Genetic Introgression and Morphological Variation in Naked-Back Bats (Chiroptera: Mormoopidae: Pteronotus Species) along Their Contact Zone in Central America

Round 1

Reviewer 1 Report

Dear Authors,

Studies investigating hybridization at the contact zone for closely related species are pivotal for increasing our knowledge of biological diversity and evolutionary process. Your work is well done and provides another useful case study in that regard. Thank you for doing this work.

The methods are largely valid and the manuscript is well written for the most part (especially the Discussion). I have few substantive comments.

That said, I believe that further clarification of the Methods is required in places, and the the manuscript requires more polishing in places, before it is acceptable for publication. Moreover, I have substantive concern about the validity of your morphological analyses. I note these concerns and comments in detail below.

Best of luck in revising your manuscript.

Comments by Line:

Line 3: Suggest removing “(Chiroptera: Mormoopidae)” and replacing with “(Pteronotus spp.)”. Also, do not hyphen “Central” in the title.

Line 24: Delete “Morphological variation and genetic relationships between” as well as “are evaluated in this study. These species”

Line 29: place “markers” after “mitochondrial”, not “DNA”

Line 30: Replace “the” with “these”

Line 33: Replace “The genetic study” with “We”

Line 35: delete “in this study”

Line 38: Change to “(Mexico to Costa Rica)”

Line 40: Round to 3.0%

Line 46: Replace “to caution” with “for caution” and delete “the” before “forearm”

Line 49: Delete “Mexico” given the study is not specific to that country alone.

Lines 54-90: These paragraphs are very well written and informative; however, they are very general and not specific to the study. As such, they could be deleted to reduce what is a long paper. Just a suggestion. No harm in keeping them.

Lines 98-99. Insert “Mormoopidae” after “family” in the second sentence and you can delete the first sentence.

Line 101: Delete “to this day”

Line 119: While FA = 48.3 is cause for concern for overlap, FA = 46.1 or 46.5 does not.

Line 137: Okay, but based on Table 1, bats were from other countries as well. Its not clear to me if the authors captured all these bats (and those in Table 1) or they relied on data or collections from others as well (i.e., museum specimens). Please clarify.

Table 1. Can Table 1 and Table S1 be combined to remove this long table from the manuscript?

Line 141: What was measured, beyond FA length. Please be specific here.

Line 142: Replace “puncher” with “punch”. Was it 2 mm? Specify please.

Figure 1. Great maps. Most helpful. Well done.

Line 143: How many as vouchers? Please be specific.

Line 147: Where permits for collection obtained from each country?

Table 1: For Be, the location is called “Gallon Jug” not “Gallon Jung”, I believe.

Line 182: why only n = 199 when earlier n = 247 (Line 136)?

Lines 184-188: What specifically did you test? FA length? If so, that is incorrect because you already binned them by FA length, so they are going to be different. Please clarify. Were these binned based on post-hoc based on genetic assignment or by FA length before molecular analyses?

Line 187 (and elsewhere in text): Delete “, using a significance level p = 0.05” – it is unnecessary.

Line 194: I worry about different measurements being taken by different observers being a problem here. How did you control or test for this?

Line 218: Provide English common name here too please.

Line 259: It is not necessary to name all of the source countries and the population codes already provided here. Delete one or the other please.

Lines 305-337 (including Figure 2): There is a big problem here. You cannot bin these species and the “intermediate” group by forearm length then test for difference in FA length! Unless I am missing something, this is not a valid analyses and must be deleted, if that is the case. You can only do this if binned based on genetic assignments, which I did not read as the case in the Methods.

Figure 3: The caption legend is insufficient to “stand alone”. Please provide more detail so that the reader can understand this figure from the caption alone.

Line 384: Replace “Northern” with “northern” – same with “Southern” on Line 390 (and anywhere else in the text).

Line 498: The Discussion is very well written. Good work.

Line 541: Delete the first “the” in this line.

Line 642: There is an extra space in “A. M.R.”

Author Response

Dear Editor,

Please find attached the new versión of our MS entitled: “Genetic introgression and morphological variation in naked-back bats (Chiroptera: Mormoopidae) along their contact zone in Central America”. In this new version, most of the comments and suggestions of the two referees have been included and we want to acknowledge their work since they have clearly improved the final paper.

Find attached responses to the comments together with the revised version of the MS, that we hope now will be acceptable for publication.

On behalf of all the authors,

REVIEWER 1  

Line 3: Suggest removing “(Chiroptera: Mormoopidae)” and replacing with “(Pteronotus spp.)”. Also, do not hyphen “Central” in the title.

 Response 1: We do not agree with the suggestion and prefer keeping the original title of the MS as it is since it put the studied species clearly in their systematic frame, which it can be more useful for readers non-familiar with the American bat fauna

Line 24: Delete “Morphological variation and genetic relationships between” as well as “are evaluated in this study. These species”

 Response 2: done

Line 29: place “markers” after “mitochondrial”, not “DNA”

 Response 3: done

Line 30: Replace “the” with “these”

 Response 4: done

Line 33: Replace “The genetic study” with “We”

 Response 5: done

Line 35: delete “in this study”

 Response 6: done

Line 38: Change to “(Mexico to Costa Rica)”

 Response 7: done

Line 40: Round to 3.0%

 Response 8: done

Line 46: Replace “to caution” with “for caution” and delete “the” before “forearm”

 Response 9: done

Line 49: Delete “Mexico” given the study is not specific to that country alone.

 Response 10: done

Lines 54-90: These paragraphs are very well written and informative; however, they are very general and not specific to the study. As such, they could be deleted to reduce what is a long paper. Just a suggestion. No harm in keeping them.

 Response 11: We appreciate the comment but still, we think that is useful to have these general paragraphs as a kind of introduction to frame the study

Lines 98-99. Insert “Mormoopidae” after “family” in the second sentence and you can delete the first sentence.

 Response 12: Again, and for the same reason that in response 1, we consider important to mention the distribution of the family Mormoopidae in America

Line 101: Delete “to this day”

 Response 13: done

Line 119: While FA = 48.3 is cause for concern for overlap, FA = 46.1 or 46.5 does not.

 Response 14: These three individuals are important to mention because they were considered as a new subspecies. Moreover, these three length values are unusually large in relation to the locality where they were found.

Line 137: Okay, but based on Table 1, bats were from other countries as well. Its not clear to me if the authors captured all these bats (and those in Table 1) or they relied on data or collections from others as well (i.e., museum specimens). Please clarify.

Response 15: For this study we have carried out the samples collection in Mexico, Costa Rica, Nicaragua and Brazil. Besides, we have used sequences downloaded from GeneBank. We have modified the text in sake of clarity.

Table 1. Can Table 1 and Table S1 be combined to remove this long table from the manuscript?

 Response 16: Thanks for the suggestion but the fact is that we prefer to leave them separately since Table 1 allows a quick location of the sampling, while Table S1 gives more information about each of the samples such as Gene Bank accession number, the amplified fragment and the citation.

Line 141: What was measured, beyond FA length. Please be specific here.

 Response 17: done. We have better specified the handling of the bats,

Line 142: Replace “puncher” with “punch”. Was it 2 mm? Specify please.

 Response 18: done

Figure 1. Great maps. Most helpful. Well done.

 Response 19:  We appreciate very much the recognition of the work done!

Line 143: How many as vouchers? Please be specific.

Response 20: 177 vochers: 30 of P. gymnonotus and 77 of P. fulvus.

Line 147: Where permits for collection obtained from each country?

 Response 21: Yes and this information is now provided in Mat and Methods.

Table 1: For Be, the location is called “Gallon Jug” not “Gallon Jung”, I believe.

 Response 22: done

Line 182: why only n = 199 when earlier n = 247 (Line 136)?

 Response 23: Only individuals that we captured in Mexico were considered since, they were the only one to present enough sample size for each locality.

Lines 184-188: What specifically did you test? FA length? If so, that is incorrect because you already binned them by FA length, so they are going to be different. Please clarify. Were these binned based on post-hoc based on genetic assignment or by FA length before molecular analyses?

 Response 24: The fact is that we were not testing the significance of the differences in FA length between the two species, but to which species could be considered closer the individuals with intermediate FA. But we understand the risk of a circular reasoning and have deleted this analysis.

Line 187 (and elsewhere in text): Delete “, using a significance level p = 0.05” – it is unnecessary.

 Response 25: done

Line 194: I worry about different measurements being taken by different observers being a problem here. How did you control or test for this?

 Response 26: Unfortunately, This is a potential risk although FA length is a quite robust and straightforward measurement. Still, we preferred to add all the available information to increase the sample size and cover the whole distribution area. We have added a cautionary comment in the discussion on this regards.

Line 218: Provide English common name here too please.

 Response 27: done

Line 259: It is not necessary to name all of the source countries and the population codes already provided here. Delete one or the other please.

 Response 28: done

Lines 305-337 (including Figure 2): There is a big problem here. You cannot bin these species and the “intermediate” group by forearm length then test for difference in FA length! Unless I am missing something, this is not a valid analyses and must be deleted, if that is the case. You can only do this if binned based on genetic assignments, which I did not read as the case in the Methods.

 Response 29: Following the referee’s comment we have left out this comment (but see Response 24) but we leave Fig.2 which is just showing the difference in size between groups.

Figure 3: The caption legend is insufficient to “stand alone”. Please provide more detail so that the reader can understand this figure from the caption alone.

 Response 30: Done. We have added some information and a call to the text for more details about the analysis.

Line 384: Replace “Northern” with “northern” – same with “Southern” on Line 390 (and anywhere else in the text).

 Response 31: done

Line 498: The Discussion is very well written. Good work.

 Response 32: We appreciate very much the referee’s comment.

Line 541: Delete the first “the” in this line.

 Response 33: done

Line 642: There is an extra space in “A. M.R.”

Response 34: done

Reviewer 2 Report

The manuscript is well written and I only have some minor requirements before acceptance.

Line 66. This is true, but it has also brought to a clumping of some species into one: see Mori E., Nerva L., Lovari S. (2019). Reclassification of the serows and the gorals: the end of a neverending story? Mammal Review 49: 256-262.

Lines 91-97. Dr. Andrea Galimberti has proposed some genetic differences to disentangle amongst different species. Please, consider this papers: Galimberti, A., Spada, M., Russo, D., Mucedda, M., Agnelli, P., Crottini, A. et al. (2012). Integrated operational taxonomic units (IOTUs) in echolocating bats: A bridge between molecular and traditional taxonomy. PLoS ONE, 7(6), e40122. https://doi.org/10.1371/journ al.pone.0040122

Lines 128-134. Authors should add predictions after the aims.

Methods are clear and replicable.

Figure 3. Please, avoid the abbreviations “pma” and “pqu” or clarify what do they mean in the figure caption.

Discussion. I suggest the author to add some comments on conservation issues, given that bats are often threatened of extinction and different species may require different efforts. What about hybrids?

Author Response

Dear Editor,

Please find attached the new version of our MS entitled: “Genetic introgression and morphological variation in naked-back bats (Chiroptera: Mormoopidae) along their contact zone in Central America”. In this new version, most of the comments and suggestions of the two referees have been included and we want to acknowledge their work since they have clearly improved the final paper.

Find attached responses to the comments together with the revised version of the MS, that we hope now will be acceptable for publication.

On behalf of all the authors,

Aline

REVIEWER 2

Line 66. This is true, but it has also brought to a clumping of some species into one: see Mori E., Nerva L., Lovari S. (2019). Reclassification of the serows and the gorals: the end of a neverending story? Mammal Review 49: 256-262.

Response 1: Done. We have added this other possible clumping effect.

Lines 91-97. Dr. Andrea Galimberti has proposed some genetic differences to disentangle amongst different species. Please, consider this papers: Galimberti, A., Spada, M., Russo, D., Mucedda, M., Agnelli, P., Crottini, A. et al. (2012). Integrated operational taxonomic units (IOTUs) in echolocating bats: A bridge between molecular and traditional taxonomy. PLoS ONE, 7(6), e40122. https://doi.org/10.1371/journ al.pone.0040122

Response 2: We consider that the concept of Integrated Operational Taxonomic Unit (IOTU), are not the most suitable for this study but we have made reference to its use.

Lines 128-134. Authors should add predictions after the aims.

 Response 3: We prefer to leave this final paragraph as it is, indicating the main goals of the paper. The two possible explanations (environmental and genetic) to the intermediate forms are already described after the background introduction.

Methods are clear and replicable.

Response 4: We appreciate the referee’s comment.

Figure 3. Please, avoid the abbreviations “pma” and “pqu” or clarify what do they mean in the figure caption.

 Response 5: done

Discussion. I suggest the author to add some comments on conservation issues, given that bats are often threatened of extinction and different species may require different efforts. What about hybrids?

Response 6: done. We have added a final comment on conservation aspects.