Phylogeography of Dolichophis Populations in the Aegean Region (Squamata: Colubridae) with Taxonomic Remarks

Round 1

Reviewer 1 Report

Abstract:

Abstract is clearly organised and written. However, it should be of 200 words maximum. It was 218 words my suggestion “So far, the genus Dolichophis has been inadequately studied in the Aegean region.  This study investigates phylogeographic patterns of Dolichophis species in the Aegean area, aiming to elucidate their genetic diversity and historical colonization routes through mitochondrial and nuclear DNA data. Our findings revealed…”

Comments on style:

References in brackets should be in normal rather than bold style.

It should be division of 2. Material and Methods and 3. Results sections into subsections, please check papers published in MDPI Diversity

Instead of (see Fiugure ..) it should be (Figure …)

 Introduction:  

the problem and significance of the study are adequately covered

L41 delete “and references therein”

L75-78 use the past tense rather than the present tense

L75-78 you should mention that genetic research was based on mtDNA and nuclear sequence analysis

 Methods:

Some information is missing and should be clarified.

L89-90 company, city and state of DNA extraction should be indicated in brackets

Table 1 is too long for the manuscript, I suggest to move it into supplementary material; please add GenBank numbers

L100-111 PCR conditions, polymerase used, quantities of materials, temperature profile are missing  

L108-109 only to check loci but not species?

L111-112 what algorithm was used for alignment?

L126 haplotype diversity was not assessed?

L141 reference is missing

 Results:

Presentation of results is unstructured and unclear

Figure 1 caption. What does the asterisk in the yellow circles mean? It is confusing than “A small black circle indicates a missing or hypothetical allele” is the same as for one of the analyzed speices, use other colour, for example red. In figures 1-3 it is not clear what does notes, such as 493b-Kos means?

Figures 2 and 3. Part A is on the top left and part B is on the bottom, make it the same.

My suggestion is to divide Figure 1 into two images i) A ii) B + C, thus giving a clearer insight into what the authors are depicting, now the lettering is in very small font

L190-192 I do not understand low intraspecific diversity, but authors provide comparison between taxa. Nucleotide diversity π should not be evaluated using percentages, what are standard deviation of nucleotide diversity values.

It would be better to present π and Hd values in table.

L209-211 I do not agree, you have obtained not differing values for both speices 0.88 and 0.86, networks show two genetic lineages for both species (L22-23) “contrasting with D. jugularis, which displayed low genetic variability“ the sample is too small to make such conclusion.

What is explanation that Dolichophis caspius comparing to D. jugularis had more haplotypes within mtDNA but less alleles in analysed nDNA loci?

Discussion

The discussion needs to be more structured. Please divide the discussion into more subsections, now there are only two subsections and each one is about two pages long. Since discussion is rather long and complicated, CONCLUSIONS should be included in the manuscript.

Author Response

Responses to Review 1

Dear colleague, thank you for your careful review of our work. Please find below our responses to your review report.

Abstract:

Abstract is clearly organised and written. However, it should be of 200 words maximum. It was 218 words my suggestion “So far, the genus Dolichophis has been inadequately studied in the Aegean region.  This study investigates phylogeographic patterns of Dolichophis species in the Aegean area, aiming to elucidate their genetic diversity and historical colonization routes through mitochondrial and nuclear DNA data. Our findings revealed…”

• Thank you. We have shortened the abstract based on your recommendations and grammatical corrections from the second reviewer.

Comments on style:

It should be division of 2. Material and Methods and 3. Results sections into subsections, please check papers published in MDPI Diversity

• Thank you, these sections are re-structured now.

Instead of (see Figure ..) it should be (Figure …)

• corrected

 Introduction:  

References in brackets should be in normal rather than bold style.
L41 delete “and references therein”
L75-78 use the past tense rather than the present tense
L75-78 you should mention that genetic research was based on mtDNA and nuclear sequence analysis.

• Thank you for all your comments. We have incorporated them all into the new version.

Methods:

Some information is missing and should be clarified.

Table 1 is too long for the manuscript; I suggest moving it into supplementary material; please add GenBank numbers.

• Thank you for your suggestion. While we generally agree, we prefer to keep the table in the main body of the manuscript for better and faster connection between information, as it contains initial data on the genetic diversity of the Aegean populations of the genus, as well as GenBank data, and is closely related to Figures 1-3.

L100-111 PCR conditions, polymerase used, quantities of materials, temperature profile are missing. 

• We added information about used polymerase. The PCR condition, etc are presented in Table S1.

L108-109 only to check loci but not species?

• improved

L111-112 what algorithm was used for alignment?

• The default Geneious alignment algorithm was used. We have added this information into the manuscript.

L126 haplotype diversity was not assessed?

• Thank you for pointing this. We corrected it.

L141 reference is missing

• We have added references, thank you.

L89-90 company, city and state of DNA extraction should be indicated in brackets

• Thank you, we have incorporated all these comments into the manuscript.

Results:

Presentation of results is unstructured and unclear

Figure 1 caption. What does the asterisk in the yellow circles mean? It is confusing than “A small black circle indicates a missing or hypothetical allele” is the same as for one of the analyzed speices, use other colour, for example red. In figures 1-3 it is not clear what does notes, such as 493b-Kos means?

Thank you. We have improved the figure by replacing missing haplotypes with empty circles. Additionally, we have added information about the asterisk (*) to the figure legend and to the text (Lines 167-168). Furthermore, we have included explanations for the codes.

Figures 2 and 3. Part A is on the top left and part B is on the bottom, make it the same.

• Corrected

My suggestion is to divide Figure 1 into two images i) A ii) B + C, thus giving a clearer insight into what the authors are depicting, now the lettering is in very small font

• Thank you for the suggestion, but we prefer to keep figure as is. The figure is complex and illustrates the main comparisons between the distribution of species/lineages based on mtDNA and the divergence in nDNA. All in one view provides good comparison. However, we will ask the production team to make figure in the manuscript enlarged.

L190-192 I do not understand low intraspecific diversity, but authors provide comparison between taxa. Nucleotide diversity π should not be evaluated using percentages, what are standard deviation of nucleotide diversity values.

• Thank you, we corrected this part. These values are related to average intraspecific distances.

It would be better to present π and Hd values in table.

• There are only a few resulting numbers, and this part represents a minor portion of the manuscript. That's why we suggest mentioning these data directly in the text.

L209-211 I do not agree, you have obtained not differing values for both speices 0.88 and 0.86, networks show two genetic lineages for both species (L22-23) “contrasting with D. jugularis, which displayed low genetic variability“ the sample is too small to make such conclusion.

• Thank you, we corrected this part.

What is explanation that Dolichophis caspius comparing to D. jugularis had more haplotypes within mtDNA but less alleles in analysed nDNA loci?

• Good question - As for the higher nDNA divergence of jugularis compared to mtDNA for caspius, we find it reasonable that the former would exhibit overall higher genetic differentiation than the latter, given their respective distributions and the fact that the former represents a species complex with undetermined taxonomy. It's challenging to determine why this pattern doesn't manifest in our sample, but several factors could contribute, including genetic drift on a specific marker. Other potential reasons might include gene flow patterns, founder effects, sex-biased dispersal, and natural selection acting on either the genome or the species. However, all this need better testing in the future and currently it is not needed to develop it in the text.

Discussion

The discussion needs to be more structured. Please divide the discussion into more subsections, now there are only two subsections and each one is about two pages long. Since discussion is rather long and complicated, CONCLUSIONS should be included in the manuscript.

• We have made some improvements to the discussion section. Since the conclusion is not typical for Diversity papers and our discussion provides all the necessary information, we decided to avoid more text.

Reviewer 2 Report

This MS showing the phylogeography and taxonomic study on the Aegean Dolichophis populations is of scientific value and should be considered for publication after some major minor improvements.

Major: as you have a sufficient molecular dataset, I strongly recommend a phylogenetic tree that shows the divergent time between the different populations, since your MS is focused on the phylogeographic study.

Minor: 

Table1: Use abbreviations of the genus name to save the space of the tables：D. jugularis...

Some small grammar mistakes should be improved (see below...)

L19：No not use "So far" to begin a sentence, it's not good English,

L49: Since Miocene

L 65: has not been

L 86 ：It seems meaningless to emphasize the "new" sample...

L 87 sample codes 

L 254 and diversification remain relatively...

L261: species-leveled...

Author Response

Responses to Review 2

Dear colleague, thank you for your detailed work and review reports. Please find our responses below.

Major: as you have a sufficient molecular dataset, I strongly recommend a phylogenetic tree that shows the divergent time between the different populations, since your MS is focused on the phylogeographic study.

Thank you for bringing up this point. While we generally agree that molecular dating could serve as a useful data source for connecting the evolution of the genus/species with biogeography, in our dataset, it would be rather impractical for several reasons:

• We do not have a well-represented DNA dataset, particularly regarding nDNA data, which are limited.
• On the intrapopulation level, these analyses would not provide significant insights due to the relatively short time of diversification. Molecular clock analysis is typically more suitable for deeper divergences.
• Our dataset covers only a portion of the entire genus range and the expected diversity within it.
• Previous studies, such as Kyriazi et al. (2013) and Jablonski et al. (2023), have already tested the molecular dating phylogeny where different colubrid snakes, including Dolichophis, were included. Also, Nagy et al. (2010) provided estimations based on genetic distances. Any additional molecular clock estimations would likely yield intervals ranging from the present (0.0 MYA) to somewhere in the Pliocene, resulting in inconclusive results without significant implications.

Therefore, while we appreciate the suggestion, we believe that molecular dating analysis would not contribute substantially to the findings of our study.

Table1: Use abbreviations of the genus name to save the space of the tables：D. jugularis...

• Thank you, we used abbreviations in the table.

Comments on the Quality of English Language
L19: No not use "So far" to begin a sentence, it's not good English,
L49: Since Miocene
L65: has not been
L86: It seems meaningless to emphasize the "new" sample...
L87: sample codes 
L254: and diversification remain relatively...
L261: species-leveled...

• Thank you, we have incorporated all grammatical corrections into the manuscript.

Round 2

Reviewer 1 Report

Authors have significantly improved their manuscript and I have noticed only few technical errors.

L56 these two snake species

L61-62 at first mentioning please use nuclear (nuDNA) and mitochondrial (mtDNA) genetic data

L90 some information is missing in this sentence “Was used Red Taq 2X Master Mix 2.0 mMgCl2.”

L159 Mitochondrial DNA Data

L179 Nuclear DNA Data

In the future research I advise to choose simpler data encoding (not such as 492, 493, 12199, 763 (ZMUP)) that readers could get an idea of the species or geographical location of a sample without having to look at the detailed tables

Author Response

Dear Reviewer, thank you again for your careful review of our work. We have addressed the issues you raised and made the necessary corrections.