Review Reports - Recent Changes in Genetic Diversity, Structure, and Gene Flow in a Passerine Experiencing a Rapid Population Decline, the Dupont’s Lark (<i>Chersophilus duponti</i>)

Round 1

Reviewer 1 Report

This manuscript describes the population genetics of a Dupon't lark population across most of its geographic range.

Title - I recommend changing the title. ...strongly declining.... is a bit vague. maybe ..... experiencing a rapid population decline.....?

Line 48. Small isolated populations theoretically experience stronger genetic drift and inbreeding than large panmictic populations, accompanied by decreases in genetic diversity. [note, there is not necessarily a increased chance of extinction from these two factors- there are other genetic factors that may increase, like fixation of deleterious alleles, inability to adapt to changes as drift dominates adaptive evolution, etc.

I have some minor grammatical corrections below. I think the study is well done and well presented. The analysis is a standard population genetics analysis of populations from the wild using microsatellites. While the sample of genetic markers is small, the results are meaningful and the interpretation is fair. I am a bit surprised that the authors did not analyze the data using a baysian analysis such as STRUCTURE. But they did thoroughly analyse HWE, discussed their decisions for keeping markers and dispensing one, which are fair.

I think for this journal this is a nice paper that will add meaningfully to conservation of small natural populations where sampling is a significant issue to help make conservation decisions. I think that the paper is publishable as is and the authors can use my minor suggestions if they like.

Line 50. I think you can start the sentence with "The main criteria used by...." and delete the previous part of that sentence. Its redundant with the previous paragraph.

Line 67. remove work 'utility'

Line 69. Replace "..seem crucial" with ".... are crucial"

Line 88. remove 'as well as' and replace with 'and'

Author Response

Response to Reviewer 1 Comments

This manuscript describes the population genetics of a Dupon't lark population across most of its geographic range.

Title - I recommend changing the title. ...strongly declining.... is a bit vague. maybe ..... experiencing a rapid population decline.....?

Thanks!. Following the suggestion of the reviewer, we have modified the title of the manuscript in the revised version.

Thank you very much for your comment, with which we agree. We have modified the sentence in order to be less restrictive. Lines 50-52: “As a consequence, they have a higher possibility of losing genetic diversity and accumulate deleterious mutations which can contribute (among other factors) to extinction risks”.

Thank you for your positive remarks. We did run STRUCTURE analysis to test the genetic structure. However, results of such analyses were not conclusive for the aim of this study. STRUCTURE analyses only found two main cluster defined by each one of the countries sampled, with no substructure at any of them. Therefore, our aim was to compare genetic temporal trends within each region (in order to identify potential conservation units, since each region is under different human pressures and impacts) rather than to study the genetic structure of Dupont´s lark. So, we finally decided to exclude the Structure analysis from the manuscript.

Line 50. I think you can start the sentence with "The main criteria used by...." and delete the previous part of that sentence. Its redundant with the previous paragraph.

Ok. Modified

Line 67. remove work 'utility'

Ok. Modified

Line 69. Replace "..seem crucial" with ".... are crucial"

Ok. Modified

Line 88. remove 'as well as' and replace with 'and'

Ok. Modified

We have considered all annotations provided by the reviewer in the new version. Changes can be seen in the main text.

Author Response File: Author Response.docx

Reviewer 2 Report

The draft investigates the temporal variation in the genetic structure, diversity, and gene flow of the Dupont´s lark in its distribution range, in Spain and Morocco.

Particularly, the draft focuses on the critical and important issue dealing with the loss of genetic variability due to habitat reduction or fragmentation and furnishes concrete data on variability changes in a time span.

The manuscript is well-organized and fluent. Sampling, protocols, and statistical analysis are complete and adequate for addressing the proposed aims. In the discussion, results, and current and future scenarios are correctly debated.

I have only a few comments:

P L.120 The authors should explain why they retained only males and how females can affect the reliability of the results;

L.134 Ethanol is not the best way to conserve blood. This is only a suggestion for future analysis;

L.143 The authors should detail if they performed simplex or multiplex amplifications and something about the use of primer dyes;

L.155 The author could perform a bottleneck test to check for recent bottlenecks;

L.223 Some current or past populations show a departure from HW equilibrium. The authors should describe in the text and discuss these observations;

L.468 I suggest more caution in discussing Ne and Nc as from the table is evident that data are not very reliable. Too many discrepancies and forks too much wide;

Table 1. Table legend is too long and many theoretical details should be moved in the text or in the supplementary (e.g L.242-247)

Author Response

Response to Reviewer 2 Comments

The draft investigates the temporal variation in the genetic structure, diversity, and gene flow of the Dupont´s lark in its distribution range, in Spain and Morocco.

Thank you for your review and positive remarks. We have considered all your suggestions to improve the original version.

I have only a few comments:

P L.120 The authors should explain why they retained only males and how females can affect the reliability of the results;

Following the reviewer´s recommendation, we have included an explanation on this issue in the method section (lines 156-159). Basically, considering that the species have two or three clutches per season and that shows very limited dispersal (data from radiotracking and capture-recapture methods) we decided to focus only in territorial males, to exclude the potential presence in the sample of related individuals (unmoulted juveniles and daughters of previous broods that have not dispersed).

L.134 Ethanol is not the best way to conserve blood. This is only a suggestion for future analysis;

Ok. Thank you very much for the recommendation. However, we routinely use 99% ethanol because other preservation methods are not always a viable alternative, especially during long and remote collection campaigns (e.g. morocco).

L.143 The authors should detail if they performed simplex or multiplex amplifications and something about the use of primer dyes;

We have included the information mentioned in the text. Lines 168-171: “Twelve microsatellite loci isolated in Dupont´s larks (A112, B107, D115, D109, B9, C119, A7, C112, B10, D10, A113, D112; [51]) were amplified through a multiplex PCR using four dyes (FAM, PET, NED, VIC) in a total volume of 10µl”

L.155 The author could perform a bottleneck test to check for recent bottlenecks;

Following your suggestion, we have included the bottleneck test. However, considering that the power of these tests is very low when populations are sampled just few generations after the bottleneck, and that the time lapse between our two time points is 5-7 generations, we perform the tests using the pooled data (considering the samples of both periods in each region). We have modified the text in the method (lines 228-237) and discussion sections (lines 483-486) of the revised version.

L.223 Some current or past populations show a departure from HW equilibrium? The authors should describe in the text and discuss these observations;

Following your suggestion, information about HWE tests per region and period is now included in Table 1, and in the main text (Lines 185-188; 322-326)

L.468 I suggest more caution in discussing Ne and Nc as from the table is evident that data are not very reliable. Too many discrepancies and forks too much wide;

Following reviewer´s recommendation, we have added a statement in the discussion where we mention the caution required when interpreting such parameters according to the results obtained (Lines 512-517).

Table 1. Table legend is too long and many theoretical details should be moved in the text or in the supplementary (e.g L.242-247)

We agree with the reviewer that the Table legend was too long. We moved some theorical information to the main text (Lines 217-227) and simplified the Table legend in the revised version.

Author Response File: Author Response.docx

Reviewer 3 Report

Dear Authors,

This is a very interesting study on a vulnerable bird species, according to IUCN, which aims to investigate the temporal variation in genetic diversity and structure over time in Dupont’s Lark (Chersophilus duponti) across its range, using two time-spaced samples.

I have only few observations:

To make the map more explicit is better to add the species distribution area for Spain and Morocco. Like this we can see how your sample location covers the species distribution area.

Can you give more information regarding the steppe areas? About their managements in Spain and in Morocco?

Can you compare the genetic indexes with the same ones, from other species/studies (preferable close to this one, or at least sedentary species)?

Best regards,

Author Response

Response to Reviewer 3 Comments

Dear Authors,

This is a very interesting study on a vulnerable bird species, according to IUCN, which aims to investigate the temporal variation in genetic diversity and structure over time in Dupont’s Lark (Chersophilus duponti) across its range, using two time-spaced samples.

Thank you very much for your comments, that we have considered in the revised version

I have only few observations:

To make the map more explicit is better to add the species distribution area for Spain and Morocco. Like this we can see how your sample location covers the species distribution area.

Following reviewer´s recommendation, we have added the species distribution area in Spain and Morocco in Figure 1.

Can you give more information regarding the steppe areas? About their managements in Spain and in Morocco?

We have included further information regarding steppe areas (description and basic management in both countries) in the method section (lines 112-133)

Can you compare the genetic indexes with the same ones, from other species/studies (preferable close to this one, or at least sedentary species)?

Thank you for your suggestion. We have tried to obtain similar (comparable) information on other lark species but, as far as we know, this information is missing. Not only on temporal changes in diversity parameters, but even diversity estimates in single time points based on nuclear markers. And we honestly don’t think that comparison with species from unrelated bird families (e.g. Callens et al., 2011. Molecular Ecology, 20(9), 1829-1844) is really helpful.

Author Response File: Author Response.docx