The Bear Truth: Analyzing Genetic Variability and Population Structure in Sloth Bear across the Vidarbha Landscape Using Microsatellite Markers
Round 1
Reviewer 1 Report (Previous Reviewer 1)
Comments and Suggestions for AuthorsDear Authors,
The proposed manuscript "The Bear truth: Analyzing genetic variability and population structure in sloth bear across Vidarbha Landscape using microsatellite markers" should be revised to be suitable for publication in the journal Diversity.
The suggestions are as follows:
The abstract needs significant improvement. It has not been revised and does not contain all the necessary elements.
In the chapter"Materials and methods", the description of the methodology needs to be significantly shortened and clarified, especially the parts related to sample collection, DNA isolation and statistical processing. I suggest that Table 1 be moved to the appendix. Some sentences in the chapter are not clear, e.g. line 243 – 244 "Genepop was used to calculate F-statistics and Nm for the samples [41,42,43,44]".
In the "Results" chapter, Figure 2 and Figure 3 are not legible or fully explained. Table 2 is not transparent (some parameters can be omitted).
The "Discussion" chapter needs to be conceptually revised, refined and clarified.
The references listed in the "References" chapter need to be revised. They are not written according to the journal's instructions.
Author Response
Reviewer 1
Dear Reviewer 1,
Thank you so much for your valuable inputs. We have gone through each comment and tried our level best to accommodate the suggestions to make this MS better.
The abstract needs significant improvement. It has not been revised and does not contain all the necessary elements.
Answer: We have re-structured the abstract and outlined the most crucial outcomes from the study in the new version.
In the chapter"Materials and methods", the description of the methodology needs to be significantly shortened and clarified, especially the parts related to sample collection, DNA isolation and statistical processing. I suggest that Table 1 be moved to the appendix. Some sentences in the chapter are not clear, e.g. line 243 – 244 "Genepop was used to calculate F-statistics and Nm for the samples [41,42,43,44]".
Answer: We have tried to balance the amount of information in the, Materials and method section. It was short in our previous versions. Upon review it was suggested that we elongate and provide in-depth explanation of each step. Hence the “Materials and Methods” have been elongated.
We feel table 1 may be included in the text to allow readers to check the primers and their grouping at hand.
We have re-organized the sentences in the chapter to make the meaning of the sentences clearer as suggested.
In the "Results" chapter, Figure 2 and Figure 3 are not legible or fully explained. Table 2 is not transparent (some parameters can be omitted).
Answer: Thank you for your input. The legibility of the figures could be because of the PDF quality given to reviewers. We do attach JPEG of the images as asked by journal as well along with manuscript.
We feel the figures are mostly self-explanatory. We have added the meaning of the figures to make it easier for the readers who might be unfamiliar with the software and their results.
We are unsure what “transparent” here indicates since we have tried to include as much information as possible and available. More information should mean more transparency. Also, apologies since we do not understand how omitting parameters can make it less transparent. We would be glad if this could be clarified if deemed important.
The "Discussion" chapter needs to be conceptually revised, refined and clarified.
Answer: Thank you for the comment. We have re-read the discussion part and have tried our best to refine the same.
The references listed in the "References" chapter need to be revised. They are not written according to the journal's instructions.
Answer: We have tried to clarify this with the editors and have tried our best to comply with the required instructions. We have re-done the entire reference list based on ACS.
Reviewer 2 Report (Previous Reviewer 2)
Comments and Suggestions for AuthorsThe authors tested and standardised a panel of 13 microsatellite markers and did preliminary population genetics analyses of sloth bears in India. The description of the methods is quite detailed and the results presented are interesting. The manuscript's structure is fairly deliberate, the text is generally clear and concise, although some sentences are awkwardly worded or confusing. The use of spaces linked to punctuation is inconsistent, some gaps between the punctuation and the adjacent text are prominent, which may cause distraction. The tables and figures are clear and generally easy to understand, however, some titles and legends might be too vague.
Line 35: The scientific name Melusus ursinus has been already used in the previous sentence, it is pointless here.
Line 78: Consider using something like “focal area” instead of “study landscape”.
Line 79: The abbreviation PIDsibs precedes the term ‘probability of identity between siblings’ mentioned in lines 84-85. The abbreviation should be resolved here, so you can later use simply PIDsibs.
Lines 80-81: Please change this to something like “genotyping with less than 11 or 12 markers results in significantly changed estimates of population structure and genetic variation”.
Line 83: Probably “being sampled [24, 25]”.
Line 89: Consider “create a baseline data of sloth bear population genetics parameters for eastern Vidarbha landscape”.
Line 98: Tadoba Andhari National Tiger Reserve is misspelt. Additionally, this site is denoted as Tadoba Andhari Tiger Reserve in Figure 1. The names should be consistently used.
Line 103: Consider changing to “In total, 565 fecal samples of sloth bears had been collected” and deleting “from the sampling sites”.
Line 119: Maybe “locality-associated data”?
Line 153: Simply “until further processing”.
Line 160: Consider “Following an initial screening of the markers”.
Line 164: The title should be “Microsatellite markers selected for the study of sloth bears”.
Line 186: Simply “with a few modifications”. Also “An overnight…”.
Line 188: Use “according to the manufacturer’s instructions” instead of “following the steps provided by the manufacturer”.
Lines 216-217 and generally: Please use the name and locality of manufacturers consequently.
Lines 218-221: This part is duplicated in lines 224-225. The latter is probably a better placement. Additionally, please use “three times” instead of thrice.
Line 234: What did you mean by “and were also done.”?
Line 249: Please add which version of BayesAss was used, and the running parameters.
Lines 254-259: This part is a reiteration of the methods, it should be deleted.
Line 295: Maybe “Variability of microsatellite markers …”, and annealing temperatures would fit better in Table 1. Additionally, use either a superscript or the abbreviation itself to resolve it in the table legend, but please be consequent.
Lines 311-314: The phrasing of this part is a bit strange. You could rephrase it something like: “The neighboring populations of TATR and UKWLS were assigned to one genetic cluster with UKWLS sharing some genetic signature with the adjacent NNTR population. The populations of NNTR and PTR formed the next cluster with PTR showing some links to the MTR population. The majority of samples from MTR were assigned to a third genetic cluster, while a small part of the MTR population grouped with samples from STR into a common cluster and the remaining part of the STR population was assigned to a separate group.”.
Line 321: Consider changing the title of Figure 2 to “TESS assignment plot of sloth bear across Vidarbha Landscape, India.” Also, the arrangement of the samples/population in Figure 2 is odd. It would be easier to see the clusterings if the order of the populations would resemble their geographic location, i.e. STR -> MTR -> PTR -> NNTR -> UKWLS -> TATR.
Line 328: A fairly high migration rate was also found between two fair away populations, STR and MTR. Why did you not mention it here? Especially because you discuss the link between the two later on.
Line 344: Consider changing the title of Figure 3 to “Circos plot of migration rates between different protected areas of the Vidarbha Landscape, India.”
Line 353: Probably “from degraded samples”.
Lines 353-355: “Based on reproducibility and consistency, 13 markers were selected for the study. The final panel was divided into six multiplex reactions to make the entire process cost effective and efficient.”
Line 402: This sentence is pointless, please delete it.
Lines 417-418: This sentence is pointless, please delete it.
Author Response
Reviewer 2
Dear Reviewer 2,
Thank you so much for your valuable time and input. We have tried our best to understand the gaps and implement appropriate changes to ensure the quality of the MS at hand increases.
Line 35: The scientific name Melursus ursinus has been already used in the previous sentence, it is pointless here.
Answer: The scientific name of the species was removed since it was already mentioned in the previous sentence.
Line 78: Consider using something like “focal area” instead of “study landscape”.
Answer: Thank you for the comment. We however do feel that the term “landscape” suits better because of the enormity of the area. Geographically too it is named “Vidarbha Landscape” , hence the usage in the MS.
Line 79: The abbreviation PIDsibs precedes the term ‘probability of identity between siblings’ mentioned in lines 84-85. The abbreviation should be resolved here, so you can later use simply PIDsibs.
Answer: Thank you. This abbreviation misplacement has been rectified as pointed out and suggested.
Lines 80-81: Please change this to something like “genotyping with less than 11 or 12 markers results in significantly changed estimates of population structure and genetic variation”.
Answer: Thank you for the suggestion. We have considered and changed it accordingly.
Line 83: Probably “being sampled [24, 25]”.
Answer: Thank you for the proofreading. We have rectified our mistake.
Line 89: Consider “create a baseline data of sloth bear population genetics parameters for eastern Vidarbha landscape”.
Answer: Thank you for your suggestion. We deemed that the suggestion made was better and have changed it accordingly.
Line 98: Tadoba Andhari National Tiger Reserve is misspelt. Additionally, this site is denoted as Tadoba Andhari Tiger Reserve in Figure 1. The names should be consistently used.
Answer: Thank you for pointing this out. We have re-checked and kept the names consistent in the document.
Line 103: Consider changing to “In total, 565 faecal samples of sloth bears had been collected” and deleting “from the sampling sites”.
Answer: Thank you. The change as been made as suggested.
Line 119: Maybe “locality-associated data”?
Answer: Thank you for the suggestion, the change has been made.
Line 153: Simply “until further processing”.
Answer: Thank you. The change as been made as suggested.
Line 160: Consider “Following an initial screening of the markers”.
Answer: Thank you. We have not made this particular change because the present wording seemed appropriate.
Line 164: The title should be “Microsatellite markers selected for the study of sloth bears”.
Answer: Thank you for the suggestion. The title of the para (if that is what is being suggested), semed appropriate to us since the contents of the paragraphs as put in a gist to make sense.e
Line 186: Simply “with a few modifications”. Also “An overnight…”.
Answer: Thank you for the comments. We have made the changes.
Line 188: Use “according to the manufacturer’s instructions” instead of “following the steps provided by the manufacturer”.
Answer: Thank you for the comments. We have made the changes.
Lines 216-217 and generally: Please use the name and locality of manufacturers consequently.
Answer: Thank you for the comment. We have added the appropriate locality of the manufacturers.
Lines 218-221: This part is duplicated in lines 224-225. The latter is probably a better placement. Additionally, please use “three times” instead of thrice.
Answer: Thank you the changes have been made as suggested.
Line 234: What did you mean by “and were also done.”?
Answer: Thank you for pointing this out. We rechecked and understood that the sentence was misplaced and rectified it.
Line 249: Please add which version of BayesAss was used, and the running parameters.
Answer: Thank you. The details have been added as directed.
Lines 254-259: This part is a reiteration of the methods, it should be deleted.
Answer: Thank you. The lines have been placed there not to reiterate but to talk about the consistency. We adjudged that by citing the numbers in these lines it would be easier for the reader to comprehend.
Line 295: Maybe “Variability of microsatellite markers …”, and annealing temperatures would fit better in Table 1. Additionally, use either a superscript or the abbreviation itself to resolve it in the table legend, but please be consequent.
Answer: Thank you. We considered putting the terms however Ta is an universal term hence in the table we put Ta. We have however added Ta in table legend.
Lines 311-314: The phrasing of this part is a bit strange. You could rephrase it something like: “The neighboring populations of TATR and UKWLS were assigned to one genetic cluster with UKWLS sharing some genetic signature with the adjacent NNTR population. The populations of NNTR and PTR formed the next cluster with PTR showing some links to the MTR population. The majority of samples from MTR were assigned to a third genetic cluster, while a small part of the MTR population grouped with samples from STR into a common cluster and the remaining part of the STR population was assigned to a separate group.”.
Answer: Thank you for your input. We have made the changes as directed since paragraph suggested made equal sense.
Line 321: Consider changing the title of Figure 2 to “TESS assignment plot of sloth bear across Vidarbha Landscape, India.” Also, the arrangement of the samples/population in Figure 2 is odd. It would be easier to see the clusterings if the order of the populations would resemble their geographic location, i.e. STR -> MTR -> PTR -> NNTR -> UKWLS -> TATR.
Answer: Thank you so much for your suggestion.The TESS figure has been renamed as suggested. We agree that even though placement of similar coloured bloack would be easier, we did not make changes to this since we wanted the populations to appear distinct. Hopfully this is comprehensible as well.
Line 328: A fairly high migration rate was also found between two fair away populations, STR and MTR. Why did you not mention it here? Especially because you discuss the link between the two later on.
Answer: Thank you. We did not mention it here because we only mentioned the highest rates of migration in this para. The programming was also done in a way to show just the highest migration because of which 2 migration routes have been highlighted here. We will however, add the BayesAss table to the appendix if that is appropriate and sufficient.
Line 344: Consider changing the title of Figure 3 to “Circos plot of migration rates between different protected areas of the Vidarbha Landscape, India.”
Answer: Thank you for the comments. The change has been made.
Line 353: Probably “from degraded samples”.
Answer:Thank you. The change has been made.
Lines 353-355: “Based on reproducibility and consistency, 13 markers were selected for the study. The final panel was divided into six multiplex reactions to make the entire process cost effective and efficient.”
Answer:Thank you. The change has been made as suggested by you.
Line 402: This sentence is pointless, please delete it.
Answer:Thank you for the suggestion. Even though we have not deleted it completely we have re-worded the same.
Lines 417-418: This sentence is pointless, please delete it.
Answer:Thank you for the suggestion. Even though we have not deleted it completely we have re-worded the same
Reviewer 3 Report (Previous Reviewer 3)
Comments and Suggestions for AuthorsDear Authors,
I have now reviewed your manuscript “The Bear truth: Analyzing genetic variability and population 2 structure in sloth bear across Vidarbha Landscape using microsatellite markers”.
I have noticed that you tried to address most of the suggestions and comments. Thanks for the efforts. However there are still a need of careful editing and addition of inadvertantly missed citations at various places.
78: Change “in the study landscape” to “Eastern Vidhrbha landscape”.
Fig 3: The labels are still not visible, but I believe that is due to PDF quality and will be improved. This figure is referred as Circos plot here and Circus plot in the text?
L 413: “such as tigers [50]”. There are other studies on tiger genetic structure in this landscape. Please cite them as well.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0029827
https://onlinelibrary.wiley.com/doi/full/10.1002/ece3.432
https://royalsocietypublishing.org/doi/abs/10.1098/rspb.2013.1506
L 431-433: There is one study that enumerated all different kind of barriers to movement in this landscape in India. Please cite this study for relevance.
https://peerj.com/articles/5587/
L439-440: There is one recent study that synthesized connectivity for a large carnivore species in this landscape. This study also emphasizes the importance of stepping stones for maintaining connectivity. I suggest authors to cite it.
https://conbio.onlinelibrary.wiley.com/doi/full/
Comments on the Quality of English LanguageThis MS requires a careful editing for English.
Author Response
Dear Reviewer 3 , Thank you for your kind words and inputs. We have tried our best to understand the gaps pointed out and fill them. We believe that with the inputs suggested out MS has been polished as well.
78: Change “in the study landscape” to “Eastern Vidhrbha landscape”.
Answer: Thank you for your input. We have made the change to make the sentence more specific to evl.
Fig 3: The labels are still not visible, but I believe that is due to PDF quality and will be improved. This figure is referred as Circos plot here and Circus plot in the text?
Answer: Thank you for pointing this out and understanding. We believe that the quality of PDF given for review might not be very clear. However, we would like to say that while submitting the quality seems good, comprehensible and we also submit JPEG images of the same.
We have also added a small part regarding visualization of the bayesAss results in the text using circus plot.
L 413: “such as tigers [50]”. There are other studies on tiger genetic structure in this landscape. Please cite them as well.
Answer:Thank you for the suggestions. We have read through the papers before this too. We deemed 2 of the suggested papers to be very important with regards to the point being made and hence have included them.
The 3 rd paper regarding historical gene flow maintenance in forest corridors, though extremely important and a good read, but was not pertinent to the point being made. And hence was not included.
L 431-433: There is one study that enumerated all different kind of barriers to movement in this landscape in India. Please cite this study for relevance.
https://peerj.com/articles/5587/
Answer: Thank you for the suggestion. We had previously seen this paper but had not included it. However, now since the “Discussion” part was being refined we deemed that this paper would make a good citation even though mainly focussed on tiger connectivity.
We have included the same in the Discussion part.
L439-440: There is one recent study that synthesized connectivity for a large carnivore species in this landscape. This study also emphasizes the importance of stepping stones for maintaining connectivity. I suggest authors to cite it.
https://conbio.onlinelibrary.wiley.com/doi/full/
Answer:Thank you for the suggestion. However, the link given was probably not accurate and hence we could not open the suggested citation. We did try to find the suggestion based on the journal/ words but could not zero-in on any paper.
Round 2
Reviewer 1 Report (Previous Reviewer 1)
Comments and Suggestions for AuthorsDear Authors,
in my opinion, the manuscript 'The Bear truth: Analyzing genetic variability and population structure in sloth bear across Vidarbha Landscape using microsatellite markers' has been significantly improved after correction.
I would suggest moving Table 1 to the Appendix (Supplementary) of the manuscript.
I have no further suggestions or comments to correct the manuscript text.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsDear Authors,
As a reviewer, I have reviewed the proposed manuscript entitled "The Bear truth: Analyzing genetic variability and population structure in sloth bear across Vidarbha Landscape using microsatellite markers and its implications in management of stepping stone patches".
I believe that the proposed format contains a number of inconsistencies.
The title of the manuscript is long, and more importantly, it does not correspond to the content of the proposed manuscript. As a general principle, it's crucial to establish a clear link between the title, research objectives, methodology, discussions, and conclusions.
The section labeled "Introduction" is brief and doesn't adequately acquaint the reader with the core issue. The scientific aim of the research (as outlined in the proposed manuscript) is not explicitly stated.
The chapter "Material and Methods" does not specify the number of samples per area (which is important for the later discussion). Population 6 is represented by three individuals, which is certainly not enough to evaluate it as a population. The subchapters DNA extraction and primer standardization, PCR standardization and data validation, and Data analysis are too long. The subchapters should be numbered.
In the "Results" chapter, there are sentences that rather belong in the "Materials and Methods" chapter (e.g.: "A total of 565 scat samples of wild sloth bear had been collected from study area in Maharashtra for standardization purposes. 7 confirmed tissue samples had been provided by Nagpur Zoo (2 blood samples) and Forensic laboratory facility of Wildlife institute of India (2 hair, 1 skin, 1 meat, 1 bone). ...." It is necessary to calculate and display population parameters by population. Some information is missing in Table 2 (e.g. G1D or G10L allele size).
The chapter "Discussion" some results of the research are repeated "In this paper we have tested and standardized a panel of 13 microsatellite tagged primers to infer population structuring and genetic variability. Using a larger number of primers at the initial stage allowed us to test for appropriate panels to identify individuals with robust statistical bolstering. We have taken care to include both dinucleotide and tetranucleotide markers to balance stuttering from peaks and higher amplification success from degraded [29]. The final panel of 13 microsatellites were put into 4 different multiplex reactions based on their temperature to make the entire process cost effective and efficient. The fundamental aim in this study was to develop a streamlined and robust panel for sloth bear genetic monitoring and demonstrate their use to infer population-level genetic information in the Vidarbha landscape. ..."
Some statements are incomprehensible (e.g. "The results showed that the observed heterozygosity for most loci was greater than the expected heterozygosity thereby establishing genetic diversity [29]).
Some claims are not part of the research itself (e.g., Compared to polar bears or brown bears, whose movement have been widely studied, very few studies have investigated the movement and range of sloth bears. On an average the home range of sloth bears ranges between a 2 sq kms to more than 100 sq kms [2,36,37] based on VHF collaring data whereas dispersal studies have not been conducted. Studies have shown that stepping stones are capable of dispersal of long ranging species and can aid in connectivity [38] and the role of such fragments in the Vidarbha Landscape cannot be overlooked).
The conclusions proposed in the manuscript are not clearly related to the title of the manuscript.
The references are not written in accordance with the journal's instructions.
Author Response
please see the PDF file
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors tested and standardised a panel of 13 microsatellite markers and did preliminary population genetics analyses of sloth bears in India. The population genetics of sloth bears in India is not well examined, thus the analyses done and their results are interesting, potentially for a greater audience. The manuscript's structure seems fairly deliberate and the text is mainly clear and concise. Although it is only a stylistic issue, the use of space linked to punctuation is inaccurate, a bit more emphasis should be given.
The Introduction is too short and incomplete. The manuscript starts with some background information about the sloth bear, however, the current status or conservation and management implications are completely omitted. Not all readers interested in the study are familiar with the focal species, more information should be presented here. The Introduction of a scientific article usually ends with a paragraph describing the aims of the study, which is completely missing from this manuscript; although there is a sentence in the Discussion stating the fundamental aim.
Please change the subsection “Sampling Area Sampling” of Materials and Methods into something like “Study Area and Sampling”. The abbreviations of the sampling areas should be given here when you use abbreviations later on.
A column containing the motives (if known) or at least the repeat type (i.e. di- or tetranucleotide) could be added to Table 1. Additionally, Table 1 should be referenced in the text where the 13 microsatellites are mentioned. Line numbering would help to pinpoint suggestions.
The subsection “DNA Extraction and Primer Standardization” only details DNA Extraction, what do you mean by “Primer Standardization”? Also, please consider changing the last sentence to something like “Genomic DNA was extracted from tissue samples using the DNaesy Kit (QIAGEN) and from bone samples using the GeneiPureID DNA Isolation Kit-Bone (Merck) following the manufacturer’s protocols.”. You should be consistent with the names of manufacturers throughout the section: QIAGEN is misspelt a few times and the manufacturer of instruments used (like the ABI 3500xl Genetic Analyzer) should be also mentioned.
References [25] and [26] are in the wrong order. The correct citation for the Cervus software is [27], for gimlet [28] and for BayesAss [34].
The citation of [28] on page 6 seems odd. What did you want to underline with it?
“TESS assigned a cluster value of 5 for the populations of sloth bears based on the lowest value of DIC before reaching a plateau (Figure 2).” suggests that Figure 2 contains a diagram of DIC values according to K. It would be informative to include this diagram.
The Circos plot in Figure 3 is hard to look into. A little explanation of the results could be added to the text; at least mentioning where the highest migration rates were found would be recommended.
It is mentioned in the Discussion that the primers were multiplexed, but multiplexing was not described in the Methods section. You should at least include which primers were multiplexed together.
“STR shows two very distinct genetic signages, one of which is also seen in MTR. PTR , BRM and NNTR are also seen to share genetic signages. TATR, UKWLS and BRM also have genetic similarity.” is confusing. You probably meant that individuals belonging to two clusters were found in STR. This should be rephrased.
Interestingly, you found a fairly high migration rate between Sahyadri Tiger Reserve and Melghat Tiger Reserve, which are around 500 km away. The migration rate between the two areas seems quite higher than some between adjacent areas, which is surprising. You mention that dispersal/migration of sloth bears has not been studied well, but you missed the opportunity to discuss this surprising result. Additionally, if your title says “and its implications in management of stepping stone patches”, you should discuss management implications.
Author Response
please see the pdf file
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsDear Authors,
I have now reviewed your manuscript “The Bear truth: Analyzing genetic variability and population structure in sloth bear across Vidarbha Landscape using microsatellite markers and its implications in management of stepping stone patches”.
Your study presents an interesting research on an often-ignored species. I am sure that this study will have important conservation implications. However, I found several missing pieces in the text and interpretation of results changes without those comparisons. Below are some of my major and minor comments. I expect the authors to address all of these issues, after which their manuscript will improve considerably.
ABSTRACT:
Restructure this sentence in the abstract: “Field techniques alone are not sufficient” explain which field techniques you are referring to and why they are not sufficient?
You are talking about “population dynamics” in the fourth sentence but it is more of demographic parameter and you haven’t done that work in your study, so remove that word.
Name those molecular markers in sentence 6? Microsatellite
Sentence 7: Minimal migration? What do you mean by that? It sounds very qualitative!
Last sentence: “reduce man-bear conflicts”. First I would suggest to change it to “human-bear conflicts” to be gender-neutral. Second, please explain how your finding will help in tailor-making of management strategies to reduce man-bear conflicts?
INTRODUCTION
Second para, second sentence: Please cite this recently published review paper about Asian bear monitoring.
Proctor, M.F., Garshelis, D.L., Thatte, P., Steinmetz, R., Crudge, B., McLellan, B.N., McShea, W.J., Ngoprasert, D., Nawaz, M.A., Te Wong, S. and Sharma, S., 2022. Review of field methods for monitoring Asian bears. Global Ecology and Conservation, 35, p.e02080.
Second para, third sentence: “however, studying sloth bear populations using genetic methods is sporadic in India.” Please cite those sporadic studies? To my knowledge there are two systematic population genetic studies implemented in India so far.
Second para, fifth sentence: “while no study has used genetic tools to infer population structuring and variability”. I think you might have missed something in your home-work! The first population genetic study on sloth bears in India was done by Dutta et al. (2015) * and I am surprised that neither you mention this study anywhere in your paper nor you acknowledge the only other study (Thatte et al. 2020) as being pioneering research in India.
*Dutta T, Sharma S, Maldonado JE, Panwar HS, Seidensticker J (2015) Genetic Variation, Structure, and Gene Flow in a Sloth Bear (Melursus ursinus) Meta-Population in the Satpura-Maikal Landscape of Central India. PLoS ONE 10(5): e0123384.
Sentence 6: Wang et. al. ………I see your argement about increasing the number of loci, but it’s not just about number of loci, their variability (estimated as polymorphism) and number of sampled individuals per population also matters. Landguth et al (2011) mentioned “We conclude that amplifying more (and more variable) loci is likely to increase the power of landscape genetic inferences more than increasing number of individuals.”
Please see these two papers and cite them.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3440332/
https://onlinelibrary.wiley.com/doi/full/10.1111/j.1755-0998.2011.03077.x
Page 2: line 4: Reference number 14 is not relevant here.
MATERIALS & METHODS
Please give a break-up of samples collected from each PA, along with other information that you present in table 2 for each of those PA in another table. This will help in assessing population specific genetic parameters.
First para, line 15: I don’t think sloth bears have specific latrine sites! So please rectify this.
Page 4, para 1, line 3: Please provide these different standardized protocols of DNA replication, PCR process and multiplex combination with annealing temepratures as supplementary information so that your work can be replicable.
It’s good to see that you have used stringent measures of quality control following Miquel et al.
Page 5, line 1: Incomplete sentence!
and were also done[25]. What?
I am wondering why you didn’t use STRUCTURE and used TESS instead? Please explain
RESULTS:
Line 2: Change “7 confirmed tissue samples” to “7 confirmed reference samples”.
Your sample size mathematics doesn’t add up! You collected 565 scat samples and 7 reference samples. That’s a total of 572. Now in the methods section you mentioned that you used 85 samples for standardizing and loci selection, and remaining 480 for subsequent analysis, which is a total of 565, so where did you use these 7 samples?
Table 1: Please provide an average of all those genetic parameters at the bottom of the table.
Fig 2: in the X axis, the number of populations are 6! Please mark those 5 genetic populations that you inferred below these sampled population.
Was there any logic in combining TATR with UKWLS. Looking at your map (Fig1) I presume that they are separately sampled regions.
Fig 3 is not legible at all. Please make the labels bigger. Do those colors correspond with colors in the TESS figure (Fig 2).
DISCUSSION
Para 1, last sentence on page 7: “Using 13 microsatellites the PIDsibs value obtained in this study 1.48×10-5 should be sufficient to conduct a large spatial scale study and is better than other studies conducted before this.” Please cite those other studies here.
Para 1, last sentence on page 8: “we focused on collecting samples from different individuals in the landscape for maximum heterozygosity information”. This sentence seems very odd. What maximum heterozygosity information you would expect by sampling different individuals. These diff individuals might be part of a single clan in small populations and might not have max heterozygosity due to inbreeding and drift effects.
Second para (5.2) line 5: “certain level”! be more specific.
Line 6: STR shows two genetic populations, please provide more explanation here? Looking at your Fig 1, its one of the most distant populations but it has shown admixture with MTR population as well. This needs some explanation based on your experience of sloth bear ecology and dispersal in the landscape. You might want to search for explanation and supporting statements from other studies done over other carnivores (tigers, leopards, jungle cats) in the Central Indian landscape!
I would also suggest the authors to do a comparison of their study results with those of Thatte et al (2020) and Datta et al. (2015). Are there any similarities or discrepancies in the results? Why is it so?
CONCLUSIONS
Change “This study is the first in the landscape” to “This study is the first in the Vidarbha landscape” to provide specificity to your work.
SUPP table: Please provide the labels of sampled populations (same that you used in Fig 2) instead of numerals in the column B “POP”.
Comments on the Quality of English Language
Has room for improvement.
Author Response
please see the pdf file
Author Response File: Author Response.pdf
