Phylogeography and Past Distribution of Peripheral Individuals of Large Hairy Armadillo Chaetophractus villosus

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In general, this is a well-written paper on an interesting topic. There are some copyediting issues, most notably with the formatting of the references (e.g., no Latin names in article titles are italicized), that I assume the guest editor will correct. The biggest concern is the sample size, both the number of animals sampled and the number of mitochondrial nucleotides sequenced. The authors address both of these in the Discussion and explain why the sample sizes were low. However, they do not really discuss how the small sample sizes might create problems with regard to interpreting their results. Some mention of this would be helpful. Another example of small sample size is the use of just the data from the present study plus those of Poljak et al. (2010) to do the Maxent modeling. I think every record of occurrence for the species would be desirable and make the analysis more comprehensive and informative.

 Comments keyed to line numbers:

16: “relationship” is a vague term. It would be better to state explicitly what is being studied.

19: State how many peripheral populations were sampled.

27: Is the British spelling of center (centre) acceptable? The rest of the text seems to use American English.

58: Should it be western instead of eastern? The entire paragraph is about the species’ distribution in Chile, which would be western Patagonia I think.

69-72: Mention of D. novemcinctus should be deleted. The recent taxonomic revision by Barthe et al. (2024, Systematic Biology) split novemcinctus into four species, so the biogeographical information being presented is no longer accurate.

91: Again, “relationship” is a vague term.

105: “the consequences” instead of “its consequences”.

113: What kind of carcass tissue? It would be useful to state how many live and dead animals were sampled (or perhaps add this information to Table 1).

116: Replace “it was” with “samples were”.

Figure 1: I am not convinced the right panel is necessary. If the left panel was enlarged a bit, I think it would suffice. Also, in the figure caption, delete “natural”. Does the IUCN need a citation?

Table 1: It should be stated that these are the 22 individual locations for each animal sampled in the study. I had to count the number of rows to figure this out.

134: Not clear what is meant by “different degrees of success” or how this affected the ability to sequence the mtDNA.

165-169: More information is needed here, e.g., “four strings” of what? Likewise, I am unclear why seasonality needs to be evaluated and what information is gained by doing so.

172: “outgroup” instead of “external group”?

177-180: If negative values indicate expansion and positive values represent a constant size over time, how does one identify population contraction?

182 (and 167): What is the rationale for the extremely high number of generations used in the models? Based on the fossil evidence reported on line 187, the time frame of the analyses is only 3.6 Ma, so basing the models on 3-10x as many generations does not seem realistic.

201-202: Why not use all available records instead of just the ones from this study and Poljak et al.? Also, need to explain why the sample size for Poljak et al. is given as 41 here but 76 on line 150. I assume this is because multiple animals were sampled at the same site but this was also the case in Chile and yet individual coordinates were provided for each animal. Was this not possible for the Argentine data?

As a final comment, I am not sure it is necessary here, but in most species occurrence analyses I have seen, random points are sampled to identify the environmental conditions at locations where the species is not found, and these are then compared against the conditions where the species is found. Also, it is common to “train” the model with a subset of the location points and then evaluate the accuracy with which the model predicts occurrence at the remaining locations. Was there a reason for not doing these things here?

215: I assume that HC stands for haplotype C. If so, it seems unnecessary to include the H because it creates redundancy (i.e., saying “the HC haplotype” basically means the “haplotype C haplotype”).

Table 2: Text under the table needs the following corrections: (1) Chilean population; and (2) polymorphic.

Figure 2: The figure legend needs to explain why Hap E has a box around it in panel (a). Also, vellerosus is misspelled in panel (b). Should this Latin name be italicized in both panels?

Figure 3: Should be “obtained from” not “obtained with”. Also, some explanation needs to be provided of what the curves presented at the bottom right of the figure represent.

251: Should be “Skyline”.

260: “farther” not “further”

Figure 4: Italicize species name in legend. Also, should it be “Skyline”? Finally, it should be “mtDNA”.

280-283: This is an awkward sentence that needs to be rewritten. Maybe delete “sequences of”?

298: “that reported”, not “the reported”

296-299: Break into two sentences.

299-301: This sentence is awkward and needs to be rewritten.

307: [45, 61]?

313-318: As mentioned previously, delete discussion of D. novemcinctus because of the taxonomic revision published by Barthe et al. The only relevant paper might be Arteaga et al. (2020, or maybe 2012) but I do not recall if they compared genetic diversity of what is now called the Mexican long-nosed armadillo (D. mexicanus) in the U.S. versus Mexico. Any comparisons between populations farther south than Costa Rica are irrelevant because they involve other species.

323: Make clear that “its” refers to C. villosus.

332: Insert “be” before “derived.

333: “found” instead of “distributed”.

338: “sequence” instead of “alignment”? (see also line 341).

351: “study” not “studied”

408: Patagonia, not Patagonian

410: shown, not indicated

413: delete “floor”?

416: delete “of”

423: development does not seem like the right word here. Also, insert “a” before “consequence”.

408-426: I am not clear what point the authors wish to make by presenting all this information about historical changes to the steppe-forest ecotone strip. Presumably, changes to this ecotone have affected where C. villosus was found but there is no supporting evidence to substantiate this. While plausible, this discussion seems to be largely conjectural, so I am not sure it is needed.

435: distribution does not seem like the right word here.

438: receptors does not seem like the right word here.

439-441: These sentences seem contradictory. If peripheral populations have a high likelihood of extinction, how can they also be stable and have high survival?

442-443: Delete the last sentence.

Author Response

Reviewer 1: In general, this is a well-written paper on an interesting topic. There are some copyediting issues, most notably with the formatting of the references (e.g., no Latin names in article titles are italicized), that I assume the guest editor will correct. The biggest concern is the sample size, both the number of animals sampled and the number of mitochondrial nucleotides sequenced. The authors address both of these in the Discussion and explain why the sample sizes were low. However, they do not really discuss how the small sample sizes might create problems with regard to interpreting their results. Some mention of this would be helpful. Another example of small sample size is the use of just the data from the present study plus those of Poljak et al. (2010) to do the Maxent modeling. I think every record of occurrence for the species would be desirable and make the analysis more comprehensive and informative.

 Comments keyed to line numbers:

16: “relationship” is a vague term. It would be better to state explicitly what is being studied.

Reply: we modified as follows (lines 16-17): We studied the evolutionary history of peripheral individuals of C. villosus using phylogeographic approaches and potential distribution models for the Holocene.

19: State how many peripheral populations were sampled.

Reply: This section was modified to indicate that these are individuals of peripheral distribution, in order to provide greater clarity. The number of populations was not identified prior to our genetic analysis. The results indicate that all samples correspond to one population within their surveyed area (lines 19-20).

27: Is the British spelling of center (centre) acceptable? The rest of the text seems to use American English.

Reply: it was fixed as american english.

58: Should it be western instead of eastern? The entire paragraph is about the species’ distribution in Chile, which would be western Patagonia I think.

Reply: it was fixed as reviewer suggested.

69-72: Mention of D. novemcinctus should be deleted. The recent taxonomic revision by Barthe et al. (2024, Systematic Biology) split novemcinctus into four species, so the biogeographical information being presented is no longer accurate.

Reply: The article by Barthe et al. (2024, Systematic Biology) was not utilized during the drafting of the first version of the manuscript, and the information pertaining to D. novemcinctus was not updated. Consequently, we removed the Dasypus novemcinctus citation from the Introduction and Discussion.

91: Again, “relationship” is a vague term.

Reply: we modified as follows (lines 83-84): To reconstruct the evolutionary history of individuals with peripheral distribution of C. villosus from Chile, we combined phylogeographical inference and species distribution modelling.

 

105: “the consequences” instead of “its consequences”.

Reply: it was fixed.

113: What kind of carcass tissue? It would be useful to state how many live and dead animals were sampled (or perhaps add this information to Table 1).

Reply: It was included in the man manuscript as follows (lines 111-117): Live individuals (n=2) were captured using five Tomahawk traps, installed for five days at each collection site. Live individuals were anesthetized with 25 mg/kg ketamine hydrochloride, taking blood samples from the ventral tail vein, and were preserved in 96% ethanol. The captured individuals were released at the same location of capture. The capture permit was authorized by Servicio Agrícola y Ganadero (SAG-Chile, authorization N° 2048/2012 and 6956/2013). We also collected muscle tissue (limbs) from carcass individuals (n=20) found on roads and near burrows.

116: Replace “it was” with “samples were”.

Reply: Fixed.

Figure 1: I am not convinced the right panel is necessary. If the left panel was enlarged a bit, I think it would suffice. Also, in the figure caption, delete “natural”. Does the IUCN need a citation?

Reply (lines 119-120): Panel B has been removed, and the term "natural" and the citation for the IUCN have been deleted.

Table 1: It should be stated that these are the 22 individual locations for each animal sampled in the study. I had to count the number of rows to figure this out.

Reply: We included the total number of individuals in the caption.

134: Not clear what is meant by “different degrees of success” or how this affected the ability to sequence the mtDNA.

Reply (lines 127-130): We deleted this part of the sentence to improve clarity. DNA extraction was sufficient to obtain high-quality sequences for all individuals, with no deviations from the protocol used.

165-169: More information is needed here, e.g., “four strings” of what? Likewise, I am unclear why seasonality needs to be evaluated and what information is gained by doing so.

Reply: The terms "strings" and "seasonality" were used in error and have been corrected to "chain" and "convergence," respectively, to address English language issues. We clarified as follows (lines 180-183): Bayesian inference analysis was run with 10 million generations and four Markov chain Monte Carlo (MCMC) to increase confidence of the estimate of parameters in the phylogenetic reconstruction. To evaluate convergence among the four MCMC, we plot each generation against the probability values, eliminating the first 1,000 trees as burn-in.

172: “outgroup” instead of “external group”?

Reply: Done

177-180: If negative values indicate expansion and positive values represent a constant size over time, how does one identify population contraction?

Reply: We modified as follows (lines 182-183): where negative values are interpreted as population demographic expansion, positive values as a population contraction and close to zero as constant size over time.

182 (and 167): What is the rationale for the extremely high number of generations used in the models? Based on the fossil evidence reported on line 187, the time frame of the analyses is only 3.6 Ma, so basing the models on 3-10x as many generations does not seem realistic.

Reply: We removed this analysis based on a recommendation from another reviewer.

201-202: Why not use all available records instead of just the ones from this study and Poljak et al.? Also, need to explain why the sample size for Poljak et al. is given as 41 here but 76 on line 150. I assume this is because multiple animals were sampled at the same site but this was also the case in Chile and yet individual coordinates were provided for each animal. Was this not possible for the Argentine data?

Reply: We did use and review all available records from Poljak et al. (2010), excluding repeated locations. The spatial autocorrelation observed in the Poljak et al. (2010) dataset arose because several individuals shared the same coordinates. The 41 individuals mentioned are those that remained after removing spatially autocorrelated samples

We modified as follows (lines 197-199): The occurrence points used were a combination of the genetic sampling locations in Chile (n=22 in Chile) and the locations reported by Poljak et al (2010), excluding repeated locations (n=19 in Argentina, Figure 1).

As a final comment, I am not sure it is necessary here, but in most species occurrence analyses I have seen, random points are sampled to identify the environmental conditions at locations where the species is not found, and these are then compared against the conditions where the species is found. Also, it is common to “train” the model with a subset of the location points and then evaluate the accuracy with which the model predicts occurrence at the remaining locations. Was there a reason for not doing these things here?

Reply: We mistakenly excluded information from the methodology and are adding it now. We modified as follows (lines 201-209): The quality of the model was evaluated using the area under the curve (AUC) and the continuous Boyce index (Hirzel et al., 2006). AUC values can vary from 0 to 1, where a value greater than 0.9 is considered an indicator of ‘‘good’’ discrimination skills (Peterson et al., 2011). Values of the Boyce index vary between -1 and 1, where positive values indicate a model with predictions that are consistent with the distribution of observed presences in the evaluation dataset (Boyce, 2002). For the distribution model a 30-fold cross-validation was used, with a data proportion of 25% for training and 75% for evaluation. Jackknife test was used to evaluate the importance of the environmental variables for predictive modeling (Almalki et al., 2015).

215: I assume that HC stands for haplotype C. If so, it seems unnecessary to include the H because it creates redundancy (i.e., saying “the HC haplotype” basically means the “haplotype C haplotype”).

Reply: We modified as suggested by the reviewer.

Table 2: Text under the table needs the following corrections: (1) Chilean population; and (2) polymorphic.

Reply: It was fixed

Figure 2: The figure legend needs to explain why Hap E has a box around it in panel (a). Also, vellerosus is misspelled in panel (b). Should this Latin name be italicized in both panels?

Reply: Done

Figure 3: Should be “obtained from” not “obtained with”. Also, some explanation needs to be provided of what the curves presented at the bottom right of the figure represent.

Reply: We modified as follows (lines 243-246): Network of haplotypes (A-Q) of C. villosus obtained from the mtDNA control region. The area of each circle is proportional to the number of individuals and represented by the curves of different sizes. The points on the blue lines represent mutation steps.

251: Should be “Skyline”.

Reply: We removed this analysis based on a recommendation from another reviewer.

260: “farther” not “further”

Reply: We removed this analysis based on a recommendation from another reviewer.

Figure 4: Italicize species name in legend. Also, should it be “Skyline”? Finally, it should be “mtDNA”.

Reply: We removed this analysis based on a recommendation from another reviewer.

280-283: This is an awkward sentence that needs to be rewritten. Maybe delete “sequences of”?

Reply: We deleted for clarity, and modified as follows (lines 253-256): Areas with medium/high probability of occurrence were observed in the Chilean provinces of Palena, Coyhaique, General Carrera, Capitán Prat, Última Esperanza and Magallanes, which varied between 0.6 and 1.0.

298: “that reported”, not “the reported”

Reply: Done

296-299: Break into two sentences.

Reply: We modified as follows (lines 269-272): This study represents the first evaluation of the genetic diversity and genetic structure of C. villosus at the western limit of its geographical distribution. Compared with the genetic diversity that reported in central distribution populations in Argentina [6], the peripheral population studied here exhibit low genetic polymorphism.

299-301: This sentence is awkward and needs to be rewritten.

Reply: We modified as follows (lines 272-274): This is demostrated by a single haplotype shared with one of the most frequent ones in Argentine populations

307: [45, 61]?

Reply: We corrected the bibliographic citation numbers.

313-318: As mentioned previously, delete discussion of D. novemcinctus because of the taxonomic revision published by Barthe et al. The only relevant paper might be Arteaga et al. (2020, or maybe 2012) but I do not recall if they compared genetic diversity of what is now called the Mexican long-nosed armadillo (D. mexicanus) in the U.S. versus Mexico. Any comparisons between populations farther south than Costa Rica are irrelevant because they involve other species.

Reply: The article by Barthe et al. (2024, Systematic Biology) was not utilized during the drafting of the first version of the manuscript, and the information pertaining to D. novemcinctus was not updated. Therefore, the citation concerning Dasypus novemcinctus has been removed from the Introduction and Discussion sections.

323: Make clear that “its” refers to C. villosus.

Reply: We modified as follows (lines 291-296): Sample size of C. villosus was limited by the low population density of the sampling area (peripheral condition). In Chile there is no information on its population density, however, studies in the Bolivian Chaco have estimated a minimum of 0.58 individuals per km2 (Cuéllar 2008), and up to 200 individuals per km2 in the Pampean region of Argentina (Superina and Abba 2018). Furthermore, behavioral characteristics shared with other armadillo species, such as their fossorial habits and crepuscular-nocturnal activity, make their study and the obtaining of biological samples difficult.

332: Insert “be” before “derived.

Reply: Done

333: “found” instead of “distributed”.

Reply: Done.

338: “sequence” instead of “alignment”? (see also line 341).

Reply: Done

351: “study” not “studied”

Reply: Done

408: Patagonia, not Patagonian

Reply: Done

410: shown, not indicated

Reply: Done

413: delete “floor”?

Reply: Done

416: delete “of”

Reply: Done

423: development does not seem like the right word here. Also, insert “a” before “consequence”.

Reply: Sentence deleted

408-426: I am not clear what point the authors wish to make by presenting all this information about historical changes to the steppe-forest ecotone strip. Presumably, changes to this ecotone have affected where C. villosus was found but there is no supporting evidence to substantiate this. While plausible, this

Reply: Although we have no evidence of the species in the periods mentioned, we do believe that this paragraph at least provides information on the vegetation changes that affected Patagonia in the past, and that could help understand the hypothesis of past geographic distribution derived from Maxent's analysis.

435: distribution does not seem like the right word here.

Reply: It was modified using habitat (line 360)

438: receptors does not seem like the right word here.

Reply: Modified by sink, considering source sink dynamic (line 369).

439-441: These sentences seem contradictory. If peripheral populations have a high likelihood of extinction, how can they also be stable and have high survival?

Reply: We modified as follows (lines 367-373): One implication of this hypothesis is that peripheral populations are at the limit of survival capacity as a result of the decrease of optimal conditions in all directions. Consequently, peripheral populations can act as sink rather than sources of genetic variants, but with high likelihood of extinction compared to central populations. However, their conservation value can be high because they function as a front for advance or colonization, allowing for the adjustment of a species' distribution following climate change or environmental alterations caused by anthropogenic factors.

442-443: Delete the last sentence.

Reply: Deleted.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Review

 

I have read the submitted manuscript and must note that despite some results obtained on the distribution of the species in nature, the work was done with gross methodological errors. In general, the methodological level of the study is very low. Most of the authors' conclusions are unfounded or directly contradict the demonstrated results. In general, I must recognize the study as inadequate and ask to reject this work, since in its current form it cannot be published. The authors should rethink the results obtained and apply other methods of data analysis so that the data obtained can be published.

 

See comments below and in the pdf file

Line 50

Fossil records do not and cannot say anything about the place of origin of a species. Based on fossils, one can only say that a species is known from a particular area in a particular period. Rephrase the phrase correctly. Mandatory

 

Line 56

What do you mean by anecdotal observations? Be more specific, what observations and where from and how reliable this information is. Mandatory

 

Line 90-99

This part would be better placed at Material and Methods

 

Materials and Methods

The first thing you should do in this section is to accurately indicate how many living animals and how many remains you examined. MANDATORY

 

Line 152-153

This is unacceptable! All information about the obtained nucleotide sequences, as well as about the sequences used from Genbank, must be directly indicated in the text of the article itself. Do this. Mandatory

 

Line 159-172

Please describe the phylogenetic analysis methodology more systematically. First, the general program you used for the calculations, then the methods and models you used, and finally the model parameters and algorithms you used. Mandatory

Also, you need to indicate what program you used to build the networks and what specific algorithms you used.

 

Line 215-216

Here it is necessary to directly indicate and include in the table which haplotypes and where were discovered in Chilean populations.

 

Line 234-235 Bayesian inherence

As I can see, most tree nodes are unreliable, which is unsurprising, since the DNA fragment used in the work is hypervariable and unsuitable for phylogenetic reconstructions. Therefore, it is pointless to discuss anything based on the structure of the obtained tree.

You should delete it and limit yourself to analysing the overall diversity and constructing a haplotype network.

 

Figure 3

Place a point map in Figure 3. Then, the reader will understand the distribution of haplotypes. In its current form, the figure does not allow the reader to imagine the geography of haplotype distribution.

Mandatory

Lines 253-264

A posterior probability of 0.1 is a completely unreliable result (must be 0.8 at least). The reliability does not differ from zero. It is impossible to make any assessments based on these calculations. This is a serious methodological error.

 

Line 285-290

Judging by the presented maps, the area of ​​the species distribution in the middle Holocene was even smaller than today, and the general suitability of habitats was worse than today. In this regard, the question arises: How can we explain the expansion of the range in this case? Obviously, the presented model is inadequate and does not explain the actual data on the species distribution.

Lines 310-311

Your data obtained based on the Maxent model indicate just the opposite.

Lines 3335-340

In fact, you can't say anything about the trees you've obtained because they're not reliable. Your reasoning may be based on the haplotype network alone, but you barely show it in a geographical context. And you never mention that the haplotype network is remarkably simple, and most of them differ from each other by single substitutions.

4.3. Evaluation of ancestral stage… lines 357-367

Since you have not found any distinctive haplotypes in the population you are studying, you have no basis to talk about their age. This entire section is completely baseless.

 

Lines 369-389

Why is all this here? The species under study has not been found in fossil form in the area under study. Why are you starting a lengthy discussion about the distribution of other species and genera, and even in much more distant times? This is meaningless text. Delete.

4.4. Evaluation of ancestral stage… line 392.

Why should you evaluate of ancestral stage of one haplotype second times? And for what purpose?..

The entire contents of section 4.4. are completely useless empty reasoning. The applied model showed that the conditions of existence for this species in the Holocene were even worse than today, the marginal populations are separated by extensive zones of extremely unfavourable conditions for the species, from this it follows that there are no reasons for the expansion of the range. The end. Further lengthy reasoning is empty, unfounded chatter, it is not clear why it was placed here. Delete the entire section

Comments for author File: Comments.pdf

Author Response

Reviewer 2:

I have read the submitted manuscript and must note that despite some results obtained on the distribution of the species in nature, the work was done with gross methodological errors. In general, the methodological level of the study is very low. Most of the authors' conclusions are unfounded or directly contradict the demonstrated results. In general, I must recognize the study as inadequate and ask to reject this work, since in its current form it cannot be published. The authors should rethink the results obtained and apply other methods of data analysis so that the data obtained can be published.

See comments below and in the pdf file

Line 50: Fossil records do not and cannot say anything about the place of origin of a species. Based on fossils, one can only say that a species is known from a particular area in a particular period. Rephrase the phrase correctly. Mandatory

Reply: We modified as follows (lines 49-51): The oldest fossil records are located in the Argentine Pampas region [5], suggesting that this species inhabited Patagonia after the ice retreated during the Pleistocene [6].

Line 56: What do you mean by anecdotal observations? Be more specific, what observations and where from and how reliable this information is. Mandatory

Reply: We modified “anecdotal” to “field observation”. The cited works correspond to research and books with reliable information that have contributed to the development of mammalogy in Chile.

Line 90-99: This part would be better placed at Material and Methods

Reply: We will maintain prayer because it is the focus of the work

Materials and Methods

The first thing you should do in this section is to accurately indicate how many living animals and how many remains you examined. MANDATORY

Reply: We modified as follows (lines 110-116): Live individuals (n=2) were captured using five Tomahawk traps, installed for five days at each collection site. Live individuals were anesthetized with 25 mg/kg ketamine hydrochloride, taking blood samples from the ventral tail vein, and were preserved in 96% ethanol. The captured individuals were released at the same location of capture. The capture permit was authorized by Servicio Agrícola y Ganadero (SAG-Chile, authorization N° 2048/2012 and 6956/2013). Additionally, muscle tissue (limbs) was collected from carcass individuals (n=20) found on roads and near burrows.

Line 152-153; This is unacceptable! All information about the obtained nucleotide sequences, as well as about the sequences used from Genbank, must be directly indicated in the text of the article itself. Do this. Mandatory

Reply: We have addressed the reviewer's concern regarding the accessibility of sequence information. As requested, the key details about the obtained nucleotide sequences and the GenBank accession numbers for the sequences used have now been directly incorporated into the main text of the manuscript (lines 149-150). We have retained the supplementary tables as they contain additional, more detailed information beyond what is essential for the main text.

Line 159-172: Please describe the phylogenetic analysis methodology more systematically. First, the general program you used for the calculations, then the methods and models you used, and finally the model parameters and algorithms you used. Mandatory

Also, you need to indicate what program you used to build the networks and what specific algorithms you used.

Reply: We included and modified as follows (lines 156-174): To achieve this, we applied a phylogenetic reconstruction model incorporating sequence migration and evolution [27] to our dataset, which was derived from the Maximum Likelihood (ML) algorithm used in the phylogenetic reconstruction included as input. Specifically, the phylogenetic reconstruction was performed using the ML approach implemented in PHYML 3.0 [28] with the default parameters. Model selection for the ML phylogenetic reconstruction was performed using JModelTest v.0.1.1 [29], which identified HKY + I as the best-fit evolutionary model based on the Bayesian Information Criterion (base frequencies: A = 0.336, C = 0.269, G = 0.103, T = 0.286; gamma distribution shape parameter = 0.2010).  The network visualization, generated in Haploviewer, incorporated haplotype frequency, mutational steps, and sampling location.

Phylogenetic relationships were analyzed with BEAST 2 v.2.5.0 and MEGA X v10.1.0 programs with bayesian inference and maximum likelihood methods [30, 31]. Bayesian inference analysis was run with 10 million generations and four Markov chain Monte Carlo (MCMC) to increase confidence of the estimate of parameters in the phylogenetic reconstruction. To evaluate convergence among the four MCMC, we plot each generation against the probability values, eliminating the first 1,000 trees as burn-in. The maximum likelihood analysis included the heuristic tree search, substitution model Tamura-Nei and bootstrap as a method to obtain the support of the nodes.

Line 215-216: Here it is necessary to directly indicate and include in the table which haplotypes and where were discovered in Chilean populations.

Reply: Done, information included in Table 1

Line 234-235: As I can see, most tree nodes are unreliable, which is unsurprising, since the DNA fragment used in the work is hypervariable and unsuitable for phylogenetic reconstructions. Therefore, it is pointless to discuss anything based on the structure of the obtained tree. You should delete it and limit yourself to analysing the overall diversity and constructing a haplotype network.

Reply: We appreciate the reviewer’s comment and acknowledge the limitations inherent to using a hypervariable region. However, we respectfully argue that in the context of intraspecific phylogenetic reconstruction, such markers can still yield valuable information regarding haplotype clustering and shallow genealogical structure.

Specifically: i) The purpose of the tree is not to infer deep phylogenetic relationships but to complement the haplotype network and diversity analyses by visualizing relationships among closely related haplotypes within the species. ii) We used both Bayesian inference (Figure 2a) and Maximum Likelihood (Figure 2b) approaches to evaluate the consistency of haplotype clustering. Interpretations are explicitly restricted to well-supported nodes (posterior probabilities > 0.95 or bootstrap values ≥ 70). iii) We revised the manuscript to clarify that the phylogeny should be interpreted with caution due to the nature of the marker (Lines 299-300), and we ensure that no conclusions are drawn from poorly supported branches.

Retaining the tree allows a more complete integrative view of the data, particularly in identifying lineages or geographically structured clusters (e.g., Hap O and P), which is relevant to our discussion. We therefore retained the phylogenetic trees in the manuscript, with the appropriate caveats and context made explicit in the text.

Figure 3: Place a point map in Figure 3. Then, the reader will understand the distribution of haplotypes. In its current form, the figure does not allow the reader to imagine the geography of haplotype distribution. Mandatory.

Reply: We understand the reviewer's suggestion to include a point map in Figure 3. While information on the general location of Argentine and Chilean populations is available in Figure 1, Table 1, and the corresponding annexes, we opted for the current representation in Figure 3 because we believe that the current figure, in conjunction with the information provided in Figure 1 and Table 1, adequately conveys the general geographic context of the sampled populations for understanding the haplotype relationships.

However, we are open to reconsidering this if the editor feels strongly that a point map is essential for clarity.

Lines 253-264 A posterior probability of 0.1 is a completely unreliable result (must be 0.8 at least). The reliability does not differ from zero. It is impossible to make any assessments based on these calculations. This is a serious methodological error.

Reply: We removed this analysis.

Line 285-290: Judging by the presented maps, the area of the species distribution in the middle Holocene was even smaller than today, and the general suitability of habitats was worse than today. In this regard, the question arises: How can we explain the expansion of the range in this case? Obviously, the presented model is inadequate and does not explain the actual data on the species distribution.

Reply: We respectfully disagree with the reviewer's assessment. Although we recognize the inherent limitations of this analysis, the resulting hypothesis of past geographic distribution for the species offers a crucial framework for interpreting the current distribution of peripheral populations in Chilean and Argentine Patagonia.

Lines 310-311: Your data obtained based on the Maxent model indicate just the opposite.

Reply: We respectfully disagree with the reviewer's assessment. Although we recognize the inherent limitations of this analysis, the resulting hypothesis of past geographic distribution for the species offers a crucial framework for interpreting the current distribution of peripheral populations in Chilean and Argentine Patagonia.

Lines 335-340: In fact, you can't say anything about the trees you've obtained because they're not reliable. Your reasoning may be based on the haplotype network alone, but you barely show it in a geographical context. And you never mention that the haplotype network is remarkably simple, and most of them differ from each other by single substitutions.

We appreciate the reviewer's comment and acknowledge the inherent limitations of low node support. However, we respectfully argue that, in the context of intraspecific phylogenetic reconstruction, such trees can still provide valuable information on haplotype clustering and surface genealogical structure.

Specifically: i) The purpose of the tree is not to infer deep phylogenetic relationships, but rather to complement network and haplotype diversity analyses by visualizing the relationships between closely related haplotypes within the species. ii) We used Bayesian inference (Figure 2a) and maximum likelihood (Figure 2b) approaches to assess the consistency of haplotype clustering. Interpretations are explicitly limited to nodes with good support (posterior probabilities > 0.95 or bootstrap values ​​≥ 70). iii) We revised the manuscript to clarify that the phylogeny should be interpreted with caution due to the nature of the marker, and we made sure not to draw conclusions from poorly supported branches.R

4.3. Evaluation of ancestral stage… lines 357-367

Since you have not found any distinctive haplotypes in the population you are studying, you have no basis to talk about their age. This entire section is completely baseless.

Reply: We have removed this analysis, its results, and related discussion.

Lines 369-389: Why is all this here? The species under study has not been found in fossil form in the area under study. Why are you starting a lengthy discussion about the distribution of other species and genera, and even in much more distant times? This is meaningless text. Delete.

Reply: Deleted.

4.4. Evaluation of ancestral stage… line 392.

Why should you evaluate of ancestral stage of one haplotype second times? And for what purpose?..The entire contents of section 4.4. are completely useless empty reasoning. The applied model showed that the conditions of existence for this species in the Holocene were even worse than today, the marginal populations are separated by extensive zones of extremely unfavourable conditions for the species, from this it follows that there are no reasons for the expansion of the range. The end. Further lengthy reasoning is empty, unfounded chatter, it is not clear why it was placed here. Delete the entire section

Reply: We modified as follows (lines 327-356): The maximum entropy algorithm predicted that areas with medium-high environmental conditions (range 0.5 to 1.0 probability), were approximately 50% lower in warm periods of the middle Holocene than the current distribution conditions of the species. Areas with high habitability conditions were observed from Neuquén province southward during the middle Holocene (Figure 5b). The extensive area with low-medium levels of habitability between Chubut and Santa Cruz provinces suggest a connection between eastern populations and those of the Atlantic coast. Also, due to climatic similarities, medium-high habitability conditions were observed in areas currently occupied by the peripheral population of C. villosus distributed between Aysén and Magallanes.

The resemblance of past climate in Chilean and Argentine Patagonia is supported by palynological studies carried out in ecotone areas of steppe-forest. In the Mallín Pollux location (Aysén district 45º41'30''S 71°50'30''W), it has been shown that 6,300 years ago grasses dominated the steppe, suggesting an arid climatic condition [52]. Southward, lithological and palynological evidence obtained in Lake Casanova (47°38'36.86''S; 72° 58'30.81''W), suggest that between 8,480 and 5,360 years ago the vegetation was mainly composed of a Fagaceae forest.  Nonetheless, higher pollen percentages of bushes and shrubs, allow us to suppose that a diversity of plants associated with open environments also developed [53]. Northward, the Argentine Mosquito Lake (42°29'37.89''S; 71° 24'14.57''W) and Cóndor Lake (42°20'47''S; 71°17'07.62''W) currently occupy the steppe-forest ecotone strip. However, between 9000 and 5230 years ago there was a high incidence of forest fires, suggesting a predominance of dry climatic conditions dominated by sclerophyllous shrubs because of lower temperatures, with expansion of forests of Fagaceae and other taxa towards steppe environments [54]. Thus, the vegetation changes indicate that climatic conditions have affected the position of the steppe-forest ecotone, which is key to the habitat dynamics of C. villosus today. This could be a consequence of changes in the conditions of insolation, as well as the humidity derived from the advance and retreat of glaciers that affected the southern center of Patagonia, mainly the valleys and adjacent plains of the Andes [53, 54].

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript reports on the phylogeography of peripheral populations of hairy armadillos (Chaetophractus villosus). The topic is interesting, but in my opinion the title is a bit misleading as the study is limited to the peripheral populations in Chile although the species has a wider distribution and, thus, several other peripheral populations. Additional comments can be found below.

 

L53: Yes, the species was originally found from Biobio south to Magallanes. However, recent records seem to be restricted to Aisén and Magallanes. See Pasutti, R. 2017. Actualización preliminar en el conocimiento de las 3 especies de armadillos presentes en Chile. Undergraduate thesis, Universidad de Chile. 76 pp. The same distribution is mentioned in the following document:  Centros de Estudios Agrarios (CEA), 2011. Ficha de antecedente de especie: Chaetophractus villosus. Available at: http://www.mma.gob.cl/clasificacionespecies/fichas12proceso/pac/Chaetophractus_villosus_12RCE_INICIO.pdf, 6.

L91: It might be appropriate to specify that you are referring to peripheral populations in Chile; otherwise you might give the impression that you are looking at peripheral populations in different parts of the species range, which is not true. Also, did you deliberately exclude the introduced population in Tierra del Fuego?

L113: This description of the methodology is too short. How did you find and capture the live animals? What was your sampling effort? You mention that you collected them between 2012 and 2014, but how many field-days did you invest? This is important to know because in the Discussion (L321), you mention that  sample size was limited due to the low population density in the sampling area – but maybe it was due to a low sampling effort? Are there really data on population densities of armadillos in your area?
Did you release them at their capture site once they recovered from anesthesia? Which vein did you puncture, and what size of needle did you use? How did you preserve the blood samples, with ethyl alcohol (and in which proportion)? On L113 you also mention “carcass tissue”, but don’t provide any information about it. How did you find the carcasses? Did you only sample fresh carcasses or any dead individual you found (or that was brought to you?)? Which tissue did you collect, and how did you store it? Which samples in Table 1 correspond to blood, and which ones to tissue samples?

L117: For future studies, please do not use Ketamine hydrochloride alone. There are plenty of combined anesthesia protocols that are more appropriate and safer for the animals than Ketamine alone. 

L132: Please briefly explain the extraction method or at least mention that you used phenol-chloroform extraction.

L134: What do you mean by “fixed and old material”? This should be clarified in the section 2.1.

L135: It might be helpful to mention that this primer amplifies the mitochondrial control region D-loop

L190: There are way more species records than you used for your model; why did you limit the model to this relatively small number of records? For instance, you excluded records from the northernmost range (Bolivia and Paraguay), so I doubt the results you obtained are representative. Indeed, see comment on L279. 

Table 2: please correct polymorphic to polymorphic, and chilean to Chilean (Chilean should also be capitalized in the title 3.3)

Figure 2: You may want to explain in the caption why Hap_E is marked with an orange rectangle. (captions should be self-explanatory) 

L240: Please check this information. The northernmost part of La Pampa province is at approx. 36 degrees LS, not 27. 27 LS roughly corresponds to Tucumán. Accordingly, the distance of 2,800 km is incorrect. 

Figure 4: Please write the scientific name in italics

L279: No, this is incorrect. Your model shows all of northern Argentina, Bolivia, and Paraguay as unsuitable, although these areas are included in the IUCN distribution. 

L296: Please rephrase this first sentence, as it is rather long and complicated. I’m not sure if you wanted to say “with those reported in populations…”? Maybe you could divide it into two sentences, one ending with “of its geographical distribution” and then starting a new one “Compared with the populations in central Argentina, the peripheral populations…”
L299: Please carefully revise the language of the discussion, as it is not consistent with the rest of the manuscript (which is very well-written!). For instance, “one not new haplotype” sounds strange.

L313: Dasypus novemcinctus has been split into 4 species, so you will have to revise this paragraph carefully. See Barthe, M., Rancilhac, L., Arteaga, M.C., Feijó, A., Tilak, M.-K., Justy, F., Loughry, W.J., McDonough, C.M., de Thoisy, B., Catzeflis, F., Billet, G., Hautier, L., Nabholz, B., Delsuc, F., 2024. Exon capture museomics deciphers the nine-banded armadillo species complex and identifies a new species endemic to the Guiana Shield. Systematic Biology. https://doi.org/10.1093/sysbio/syae027

L322: It is rather difficult to assess if this is correct because you did not provide any data on sampling effort or the technique you used to find armadillos, nor are you citing any paper about population density of hairy armadillos in your study area.

L330: I’m not convinced about this explanation. Tucumán is a very small province, and simply one of many on the (very long) western limit of the species distribution. Why should it be considered a marginal distribution?

L350: are you referring to the distribution in Chile? The Patagonian steppe is not an ecotone but an ecoregion.

L351: replace studied by study 

L354: delete duplicated “of” (and please make sure you carefully revise the language of this Discussion)

L379&388: This reminds me of the history of armadillos in North America, where Dasypus bellus went extinct long before the colonization by nine-banded armadillos. Is it possible that there were several geographic expansions (and local extinctions) of armadillos from Argentina to Chile?

L408: Replace Patagonian by Patagonia

L423: I’m not sure “development” is the correct word here. In this context, I suggest to revise the language/wording of the paragraph starting on L408, as it is different to that of the rest of the manuscript.

L430: Should this be “its population is stable”, referring to the armadillo C.villosus? Same for the rest of the sentence.

L439: I’m confused. Here you say that the likelihood of extinction of peripheral populations is high, but in the next sentence you state that they can remain stable and with a high survival rate to climatic fluctuations. Isn’t that contradictory? Also, why would these peripheral populations be important for the long-term conservation of genetic diversity if they have a lower genetic diversity than the main population? 

L442: delete this sentence.

References: Please make sure all scientific names are written in italics.

Table S1: Please correct Córdova to Córdoba (same in Figures 3&4)

Author Response

Reviewer 3:

This manuscript reports on the phylogeography of peripheral populations of hairy armadillos (Chaetophractus villosus). The topic is interesting, but in my opinion the title is a bit misleading as the study is limited to the peripheral populations in Chile although the species has a wider distribution and, thus, several other peripheral populations. Additional comments can be found below.

L53: Yes, the species was originally found from Biobio south to Magallanes. However, recent records seem to be restricted to Aisén and Magallanes. See Pasutti, R. 2017. Actualización preliminar en el conocimiento de las 3 especies de armadillos presentes en Chile. Undergraduate thesis, Universidad de Chile. 76 pp. The same distribution is mentioned in the following document:  Centros de Estudios Agrarios (CEA), 2011. Ficha de antecedente de especie: Chaetophractus villosus. Available at: http://www.mma.gob.cl/clasificacionespecies/fichas12proceso/pac/Chaetophractus_villosus_12RCE_INICIO.pdf, 6.

Reply: Done. We modified as follows (lines 52-55): C. villosus is distributed mainly in Argentina and Paraguay, with less representation in Bolivia and Chile (Figure 1). In Chile this species has a peripheral distribution in the Andean plains and valleys from Aysén to Magallanes district (44° to 53°S), and the historical record of sightings is scarce, corresponding mainly to observations fields.

L91: It might be appropriate to specify that you are referring to peripheral populations in Chile; otherwise you might give the impression that you are looking at peripheral populations in different parts of the species range, which is not true. Also, did you deliberately exclude the introduced population in Tierra del Fuego?

Reply: We changed the term "peripheral populations" in the text to "individuals with peripheral distribution." The Tierra del Fuego populations were not considered due to economic resource issues; furthermore, they are an introduced population (Abba et al. 2014).

L113: This description of the methodology is too short. How did you find and capture the live animals? Trampeo y captura manual. What was your sampling effort?

Reply: We modified as follow (lines 106-117): Biological samples were obtained in 2012 and 2014 in Chilean localities between 44° to 53° South (Aysén and Magallanes districts). In the Aysén district the collection locations were the provinces of Coyhaique, General Carrera and Capitán Prat; in Magallanes district it was collected in Última Esperanza and Magallanes provinces (Figure 1, Table 1). Live individuals (n=2) were captured using five Tomahawk traps, installed for five days at each collection site. Thes individuals were anesthetized with 25 mg/kg ketamine hydrochloride, taking blood samples from the ventral tail vein. The captured individuals were released at the same location. The capture permit was authorized by the Chilean Agricultural and Livestock Service (authorization documents 2048/2012 and 6956/2013). (capture permits 2048/2012 and 6956/2013). Additionally, muscle tissue (limbs) was collected from carcass individuals (n=20) found on roads and near burrows.

You mention that you collected them between 2012 and 2014, but how many field-days did you invest?

Reply: We modified as follow (lines 110-111): Live individuals (n=2) were captured using five Tomahawk traps, installed for five days at each collection site.

This is important to know because in the Discussion (L321), you mention that  sample size was limited due to the low population density in the sampling area – but maybe it was due to a low sampling effort? Are there really data on population densities of armadillos in your area? Did you release them at their capture site once they recovered from anesthesia?

Reply: The individuals were released at the same capture site.

Reply: We have included bibliographical references on the population density of the species (lines 291-294). In Chile there is no information on its population density in C. villosus, however, studies in the Bolivian Chaco have estimated a minimum of 0.58 individuals per km2 [46], and up to 200 individuals per km2 in the Pampean region of Argentina [47].

Which vein did you puncture, and what size of needle did you use?. How did you preserve the blood samples, with ethyl alcohol (and in which proportion)? On L113 you also mention “carcass tissue”, but don’t provide any information about it. How did you find the carcasses? Did you only sample fresh carcasses or any dead individual you found (or that was brought to you?)? Which tissue did you collect, and how did you store it? Which samples in Table 1 correspond to blood, and which ones to tissue samples?

Reply: We modified as follow (lines 111-116). These individuals were anesthetized with 25 mg/kg ketamine hydrochloride, taking blood samples from the ventral tail vein, and were preserved in 96% ethanol. The captured individuals were released at the same location of capture. The capture permit was authorized by Servicio Agrícola y Ganadero (SAG-Chile, authorization N° 2048/2012 and 6956/2013). Additionally, muscle tissue (limbs) was collected from carcass individuals (n=20) found on roads and near burrows.

L117: For future studies, please do not use Ketamine hydrochloride alone. There are plenty of combined anesthesia protocols that are more appropriate and safer for the animals than Ketamine alone.

Reply: We appreciate the suggestion

L132: Please briefly explain the extraction method or at least mention that you used phenol-chloroform extraction.

Reply: We modified as follow (lines 129-130): Briefly, this protocols included a digestion of tissue with PK, then separation of organic proteins by phenol chloroform, and precipitated with ethanol.

L134: What do you mean by “fixed and old material”? This should be clarified in the section 2.1.

Reply: We deleted this part for clarity for the readers, as suggested by the reviewer 1.

L135: It might be helpful to mention that this primer amplifies the mitochondrial control region D-loop

Reply: It was included (line 131).

L190: There are way more species records than you used for your model; why did you limit the model to this relatively small number of records? For instance, you excluded records from the northernmost range (Bolivia and Paraguay), so I doubt the results you obtained are representative. Indeed, see comment on L279.

Reply: We limited the occurrence points to those obtained during the sampling conducted in Chile and incorporated the sampling points included in the work of Poljak et al. 2009, excluding those repeated in the same localities. We did not consider the species' occurrence points in Bolivia and Paraguay because we are primarily interested in modeling the species' past geographic distribution in the southern part of its range.

Table 2: please correct polymorphic to polymorphic, and chilean to Chilean (Chilean should also be capitalized in the title 3.3)

Reply: Done

Figure 2: You may want to explain in the caption why Hap_E is marked with an orange rectangle. (captions should be self-explanatory)

Reply: We removed the orange rectangle and corrected the italic name of the species.

L240: Please check this information. The northernmost part of La Pampa province is at approx. 36 degrees LS, not 27. 27 LS roughly corresponds to Tucumán. Accordingly, the distance of 2,800 km is incorrect.

Reply: We modified this as suggested.

Figure 4: Please write the scientific name in italics

Reply: We modified this as suggested.

L279: No, this is incorrect. Your model shows all of northern Argentina, Bolivia, and Paraguay as unsuitable, although these areas are included in the IUCN distribution.

Reply: We deleted this part for clarity for the readers

L296: Please rephrase this first sentence, as it is rather long and complicated. I’m not sure if you wanted to say “with those reported in populations…”? Maybe you could divide it into two sentences, one ending with “of its geographical distribution” and then starting a new one “Compared with the populations in central Argentina, the peripheral populations…”

Reply: We splited in two sentences (lines 269-272): This study represents the first evaluation of the genetic diversity and genetic structure of C. villosus at the western limit of its geographical distribution. Compared with the genetic diversity that reported in central distribution populations in Argentina [6], the peripheral population studied here exhibit low genetic polymorphism.

L299: Please carefully revise the language of the discussion, as it is not consistent with the rest of the manuscript (which is very well-written!). For instance, “one not new haplotype” sounds strange.

Reply: We rewrite this part as follows (lines 272-274): This is demostrated by a single haplotype shared with one of the most frequent ones in Argentine populations [6]. This result agrees with the center-periphery model that relates lower genetic diversity in marginal areas of distribution to a reduction in gene flow and an increase in selective pressures [45, 46].

L313: Dasypus novemcinctus has been split into 4 species, so you will have to revise this paragraph carefully. See Barthe, M., Rancilhac, L., Arteaga, M.C., Feijó, A., Tilak, M.-K., Justy, F., Loughry, W.J., McDonough, C.M., de Thoisy, B., Catzeflis, F., Billet, G., Hautier, L., Nabholz, B., Delsuc, F., 2024. Exon capture museomics deciphers the nine-banded armadillo species complex and identifies a new species endemic to the Guiana Shield. Systematic Biology. https://doi.org/10.1093/sysbio/syae027

Reply: The article by Barthe et al. (2024, Systematic Biology) was not utilized during the drafting of the first version of the manuscript, and the information pertaining to D. novemcinctus was not updated. Consequently, we removed the Dasypus novemcinctus citation from the Introduction and Discussion.

L322: It is rather difficult to assess if this is correct because you did not provide any data on sampling effort or the technique you used to find armadillos, nor are you citing any paper about population density of hairy armadillos in your study area.

Reply: We modified as follow (lines 291-296): In Chile there is no information on its population density, however, studies in the Bolivian Chaco have estimated a minimum of 0.58 individuals per km2 (Cuéllar 2008), and up to 200 individuals per km2 in the Pampean region of Argentina (Superina and Abba 2018). Furthermore, behavioral characteristics shared with other armadillo species, such as their fossorial habits and crepuscular-nocturnal activity, make their study and the obtaining of biological samples difficult.

L330: I’m not convinced about this explanation. Tucumán is a very small province, and simply one of many on the (very long) western limit of the species distribution. Why should it be considered a marginal distribution?

Reply: We deleted this part for clarity for the readers.

L350: are you referring to the distribution in Chile? The Patagonian steppe is not an ecotone but an ecoregion.

Reply: We modified as follows (lines 320-322): In Chile, C. villosus is mainly distributed in the Patagonian steppe ecoregion. The peripheral individuals included in this study are found in the steppe-forest ecotone, which is characterized by an arid and cold climate [55]

L351: replace studied by study

Reply: Done

L354: delete duplicated “of” (and please make sure you carefully revise the language of this Discussion)

Reply: We deleted “of” and we checked the language as suggested.

L379-388: This reminds me of the history of armadillos in North America, where Dasypus bellus went extinct long before the colonization by nine-banded armadillos. Is it possible that there were several geographic expansions (and local extinctions) of armadillos from Argentina to Chile?

Reply: Due to reviewer suggestion, we had to remove this part of discussion.

L408: Replace Patagonian by Patagonia

Reply: Done

L423: I’m not sure “development” is the correct word here. In this context, I suggest to revise the language/wording of the paragraph starting on L408, as it is different to that of the rest of the manuscript.

Reply: We rewrite this as also suggestion of the reviewer 1

L430: Should this be “its population is stable”, referring to the armadillo C.villosus? Same for the rest of the sentence.

Done

L439: I’m confused. Here you say that the likelihood of extinction of peripheral populations is high, but in the next sentence you state that they can remain stable and with a high survival rate to climatic fluctuations. Isn’t that contradictory? Also, why would these peripheral populations be important for the long-term conservation of genetic diversity if they have a lower genetic diversity than the main population?

Reply: we rewrite this part as follows (lines 365-371): A consequence of this hypothesis is that peripheral populations are situated at the limit of their survival potential due to the degradation of optimal conditions across their range. As a result, these populations may act as sinks rather than sources of genetic diversity, but with high likelihood of extinction compared to central populations. However, their conservation value can be high because they function as a front for advance or colonization, allowing for the adjustment of a species' distribution following climate change or environmental alterations caused by anthropogenic factors.

L442: delete this sentence.

Reply: it was deleted

References: Please make sure all scientific names are written in italics.

Reply: It was corrected.

Table S1: Please correct Córdova to Córdoba (same in Figures 3&4)

Reply: It was modified in the table S1 and Figures 3 and 4.

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear colleagues.
I have read the corrected version of the manuscript sent to me and must admit that it has definitely become better. The authors have done a great job and have put the description of the methodological part of the article in order, and have also systematized the data on the study area and the materials obtained. And although I still consider some of the authors' conclusions to be not very well founded, this does not prevent the publication and expression of the authors' original point of view. I believe that the article can be published.
With respect