Genetic Evidence Reveals Unexpected Diversity and Genetic Exchange Between White-Fringed Weevils (Coleoptera, Curculionidae)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

L80-85 change stylistically, “The aim” and “our goal” is the same, so my suggestion is to raise two main objectives of the study the first one “elucidate the taxonomic assignment” and the second one to infer phylogenetic relationships, delineate species boundaries and investigate possible interspecific gene flow.

The table 1 should appear immediately after its mention in the text, not after another page.

In Table 1

L131-142 Table footer should be organised in different format, please check other published DIVERSITY articles and template file.

In Table 1 “sp ” have to be changed to “sp. [normal style, not italic]”…” Acc. No.* ” this is not explained in table footer

L95 “Of particular interest were three male individuals”  change this not very clear English expression

L99-100 from chemical point of view 100% ethanol is practically impossible. I suggest to leave “…absolute ethanol and stored..”

L88, L101 and … please check MDPI 2.2 Heading2 format, ant DIVESRITY template, now it is incorrect

L107 here ITS1 should be in italic

L109-110 in ethanol

L116 for easy access and replication of the study, provide the primer sequences in this article as well, it doesn't take up much space

L124 “Service of Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires” translate into English

L122-127 please check formatting

L197-198 isn’t GTR+G?

L278 for the sentence it seems that N. leucoloma and N. minor females shared some of their haplotypes. However, it is not how many of them were shared by both species, please include this information.

L280-281 “In others, we observed superimposed peaks in the electropherograms (recorded as NA in Table 1),” so how many specimens and how many sites?

L296-320 too long footer, truncate to at most 8-10 lines, incorporating the most important information. The main information should be clear from the figure; such long explanations are not needed.

L292-293 improve English expression for clarity

L328-340 too long figure caption, concentrate on the most important information

Authors provided two Figures 2 in pages 10 and 11, leave one.

Figure 3C it could be presented in one graph mt DNA above diagonal and ntDNA below diagonal, or vice versa

L348-349 “likely indicating putative outcrossing events between the taxonomic units included in this analysis” move to Discussion

From results I do not understand how authors delineated species boundaries. If this objective set at the end of introduction was achieved, it should be clearly highlighted in the Results.

Results: to my deep understanding authors should clearly explain why the results obtained are not enough for the description of a new species, and data should be accumulated for the description of the new Naupactus species

L389-L423 to my opinion morphological results are presented in complicated manner, my suggestion is to include a separate new Table, presenting morphological diagnosis for species; important taxonomic morphological features and how are they presented in species examined.

L435 [41-46]?

3.1.-3.4. titles are incomplete, please change. For example, it is not clear “Morphological aspects” of what.

L430-L512 please divide Discussion into 2-3 sections (4.1., 4.2., 4.3 ?) the discussion is now not clearly structurally coherent; also avoid short one-two sentence paragraphs as in L446-448, combine to other or expand.

L514 how to understand “these”? maybe it could be rephrased

Conclusion: 1) “Otiorhynchus scaber” appears only in Conclusions, this is one of the critical general mistakes to include new results, considerations that have not been provided in Results and or Discussion.  I see two alternatives, exclude Otiorhynchus scaber or add some information on it in Discussion. 2) Conclusions is too long, please emphasise the most important points rather than debating 3) one of the general mistakes is to include citations in conclusions, if so this is not conclusions, but discussion 4) in summary, conclusions are not appropriate, please re-write.

L560-573 there is  no need for such long and complex figure caption, please concentrate text.

L582 “Naupactus ” have to be in italic

References: titles of articles are presented using either Sentence or Capitalized Each Word formats; it should be unified.

 

 

 

 

 

 

 

Author Response

Comment 1: L80-85 change stylistically, “The aim” and “our goal” is the same, so my suggestion is to raise two main objectives of the study the first one “elucidate the taxonomic assignment” and the second one to infer phylogenetic relationships, delineate species boundaries and investigate possible interspecific gene flow.

Authors’ response 1: We rephrased this sentence to make it simpler.

 

Comment 2: The table 1 should appear immediately after its mention in the text, not after another page.

Authors’ response 2: We agree with Reviewer #1. We solved this problem by moving the figure to the place indicated by Reviewer #1.

 

Comment 3: L131-142 Table footer should be organised in different format, please check other published DIVERSITY articles and template file.

Authors’ response 3: We modified the table footer in agreement with the MDPI Layout Style Guide (section 7.2).

 

Comment 4: In Table 1 “sp ” have to be changed to “sp. [normal style, not italic]”…” Acc. No.* ” this is not explained in table footer.

Authors’ response 4: We added the dot to “sp” and changed to the normal style and explained the Acc. No.  abbreviation in the table footer (GenBank Accession Numbers).

 

Comment 5: L95 “Of particular interest were three male individuals”  change this not very clear English expression

Authors’ response 5: We revised this sentence to improve clarity and academic tone.

 

Comment 6: L99-100 from chemical point of view 100% ethanol is practically impossible. I suggest to leave “…absolute ethanol and stored.”

Authors’ response 6: We deleted (100%).

 

Comment 7: L88, L101 and … please check MDPI 2.2 Heading2 format, ant DIVESRITY template, now it is incorrect.

Authors’ response 7: We checked the heading format against the Diversity template and confirmed that it complies with the current guidelines.

 

Comment 8: L107 here ITS1 should be in italic

Authors’ response 8: Done.

 

Comment 9: L109-110 in ethanol

Authors’ response 9: We replaced “100%” by “absolute”.

 

Comment 10: L116 for easy access and replication of the study, provide the primer sequences in this article as well, it doesn't take up much space

Authors’ response 10: We provided primer sequences for both DNA regions.

 

Comment 11: L124 “Service of Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires” translate into English

Authors’ response 11: Done.

 

Comment 12: L122-127 please check formatting

Authors’ response 12: We removed the bold formatting and adjusted the font size accordingly.

 

Comment 13: L197-198 isn’t GTR+G?

Authors’ response 13: Reviewer #1 is right. We replaced “GAMMA” by “+G”.

 

Comment 14: L278 for the sentence it seems that N. leucoloma and N. minor females shared some of their haplotypes. However, it is not how many of them were shared by both species, please include this information.

Authors’ response 14: The original sentence was unclear. Therefore, we first stated how many different haplotypes were found in the sample, and then specified how many haplotypes each species had. As a result, no confusion about shared haplotypes should arise.

 

Comment 15:  L280-281 “In others, we observed superimposed peaks in the electropherograms (recorded as NA in Table 1),” so how many specimens and how many sites?

Authors’ response 15: By “superimposed electropherograms,” we referred to almost the entire sequence, not just a few nucleotides. We clarified this point in the manuscript. Moreover, all individuals from each location showed superimposed peaks, as no other genotype was reported in Table 1.

 

Comment 16: L296-320 too long footer, truncate to at most 8-10 lines, incorporating the most important information. The main information should be clear from the figure; such long explanations are not needed.

Authors’ response 16: Done. We removed superfluous information about each cluster, as it is already described in the main body of the manuscript.

 

Comment 17: L292-293 improve English expression for clarity

Authors’ response 17: We noticed that the sentence had a few grammatical mistakes and corrected them accordingly.

 

Comment 18: L328-340 too long figure caption, concentrate on the most important information

Authors’ response 18: Done. We removed superfluous information about each cluster, as it is already described in the main body of the manuscript.

 

Comment 19: Authors provided two Figures 2 in pages 10 and 11, leave one.
Authors’ response 19: We deleted one of the figures.

 

Comment 20: Figure 3C it could be presented in one graph mtDNA above diagonal and ntDNA below diagonal, or vice versa

Authors’ response 20: We modified this figure accordingly to the Reviewer # 1’s suggestion.

 

Comment 21: L348-349 “likely indicating putative outcrossing events between the taxonomic units included in this analysis” move to Discussion

Authors’ response 21: Done.

 

Comment 22: Results: to my deep understanding authors should clearly explain why the results obtained are not enough for the description of a new species, and data should be accumulated for the description of the new Naupactus species

Authors’ response 22: The morphological characters of the few male specimens we had are not conclusive to distinguish it from males of Naupactus leucoloma. In addition, because most of the species from the N. leucoloma species group reproduces by parthenogenesis and males are currently absent, it would be essential to have females available in order to designate a type specimen — which is a formal requirement in taxonomy. Unfortunately, this is not the case.

Comment 23: L389-L423 to my opinion morphological results are presented in complicated manner, my suggestion is to include a separate new Table, presenting morphological diagnosis for species; important taxonomic morphological features and how are they presented in species examined.

Authors’ response 23: We included a table highlighting the key diagnostic characters used to distinguish the species under study.

 

Comment 24: 3.1.-3.4. titles are incomplete, please change. For example, it is not clear “Morphological aspects” of what.

Authors’ response 24: We changed the title of section 3.4 from “Morphological aspects” to “Morphological evaluation.” However, we believe that the title of section 3.1, “Phylogenetic analyses,” accurately reflects the content of that subsection; therefore, we have retained it.

 

Comment 25: L435 [41-46]?

Authors’ response 25: Reviewer #1 is right. We included only the first and the last references from the whole list.

 

Comment 26: L430-L512 please divide Discussion into 2-3 sections (4.1., 4.2., 4.3 ?) the discussion is now not clearly structurally coherent; also avoid short one-two sentence paragraphs as in L446-448, combine to other or expand.

Authors’ response 26: We have rewritten the Discussion to follow the order of the analyses presented in the Results section and divided it into three subsections.

 

Comment 27: L514 how to understand “these”? maybe it could be rephrased

Authors’ response 27 We have rewritten the entire section, so this ambiguity has most likely been resolved.

 

Comment 28: Conclusion: 1) “Otiorhynchus scaber” appears only in Conclusions, this is one of the critical general mistakes to include new results, considerations that have not been provided in Results and or Discussion.  I see two alternatives, exclude Otiorhynchus scaber or add some information on it in Discussion. 2) Conclusions is too long, please emphasise the most important points rather than debating 3) one of the general mistakes is to include citations in conclusions, if so this is not conclusions, but discussion 4) in summary, conclusions are not appropriate, please re-write.

Authors’ response 28: In agreement with Reviewer #1, and also following Reviewer #3's suggestion, we moved the comparison with O. scaber to the Discussion section. Additionally, we revised the conclusion to better reflect the key results of our investigation.

 

Comment 29: L560-573 there is  no need for such long and complex figure caption, please concentrate text.

Authors’ response 29: We agree with Reviewer #1’s suggestion and have summarized the content of this figure caption.

 

Comment 30: L582 “Naupactus ” have to be in italic

Authors’ response 30: Done.

 

Comment 31: References: titles of articles are presented using either Sentence or Capitalized Each Word formats; it should be unified.

Authors’ response 31: Done

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is well-written, and the research is correctly presented.

The Introduction includes all the relevant information, thence to further information are required.

The research design is correctly described, and the analyses allowed to define the species delimitation within this very troublesome taxonomic group.

The results are clearly presented, all the figures and tables are useful to furnish a better understanding of these species.

The discussion and Conclusions are supported by the results.

All the relevant references are included.

 

Author Response

No criticisms were raised by Reviewer #2.

Reviewer 3 Report

Comments and Suggestions for Authors

This study used mitochondrial and nuclear genetic markers to analyze the phylogenetic relationships of several white-fringed weevils, but the existing evidence is insufficient to support hybridization or new species hypotheses.

Some other issues are as follows:

1. Introduction: The introduction section lacks sufficient introduction to the phylogenetic relationships, taxonomic status, and evolutionary history of these white-fringed beetles.
2. Materials and Methods: The existing analysis is insufficient, and it is recommended to use Structure or Admixture for genetic structure analysis to verify whether it is the independent lineage. You can refer to: https://doi.org/10.1111/jse.13044.
3. Materials and Methods: The existing analysis is insufficient, and it is recommended to use D-statistics (ABBA-BABA) and heterozygous site analysis to clearly distinguish the possibility of hybridization from incomplete lineage sorting (ILS). You can refer to: https://doi.org/10.1038/s41467-022-29643-4.
4. Section 3.1: This study did not find any shared haplotypes, so how can this study support the hybridization hypothesis?
5. Lines 342-343, 348-349: The PCA based on the SNP dataset placed the undetermined males in the middle position does not confirm the conclusion of hybridization or new species.
6. Figure 3: The specific contribution rate of PCA axis needs to be added to Figure A, and the legend of Figure B is unclear.
7. Section 3.4: Simple morphological description is not qualified and requires morphological measurement and statistics.
8. Discussion: The discussion section did not fully address the key conflicts between morphological and molecular data, and cannot support the hypothesis of new species or hybridization. It is not clear whether these undefined males are independently heritable lineages, and the interference of introgression and incomplete lineage sorting cannot be ruled out, which does not support the key conclusions in the discussion.
9. Conclusion: The conclusion is unqualified and does not explain the key results and conclusions of this study, nor does it clearly state the title of this study.

Author Response

Comment 1: Introduction: The introduction section lacks sufficient introduction to the phylogenetic relationships, taxonomic status, and evolutionary history of these white-fringed beetles.

Authors’ response 1: We respectfully disagree with Reviewer #3, since all what is known about taxonomic status, and evolutionary history of these white-fringed beetles was explained in the Introduction section of our manuscript. However, we enlarged the description of these weevils about the phylogenetic relationships. .

 

Comment 2: Materials and Methods: The existing analysis is insufficient, and it is recommended to use Structure or Admixture for genetic structure analysis to verify whether it is the independent lineage. You can refer to: https://doi.org/10.1111/jse.13044.

Authors’ response 2: We intentionally avoided using programs such as STRUCTURE or ADMIXTURE to infer the number of panmictic units or admixture events, as their inferences rely on the maximization of allele frequencies under the assumption of Hardy–Weinberg equilibrium. Since our dataset includes three parthenogenetic species, it was impossible to meet the assumptions required by these software packages.

 

Comment 3: Materials and Methods: The existing analysis is insufficient, and it is recommended to use D-statistics (ABBA-BABA) and heterozygous site analysis to clearly distinguish the possibility of hybridization from incomplete lineage sorting (ILS). You can refer to: https://doi.org/10.1038/s41467-022-29643-4

Authors’ response 3: Following Reviewer #3’s suggestion, we included the ABBA-BABA test, but were unable to accomplish the heterozygous site analysis (because of the nature of our dataset). Instead, we included the triangle plot analyses.

 

Comment 4: Section 3.1: This study did not find any shared haplotypes, so how can this study support the hybridization hypothesis?

Authors’ response 4: Indeed, evidence of hybridization or introgression was detected in the SNP dataset, whereas the mitochondrial data did not reveal such signals. We therefore explored potential explanations for these patterns, including hybridization, introgression, and incomplete lineage sorting.

 

Comment 5: Lines 342-343, 348-349: The PCA based on the SNP dataset placed the undetermined males in the middle position does not confirm the conclusion of hybridization or new species.

Authors’ response 5: We agree with Reviewer #3's comment and have accordingly tempered our conclusions.

 

Comment 6: Figure 3: The specific contribution rate of PCA axis needs to be added to Figure A, and the legend of Figure B is unclear.

Authors’ response 6: Done.

 

Comment 7: Section 3.4: Simple morphological description is not qualified and requires morphological measurement and statistics.

Authors’ response 7: We included a table highlighting the key diagnostic characters used to distinguish the species under study and the corresponding measurements of the quantitative characters.

 

Comment 8: Discussion: The discussion section did not fully address the key conflicts between morphological and molecular data, and cannot support the hypothesis of new species or hybridization. It is not clear whether these undefined males are independently heritable lineages, and the interference of introgression and incomplete lineage sorting cannot be ruled out, which does not support the key conclusions in the discussion.

Authors’ response 8: We included additional analyses that support the occurrence of introgression and help rule out hybridization as a likely explanation. Furthermore, we considered the possibility of incomplete lineage sorting. Accordingly, we have revised the Discussion section to reflect these findings.

 

Comment 9: Conclusion: The conclusion is unqualified and does not explain the key results and conclusions of this study, nor does it clearly state the title of this study.

Authors’ response 9: In agreement with Reviewer #3, and also following Reviewer #1's suggestion, we moved the comparison with O. scaber to the Discussion section. Additionally, we revised the conclusion to better reflect the key results of our investigation.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

L191-192 please delete sentence, it does not belong to Introduction.

 

Table 1 and 1631-1632 data availability statement: why ITS1 sequences are still pending? Usually, to gain GenBank acc. nos. less time is needed. Furthermore, ITS1 could be more easily and rapidly deposited in GenBank in comparison to some other sequences. Have you some problems with verification of sequences?

 

“From results I do not understand how authors delineated species boundaries. If this objective set at the end of introduction was achieved, it should be clearly highlighted in the Results.” I'm sorry, but I didn't find an answer to my question that I had raised in the first round of the review. Would you be kind to reply to it.

 

3.4. “Morphological Evaluation”  2.7. “2.7. Morphological Analysis”  “2.5. Network and Genetic Structure Analyses“ I provided correct variant, please also change 2.1., 2.2., 2.3., 2.4., 4.1., 4.2.,4.3.

 

L1509 “see Husemann et al. [55].”, however in reference list Husemann et al is 58 (L1912-L1913). Also, please provide more details on Husemann et al. paper, what was found similar compared to present study

 

L1517 please delete “[9]”

 

“Comment 31: References: titles of articles are presented using either Sentence or Capitalized Each Word formats; it should be unified.

Authors’ response 31: Done” I am sorry, but you have not corrected mistakes in reference list.

Author Response

Comment 1: “L191-192 please delete sentence, it does not belong to Introduction.”
Authors’ response 1: Done.

Comment 2: “Table 1 and 1631-1632 data availability statement: why ITS1 sequences are still pending? Usually, to gain GenBank acc. nos. less time is needed. Furthermore, ITS1 could be more easily and rapidly deposited in GenBank in comparison to some other sequences. Have you some problems with verification of sequences?”
Authors’ response 2: Unfortunately, the submission of both COI and ITS1 sequences to GenBank was unintentionally delayed. Although we received the COI accession numbers in time to include them in the revised version of the manuscript, the ITS1 accession numbers were issued only a few hours after the submission. We are now able to include them in the second round of revision.

Comment 3: “From results I do not understand how authors delineated species boundaries. If this objective set at the end of introduction was achieved, it should be clearly highlighted in the Results.” I'm sorry, but I didn't find an answer to my question that I had raised in the first round of the review. Would you be kind to reply to it.”
Authors’ response 3: Indeed, we were unable to delineate species boundaries. We added a summary of the main results at the end of the corresponding section of the manuscript (“In short, the combination of molecular and morphological evidence prevented us from assigning the UM to any of the parthenogenetic species within the N. leucoloma species group. Morphologically, the UM exhibits intermediate characteristics among the three species, though with a closer resemblance to N. leucoloma, while the molecular data suggest that it may represent an independently evolving lineage. However, based on the currently available data, we are not in a position to describe it as a new species.”). Accordingly, we have addressed this issue at the outset of the Discussion section.
 
Comment 4: “3.4. “Morphological Evaluation”  2.7. “2.7. Morphological Analysis”  “2.5. Network and Genetic Structure Analyses“ I provided correct variant, please also change 2.1., 2.2., 2.3., 2.4., 4.1., 4.2.,4.3.”
Authors’ response 4: We unified the titles of the 'Materials and Methods' subsections with those of the corresponding subsections in the Results section.
 
Comment 5: “L1509 “see Husemann et al. [55].”, however in reference list Husemann et al is 58 (L1912-L1913). Also, please provide more details on Husemann et al. paper, what was found similar compared to present study”
Authors’ response 5: We corrected several citation errors in the Discussion section and expanded our discussion of the work by Husemann et al. (2013), which serves as an example of the discovery of high levels of genetic variability in a grasshopper species from South America.
 
Comment 6: “L1517 please delete “[9]””
Authors’ response 6: Done.
 
Comment 7: “Comment 31: References: titles of articles are presented using either Sentence or Capitalized Each Word formats; it should be unified. Authors’ response 31: Done” I am sorry, but you have not corrected mistakes in reference list.”
Authors’ response 7: Thanks for the observation. We have now carefully revised again the reference list multiple times independently and did not find any titles formatted in Capitalized Each Word style.

 

Reviewer 3 Report

Comments and Suggestions for Authors

This study used mitochondrial and nuclear genetic markers to analyze the phylogenetic relationships of several white-fringed weevils. However, this study cannot support the conclusion that undetermined males are new species or independent evolutionary lineages.

Some other issues are as follows:

1. Title: The content of the title is too empty and does not correspond to the content of the manuscript, such as insufficient research on evolutionary history.
2. Introduction: The clarification of taxonomic assignment proposed in the research objectives has not been completed.
3. Introduction: The explanation of the evolutionary process or history proposed in the title and research objectives has not been completed. It is recommended to use genomic data for population history analysis.
4. Section 2.1: The geographical distribution map of these samples is necessary.
5. The “likely indicating putative outcrossing events between the taxonomic units included in this analysis” in Section 3.2: It seems that these conclusions cannot be drawn solely from these analyses.
6. The “However, the inferred sister-group relationships are different between datasets, revealing some degree of conflict” in Section 4.1: The discussion section should explain the main conflicts and possible reasons.
7. Section 4.2: It seems that there is no explanation on how to exclude hybridization.
8. The “On the other hand, morphological analysis suggested that the UM might represent a race within N. leucoloma, a conclusion that clearly conflicts with the genetic evidence, which supports the UM as an independently evolving lineage and sister species to N. minor” in Section 4.2: This study did not explicitly state whether undetermined males are a race within N. leucoloma or an independently evolving lineage. This study only obtained a few undefined males and cannot confirm whether they can reproduce on their own and have reproductive isolation from other species. It seems that it cannot confirm whether they are new species or independent evolutionary lineages.
9. Conclusion: The conclusion is unqualified and does not elaborate on the corresponding conclusions of the research objectives proposed in this study.
10. Conclusion: The conclusion section should elaborate on the findings of this study and should not include citations.

Author Response

Comment 1: “Title: The content of the title is too empty and does not correspond to the content of the manuscript, such as insufficient research on evolutionary history.”

Authors’ response 1: We appreciate the reviewer’s observation regarding the title. Indeed, 'evolutionary history' is a broad and somewhat vague term. However, revealing unexpected levels of genetic variability and divergence, along with signatures of historical introgression among a group of species, is precisely a way of addressing their evolutionary history and uncovering how complex it might have been. Ours is not merely a systematic study; rather, we investigated the genetic structure of a group of closely related species and the genomic traces left by their past interactions, an integral part of their shared evolutionary history. However, we acknowledge that the title can be more descriptive of the main findings of the manuscript.  We replaced the former title by “Genetic evidence reveals unexpected diversity and genetic exchange between white-fringed weevils (Coleoptera, Curculionidae).”

Comment 2: “Introduction: The clarification of taxonomic assignment proposed in the research objectives has not been completed.”

Authors’ response 2: We thank Reviewer #3 for this observation, with which we agree. Unfortunately, we were not able to assign the males to any of the identified species within the N. leucoloma species group. In the current version, we state this at the end of the Results section, as a synthesis of all the findings and accordingly discussed it in the appropriate section of the manuscript. However, in response to this comment, we have clarified this point in the Introduction to more accurately state the scope of our objectives. Introduction (lines 185-186). From “Therefore, further molecular analyses are still needed to clarify the taxonomic identity of these males” to “Accordingly, to clarify whether these males correspond to one of the already described species or constitute an independently evolving lineage within the N. leucoloma species group, additional analyses integrating morphological and molecular data will need to be performed.” Even though we could not assign these specimens to any of the named species, we believe our results are valuable and worth communicating to the scientific community, as they reveal high levels of genetic diversity in a recently evolved group with a complex evolutionary history.

 

Comment 3: “Introduction: The explanation of the evolutionary process or history proposed in the title and research objectives has not been completed. It is recommended to use genomic data for population history analysis.”

Authors’ response 3: Indeed, we used genomic data obtained through a complexity reduction technique, namely the ddRAD-seq approach. This allowed us to retrieve several thousand markers broadly distributed across the genome. Moreover, our study focused on species delimitation, genetic structure, and signatures of gene flow within the N. leucoloma species group, using integrative molecular approaches. While we did not perform explicit demographic or historical reconstruction analyses (e.g., demographic modeling or divergence time estimation), it is important to highlight that the species delimitation analyses we conducted using the genomic resequencing data are based on coalescent theory, which incorporates population-level parameters (such as effective population size) to determine species boundaries. In this context, we have performed not only phylogenetic diversification analyses, but also population genomic studies aimed at examining the boundaries between populations and species—an essential approach when dealing with recent diversification processes. Thus, we revealed signals consistent with historical introgression and complex reproductive dynamics. These findings, supported by the ABBA-BABA test and triangular plot analyses, provided insights into the evolutionary processes affecting these weevils.

Comment 4: “Section 2.1: The geographical distribution map of these samples is necessary.”

Authors’ response 4: We included a map with the geographical distribution the samples as supplementary material.

 Comment 5: The “likely indicating putative outcrossing events between the taxonomic units included in this analysis” in Section 3.2: It seems that these conclusions cannot be drawn solely from these analyses.”

Authors’ response 5: We thank the reviewer for this observation. We agree that the phrase may have overstated the evidence derived from the neighbor-net network analyses. Accordingly, we have revised the whole subsection 4.2.

Comment 6: “The “However, the inferred sister-group relationships are different between datasets, revealing some degree of conflict” in Section 4.1: The discussion section should explain the main conflicts and possible reasons.”

Authors’ response 6: We thank the reviewer for this suggestion. Conflicts between datasets in the phylogenetic analyses were thoroughly discussed. Given the greater number of informative characters in the ddRAD-seq dataset, we favored the hypothesis supported by this dataset. Regarding the conflicts evidenced by the neighbor-net network, an entire subsection of the Discussion (subsection 4.2 “Causes of conflict”) was devoted to this issue. In the current version, we also included recombination and homoplasy as possible sources of conflict. Based on this analysis, we ruled out recombination, homoplasy, incomplete lineage sorting, and hybridization in favor of historical introgression. We also adapted the title to reflect the newly considered causes (homoplasy and recombination).

 

Comment 7: “Section 4.2: It seems that there is no explanation on how to exclude hybridization.”

Authors’ response 7: We appreciate this reviewer’s comment. In fact, a test specifically designed to detect hybridization yielded negative results, and this was discussed in the manuscript. However, we expanded this argument slightly to ensure it does not go unnoticed by the reader. In line 1195, we have clarified how we evaluated this possibility.

 

Comment 8: “The “On the other hand, morphological analysis suggested that the UM might represent a race within N. leucoloma, a conclusion that clearly conflicts with the genetic evidence, which supports the UM as an independently evolving lineage and sister species to N. minor” in Section 4.2: This study did not explicitly state whether undetermined males are a race within N. leucoloma or an independently evolving lineage. This study only obtained a few undefined males and cannot confirm whether they can reproduce on their own and have reproductive isolation from other species. It seems that it cannot confirm whether they are new species or independent evolutionary lineages.”

Authors’ response 8: Both sources of characters are in conflict. But, as we clearly explained in the Discussion subsection 4.2, the conflict might be attributed to historical introgression, which might account for the morphological resemblance between the UM and N. leucoloma (being these two entities independently evolving lineages that exchanged genetic information). As to the contribution of this investigation, indeed, Reviewer #1 is correct. We found a few males that were initially assigned to N. leucoloma or N. minor based on morphology. Surprisingly, barcoding using COI, as well as the ITS1 sequence, revealed that these males actually belong to a highly divergent lineage. We did not merely uncover unexpected genetic variation within this species group—as the title suggests—but rather made a finding of critical importance. Our results provide further evidence that the gallery forests of southern South America are hotspots of biodiversity, as highlighted at the end of the Discussion section in the current version of the manuscript.

 

Comment 9: “Conclusion: The conclusion is unqualified and does not elaborate on the corresponding conclusions of the research objectives proposed in this study. Conclusion: The conclusion section should elaborate on the findings of this study and should not include citations.”

Authors’ response 9: We have rewritten the conclusion as to elaborate on the findings of this study

 

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

Relevant and well performed study, I wish that authors could describe new species in the analysed group of animals.

 

L182-184 Change the end of the sentence “will need to be performed” to “are needed”

L184, L1000 N. leucoloma has to be in italic

Authors agreed that they were not able to delineate species boundaries, so please delete it from L67

Once again, sorry but your style is not according to MDPI requirements, 2.1. not “Sample collection” but “Sample Collection”, 2.2. not “Amplification and sequencing of mitochondrial and nuclear markers” but “Amplification and Sequencing of Mitochondrial and Nuclear Markers”, etc.

Comment 4: “3.4. “Morphological Evaluation” 2.7. “2.7. Morphological Analysis” “2.5. Network and Genetic Structure Analyses“ I provided correct variant, please also change 2.1., 2.2., 2.3., 2.4., 4.1., 4.2.,4.3.”
Authors’ response 4: We unified the titles of the 'Materials and Methods' subsections with those of the corresponding subsections in the Results section. 

 

Author Response

Comments #1: “L182-184 Change the end of the sentence “will need to be performed” to “are needed”.”

Authors’ response 1: Done

 

Comments #2: “L184, L1000 N. leucoloma has to be in italic.”

Authors’ response 2: Done

 

Comments #3: “Authors agreed that they were not able to delineate species boundaries, so please delete it from L67.”

Authors’ response 3: We corrected the sentence accordingly to Reviewer #1’s suggestion.

 

Comments #4: “Once again, sorry but your style is not according to MDPI requirements, 2.1. not “Sample collection” but “Sample Collection”, 2.2. not “Amplification and sequencing of mitochondrial and nuclear markers” but “Amplification and Sequencing of Mitochondrial and Nuclear Markers”, etc.”

“Comment 4: “3.4. “Morphological Evaluation” 2.7. “2.7. Morphological Analysis” “2.5. Network and Genetic Structure Analyses“ I provided correct variant, please also change 2.1., 2.2., 2.3., 2.4., 4.1., 4.2.,4.3.”
Authors’ response 4: We unified the titles of the 'Materials and Methods' subsections with those of the corresponding subsections in the Results section.”

Authors’ response 4: Thank you for your observation. We acknowledge that we misunderstood your previous comment. The revised version now conforms to MDPI formatting requirements.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have clarified most of the issues in the manuscript, but there are still a few minor issues that need further correction.

1. The abstract lacks the key results and conclusions of this study.
2. The “Our goal was to assign these unidentified males to a species, delineate species boundaries, and explore the possibility of interspecific gene flow” and “These findings uncover a complex evolutionary history within this group of Neotropical weevils” in Abstract, and “Accordingly, to clarify whether these males correspond to one of the already described species or constitute an independently evolving lineage within the N. leucoloma species group, additional analyses integrating morphological and molecular data will need to be performed. The aim of the present study is to elucidate the taxonomic assignment of these white-fringed male weevils through phylogenetic inference and species delimitation methods based on multiple datasets—including mitochondrial and nuclear DNA sequences, as well as genome-wide SNPs—and by analyzing the distribution of genetic variation among these entities as well as interspecific gene flow” in Introduction: Although this study aims to address these issues, it has never been able to clarify them. It is recommended to change the research objectives to match the research content of this study. The classification status issue that needs to be clarified can be discussed at the end for future prospects.
3. The purpose of this study is to elucidate the taxonomic assignment of these white-fringed male weevils, however, this study did not achieve this goal. It is suggested to add the shortcomings of this study and future research directions at the end of the discussion.

Author Response

Comments #1: “The abstract lacks the key results and conclusions of this study.”

Authors’ response 1: We stressed that the males conform and independently evolving lineage.

 

Comments #2: “The “Our goal was to assign these unidentified males to a species, delineate species boundaries, and explore the possibility of interspecific gene flow” and “These findings uncover a complex evolutionary history within this group of Neotropical weevils” in Abstract, and “Accordingly, to clarify whether these males correspond to one of the already described species or constitute an independently evolving lineage within the N. leucoloma species group, additional analyses integrating morphological and molecular data will need to be performed. The aim of the present study is to elucidate the taxonomic assignment of these white-fringed male weevils through phylogenetic inference and species delimitation methods based on multiple datasets—including mitochondrial and nuclear DNA sequences, as well as genome-wide SNPs—and by analyzing the distribution of genetic variation among these entities as well as interspecific gene flow” in Introduction: Although this study aims to address these issues, it has never been able to clarify them. It is recommended to change the research objectives to match the research content of this study. The classification status issue that needs to be clarified can be discussed at the end for future prospects.”

Authors’ response 2: Regarding the sentence: “Accordingly, to clarify whether these males correspond to one of the already described species or constitute an independently evolving lineage within the N. leucoloma species group, additional analyses integrating morphological and molecular data will need to be performed” we believe this objective has been fulfilled. Based on our molecular datasets, we determined that these males represent an independently evolving lineage, with evidence of interspecific gene flow. This likely explains the morphological ambiguity that hinders their taxonomic assignment. Accordingly, we have retained the sentence, although we have revised the paragraph discussing the taxonomic placement of this group, which remains unresolved.

 

Comments #3: “The purpose of this study is to elucidate the taxonomic assignment of these white-fringed male weevils, however, this study did not achieve this goal. It is suggested to add the shortcomings of this study and future research directions at the end of the discussion.”

Authors’ response 3: Lines 786–788 already address both the current shortcomings and future research directions needed to clarify the taxonomy of the group under study. Since in Revision Round 1 Reviewer #1 suggested splitting the Discussion into subsections, we believe this issue is more appropriately placed at the end of Subsection 1 rather than at the end of the entire Discussion.