Fencing Can Alter Gene Flow of Asian Elephant Populations within Protected Areas

Round 1

Reviewer 1 Report

The manuscript presents that fencing can alter gene flow of Asian elephant populations within protected areas, and found that hard PA boundaries abutting slopes can fragment elephant populations. The topic is meaningful. However, there are many problems to be further improved as well.

Organization and language:

Comment #1: Line 22-35: Please reorganize the abstract following the steps as below: object, methods, main results and conclusion.

Comment #2: Line 66-108: These four paragraphs were methods, and should be placed in “2. Materials and Methods”.

Comment #3: P48-54: Are there any related study on elephants? Please supplement.

Comment #4: P113: Change “Study area” for “Study area and sample collection”, and supplement sample information in this part.

Comment #5: Line 126-129: Move “Fresh elephant dung samples were collected between 2014 and 2018 from 1,030 locations. The geographic coordinates were noted, and the surface layer of the dung scraped into 50 ml Tarson tubes with absolute ethanol. Samples were stored at -20ºC after transportation to the laboratory.” to “Study area and sample collection” section.

Comment #6: The position of the cited literature should be normative. Please consult the literature for reference, and please check through the text.

Comment #7: The results section should be reorganized methodically.

Comment #8: There are many points with linguistic and expression errors. So there urgently needs to be considerable editing of the paper in order to make its message clear. 

Such as:

Line 55-56: Landscape genetics helps understand how geographical and environmental features 55 structure genetic variation and gene flow [15].

Line 129: DNA extraction and analysis follow [24].
Line 584-585: Fernando, P.; Ekanayaka, S.K.K.; Pastorini, J. the elephant at the fence: almsman, panhandler, friend or foe? Eur. J. Wildl. Res. 584 2020, 66, 97, doi:10.1007/s10344-020-01435-5. In this reference, “the” should be “The”.

And so on.…………

Technique:

Comment #9: Line 175: Genetic diversity and population structure

For this section, the phylogenetic analysis and the haplotype network are always important contents for population structure. I recommend to supplement.

Comment #10: Line 309-310: Please provide the p value of AMOVA analysis.

Comment #11: Where is the result of gene flow?

Comment #12: Line 301: Generally, 0.05＜Fst≤0.15 means small or moderate level genetic differentiation. Here, “A considerable level of genetic differentiation”, please clarify.

Author Response

Reviewer 1

The manuscript presents that fencing can alter gene flow of Asian elephant populations within protected areas, and found that hard PA boundaries abutting slopes can fragment elephant populations. The topic is meaningful. However, there are many problems to be further improved as well.

Organization and language:

Comment #1: Line 22-35: Please reorganize the abstract following the steps as below: object, methods, main results and conclusion.

To the best of our understanding, our abstract followed the Journal’s guidelines, unless a more specific comment can help with indicating where the problem lies.

The abstract was structured according to the Journal’s guidelines:

• Background: The Asian elephant is mostly confined to mountainous ranges, and therefore risks population fragmentation if hard protected area (PA) boundaries abutting steep slopes prevent movement.
• Methods: We tested whether elephant gene flow is (i) controlled by slope and (ii) affected by the interplay between barriers and slope. We used 176 unique genotypes obtained non-invasively from fresh elephant dung to assess individual by individual genetic distance across the Western Ghats of India, a biodiversity hotspot. To assess landscape distance, 36 resistance models were produced by transforming a slope raster. Core areas and corridors were calculated from the raster that provided the best correlation between the genetic and distance matrices. The influence of closure of PAs on gene flow was examined for one region, the Nilgiri Biosphere Reserve.
• Results: The best resistance raster obtained by transforming the slope occupancy model, was better than Euclidian distance in explaining genetic distance, indicating that slope partially controls gene flow. Fencing elephant PAs on hilly terrain reduces core areas and disconnects corridors.
• Conclusions: Hard PA boundaries abutting slopes can fragment elephant populations, but this can be ameliorated by protecting the adjacent flatter terrain.

Comment #2: Line 66-108: These four paragraphs were methods, and should be placed in “2. Materials and Methods”.

We considerably revised the paragraphs. Some parts were retained, others moved to methods, and some deleted.

Comment #3: P48-54: Are there any related study on elephants? Please supplement.

The reviewer probably referred to Lines 48-54, not to pages. The paragraph was meant to describe the difficulties faced by elephants to climb slopes. It has two references specifically on elephants (Feng and Zhang, 2005 and Wall et al. 2006), one on heat dissipation in endotherms that mentions elephants four times (Speakman and Kroll 2010). Their observations are valid for large animals, including the elephant(s). Schmidt-Nielsen’s (1984) book addresses size in general and the allometric models presented are also valid for elephants. Wheatley et al. (2021), also mentions the Asian elephant when assessing risk perception.

All references seem to be appropriate.

Comment #4: P113: Change “Study area” for “Study area and sample collection”, and supplement sample information in this part.

Done.

Comment #5: Line 126-129: Move “Fresh elephant dung samples were collected between 2014 and 2018 from 1,030 locations. The geographic coordinates were noted, and the surface layer of the dung scraped into 50 ml Tarson tubes with absolute ethanol. Samples were stored at -20ºC after transportation to the laboratory.” to “Study area and sample collection” section.

Done.

Comment #6: The position of the cited literature should be normative. Please consult the literature for reference, and please check through the text.

An example would have been welcome. Is the reviewer referring to a particular source that advises on standard(s) or to articles that have been published by MDPI?

The numbering was done automatically by Zotero we checked the order again.

We see from recent articles in MDPI that citations (numbers) are placed at the end of a phrase or in the middle of a phrase with or without punctuation to substantiate an assertion. Sometimes, authors names are provided followed by the reference number,  eg: Ali et al. [23]. Sometimes an authority appears with the reference number (ex: Inland Fisheries Advisory Commission (1973) [44]) (ref: Buyain et al. 2022). Taking these observations into consideration, we understand that we should cite the author in places. For example, Line 86 of our article, we say “as in [17]” when we should maybe say “as in Shirk et al. [17]”. We reviewed our article entirely to make such corrections. Changes were made Line 58, 66, 87, 132, 172, 194 and 435 of the original submission.

Comment #7: The results section should be reorganized methodically.

The comment #7 is not specific. We followed a presentation that is straightforward in landscape genetics, starting with the genetics, the population structure, some field data used to calibrate / verify, resistance maps, choice of the best model and calculation of corridors / core areas. As far as the general organization is concerned, we feel that our present organization is appropriate.

Comment #8: There are many points with linguistic and expression errors. So there urgently needs to be considerable editing of the paper in order to make its message clear.

Such as:

 Line 55-56: Landscape genetics helps understand how geographical and environmental features structure genetic variation and gene flow [15].

We cannot find a linguistic or expression error in this phrase. “Structure” is a verb. It simply says that landscape genetic techniques and theories help understand how the environment (eg. mountains), structure genetic variation and gene flow.

Line 129: DNA extraction and analysis follow [24].

This point has been changed into “analysis follow Parida et al. [24].”

Line 584-585: Fernando, P.; Ekanayaka, S.K.K.; Pastorini, J. the elephant at the fence: almsman, panhandler, friend or foe? Eur. J. Wildl. Res. 584 2020, 66, 97, doi:10.1007/s10344-020-01435-5. In this reference, “the” should be “The”.

We made the change.

And so on.…………

We have addressed the citation style and we have entirely reviewed the literature where we found a few typos in three references.

Technique:

 Comment #9: Line 175: Genetic diversity and population structure

For this section, the phylogenetic analysis and the haplotype network are always important contents for population structure. I recommend to supplement.

We have only used microsatellite genotyped data to check whether there was a relationship between genetic distance and landscape distance (relative to slope). We have not used any mitochondrial DNA that would allow us to make such analysis.

Comment #10: Line 309-310: Please provide the p value of AMOVA analysis.

The probability (p < 0.001) was included. It is added to the text.

Comment #11: Where is the result of gene flow?

The reviewer has considered this paper as a population genetics study, which is not really the case.The population genetics has been covered in detail in an earlier paper by De et al. (2021) that we have used as a reference. Our paper is a landscape genetic paper, where barriers to gene flow are assessed at the landscape scale by least cost path modelling comparing genetic distance with landscape distance. Landscape genetics has considerable importance for the design of protected areas and corridors and our results is useful for the conservation of the Asian elephants.

In landscape genetics, one of the objectives is to improve prediction of gene flow within a landscape for conservation purposes. Genetic distances among populations (McRae and Beier 2008) or individuals (several articles by Sam Cushman, a co-author of this paper) are correlated against landscape distances (often measured from land use, slope, elevation, etc.). Maps that represent impediments to gene flow are produced. The figures 4 and 5 of this paper represent the areas of least resistance to gene flow relative to slope (core areas in figure 4) and least-cost path (figure 5).

In our study, genetic data is used to understand whether slope influences gene flow by a connectivity analysis. This is not a paper on population genetic analyses of WG elephants where gene flow is measured by some metrics such as the Fst, and two populations are compared with no reference to space. The results of the Fst (among others) analyses have been included in De et al. 2021 and shown provided in our paper as well.

Comment #12: Line 301: Generally, 0.05＜Fst≤0.15 means small or moderate level genetic differentiation. Here, “A considerable level of genetic differentiation”, please clarify.

A moderate level of genetic differentiation (as was in Line 412) is more appropriate. (changed in the text).

Reviewer 2 Report

Fencing can alter gene flow of Asian elephant populations 2 within protected areas. Puyravaud et al.

Comments;

I have reviewed the Manuscript by Puyravaud et al. entitled “Fencing can alter gene flow of Asian elephant populations 2 within protected areas” This manuscript provide information about the terrain slope partly controls gene flow for the Asian elephant. This research highlighted the sensitivity of elephant to slope raises the question whether mountainous habitats, where most elephant reserves are found today, are best suited as PAs. Although the use of the methodology and analytical approaches seem appropriate, but there are some flaws in presentation of results author need to add suggestion.   

Some minor comments are given below

1.     Author needs to calculate allelic richness of the both population and size differ.

2.     It is bit biased to compare the presence of private allele in two different population having difference in population size. In this condition population having low sample size will show less private allele. Which clearly visible in the present study. In this condition Bayesian based method will be best way to see the allelic difference between the population

3.     Statement in line number 408-410 is not clear.  If you want to Justify this statement then you need to check the Effective population size and then you can say that Effective population size will be the possible reason for Null Allele

4.     I am not agreeing with this statement in line number 411-413“as level of genetic diversity in similar in Both Populations and no signals for genetic structure.

5.     Fst would be reflection of bit historical connectivity. It would be good if the author calculate contemporary and first-generation migration then it would be clearer to say about the level of Connectivity or gene flow.

Comments for author File: Comments.pdf

Author Response

Review # 2

Comments; 

I have reviewed the Manuscript by Puyravaud et al. entitled “Fencing can alter gene flow of Asian elephant populations 2 within protected areas” This manuscript provide information about the terrain slope partly controls gene flow for the Asian elephant. This research highlighted the sensitivity of elephant to slope raises the question whether mountainous habitats, where most elephant reserves are found today, are best suited as PAs. Although the use of the methodology and analytical approaches seem appropriate, but there are some flaws in presentation of results author need to add suggestion.   

Some minor comments are given below

1. Author needs to calculate allelic richness of the both population and size differ.

We have now calculated allelic richness and included these values in Table 1.2.    

2. It is bit biased to compare the presence of private allele in two different population having difference in population size. In this condition population having low sample size will show less private allele. Which clearly visible in the present study. In this condition Bayesian based method will be best way to see the allelic difference between the population

We agree and we have removed private alleles and included allelic richness (Ar) in Table 1 now, as the number of private alleles will differ with sample size. The allelic richness is 7.6 in NBR and 5.8 in KMTR-ATR populations. Due to sample size differences, we have also provided the mean number of alleles (MNA). MNA was adjusted for discrepancies in sample size by incorporating a rarefaction method and was estimated for each region with HP-Rare 1.0. Applying correction for variable sample sizes using rarefaction, MNA ranged between 6.27 (KMTR-ATR) and 7.26 (NBR).

3. Statement in line number 408-410 is not clear.  If you want to Justify this statement then you need to check the Effective population size and then you can say that Effective population size will be the possible reason for Null Allele

We have already explained in the text and given an example of a study that showed “Null alleles are most likely to be found only in species with large effective population sizes due to high levels of diversity in flanking regions of target microsatellites [28]”. It is a well-known fact that the past effective population size of Asian elephants has always been very large. As we have mentioned above, this is not a population genetics paper and the main objective in the study is not to estimate the past and present effective population sizes, but to assess genetic distance with regard to landscape distance. The genotyped data is included in the study to support our main hypothesis and results. We have now deleted these lines from the text.

4. I am not agreeing with this statement in line number 411-413“as level of genetic diversity in similar in Both Populations and no signals for genetic structure.

It is difficult to understand why the reviewer is not agreeing with this statement on genetic diversity. It would have been useful if the reviewer had been more specific.

The genetic diversity is almost similar in both populations. Please check Table 1. NBR: Ho:0.74, He:0.69; ATR-KMTR: Ho=0.75, He= 0.62.

In fact, we observed a moderate level of genetic differentiation (pairwise Fst = 0.08; Gst = 0.04, and Djost=0.12) between the two populations in the north and south of the Palghat Gap suggesting limited gene flow between the populations. These results are in agreement with previous De et al., 2021 who found similar values (Fst=0.07 and Djost =0.10) for the sampled elephant populations along the Western Ghats using 14 microsatellites.

5. Fst would be reflection of bit historical connectivity. It would be good if the author calculate contemporary and first-generation migration then it would be clearer to say about the level of Connectivity or gene flow.

 We agree with this point. Population-based methods (e.g., Fst), offer historical perspectives and significant time lags can exist for genetic discontinuities to develop after barrier formation and these time lags make it especially difficult to detect changes in gene flow for species with long-generation times, such as elephants. Hence, we have now included both the movement data from the field observations and first-generation migrants (calculated using Geneclass2) data to support our statement. Our analyses detected first-generation migrants in both populations and suggests ongoing bidirectional gene flow.

Reviewer 3 Report

Detailed comments are in the attachment.

Comments for author File: Comments.pdf

Author Response

please see the attachment.

Author Response File: Author Response.pdf

Reviewer 4 Report

This research article by Prof. Dr. Priya Davidar and others is well designed and nicely executed. This manuscript do require minor reference formatting adjustments as per the journal. Rest of the contents are excellent and i recommend it for publication as it is, with minor reference reformatting adjustments.

Author Response

Review #3

 This research article by Prof. Dr. Priya Davidar and others is well designed and nicely executed. This manuscript do require minor reference formatting adjustments as per the journal. Rest of the contents are excellent and i recommend it for publication as it is, with minor reference reformatting adjustments.

Thank you very much. We appreciate your comments.

Round 2

Reviewer 1 Report