 1): Define the OUT acronym in both the text and the figure.

Line 193: Does Fig. 2A report diet composition for both seasons? Please clarify in the figure caption.

Line 194: Separate spring-winter pie rings in Fig. 2B, probably with a different color line, for visibility purposes.

Lines 197-204: There is a 95% probability that the two sample means come from populations with the same mean. So, you cannot talk about differences here. Just refer to the absence of differences and avoid higher-lower means comparisons.

Lines 211-212: I cannot see a “clear separation trend”. I rather see a big mixing. I would suggest trying a cluster analysis. Perhaps spring, winter, and spring-winter groups could be formed, allowing for some meaningful interpretations.

Lines 212-214: This is a discussion. Remove.

Lines 227-232, 268, 273: Do not give results in the discussion. Remove percentages.

Lines 264-267: Refer to the need for a larger sample for more robust conclusions as a limitation.

Author Response

Comments 1: [Lines 159, 161 (Fig. 1): Define the OUT acronym in both the text and the figure.]

Response 1: [Thank you for pointing this out. We agree with this comment. Therefore, we have defined the OUT acronym in both the text and the figure. This change can be found on pages 4 and 5, lines 177-181 of the revised manuscript.]

 

Comments 2: [Line 193: Does Fig. 2A report diet composition for both seasons? Please clarify in the figure caption.]

Response 2: [Thank you for pointing this out. We agree with this comment. Therefore, we have clarified in the figure caption that Fig. 2A reports diet composition for both seasons. This change can be found on page 6, line 214 of the revised manuscript.]

 

Comments 3: [Line 194: Separate spring-winter pie rings in Fig. 2B, probably with a different color line, for visibility purposes.]

Response 3: [Thank you for pointing this out. We have revised Fig. 2B to separate the spring and winter pie rings using different colored lines to enhance visibility. This change can be found on page 6 of the revised manuscript.]

 

Comments 4: [Lines 197-204: There is a 95% probability that the two sample means come from populations with the same mean. So, you cannot talk about differences here. Just refer to the absence of differences and avoid higher-lower means comparisons.]

Response 4: [Thank you for pointing this out. We agree with this comment. As I later changed the analysis method, this part has been removed. This change can be found on page 6, lines 218-236 of the revised manuscript.]

 

Comments 5: [Lines 211-212: I cannot see a “clear separation trend”. I rather see a big mixing. I would suggest trying a cluster analysis. Perhaps spring, winter, and spring-winter groups could be formed, allowing for some meaningful interpretations.]

Response 5: [Thank you for pointing this out. We agree with this comment. Therefore, we have conducted a cluster analysis, which has led to more meaningful interpretations. This change can be found on page 6, lines 217-235 of the revised manuscript.]

 

Comments 6: [Lines 212-214: This is a discussion. Remove.]

Response 6: [Thank you for pointing this out. We agree with this comment. Therefore, we have removed the discussion from lines 212-214.]

 

Comments 7: [Lines 227-232, 268, 273: Do not give results in the discussion. Remove percentages.]

Response 7: [Thank you for pointing this out. We agree with this comment. Therefore, we have removed this content from the discussion section.]

 

Comments 8: [Lines 264-267: Refer to the need for a larger sample for more robust conclusions as a limitation.]

Response 8: [Thank you for pointing this out. We agree with this comment. Therefore, we have added this content to the discussion. This change can be found on page 8, lines 290-292 of the revised manuscript.]

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors considered all my comments and the paper is now clearer and improved in significance and impact. I have no further suggestions