The Mosquitoes of Morelos, Mexico: DNA Barcodes, Distribution, Ecology and the Resurrection of the Name Culiseta dugesi Dyar and Knab (Diptera: Culicidae)^†

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review of "The mosquitoes of Morelos, Mexico: DNA-Barcodes, distribution, ecology and the discovery of Culiseta vulcanica n. sp. (Diptera: Culicidae)" by Ortega-Morales et al.

 

This paper is a result of a long-term survey in a small Mexican state near Mexico City.  All due diligence was paid to researching background information on species that were historically reported in the area.  The new state records are important additions as well as the records of the species collected during the survey.  Barcode sequence of a subset of species is also an important addition to knowledge of mosquitoes of the area, and all of Mexico.  The addition of a species new to science is a big plus.  The descriptions of ecology for a number of species was good and the species chosen well.  I liked that more information was provided for species newly found in the state (Table 3).  Likewise, I liked that species not collected were discussed.  Also good are discussions of species that could be found in the state but haven't been yet, and the names, with references, of species removed from previous lists.

 

In general, the paper is understandable, except for a few instances where I couldn't quite tell what was meant.  I have marked these instances on the manuscript.  However understandable the language is it contains much awkward or non-standard wording.  These also are marked on the manuscript throughout.  It is not to be assumed that I have always chosen the best alternative wording and I left many questions for the authors to clarify.  Neither is it to be assumed that I found all instances of needed edits.  In other words, I didn't set out to re-write the paper, that will be up to the authors to be satisfied with their work. 

 

A few things stood out for me.  

 

The system of physiographic regions was confusing, and the reference given (number 41) did not lead me to an explanation, or anything approaching the subject.  My impression is that perhaps translation of the regions to English was not always the best.  I did try to find this online but the various "hits" were not consistent among themselves.  Please clarify.

 

Figure 1 needs a reference and if it is original or after some source (related to the above).

 

Table 1 also needs a reference or statement that it is a construct of the authors.  Note that Table 2 is quite well-referenced.

 

Figure 2.  Please clarify that this is a species accumulation curve for samples taken in this study, not all mosquitoes reported from the area.

 

Starting with line 312.  COI is most often used for discovering clusters of similar species and pointing to cryptic taxa.  It is not usually intended for phylogenetic analysis.  It's a similarity measure.  I'm sure I'm not telling the authors anything they don't know but the suggestion here is that this is a phylogenetic tree.

 

Photos.  Are the review photos to the same resolution as will be used for publication?  If so, they need to be improved.  I leave this to journal standards, however.

 

Table 4 information needs a reference of references.

 

Description.  It is up to the authors, but I would have designated the larvae as paratypes since they are associated in the minds of the authors with the holotype and were before them when they did the description.  Obviously, they were since a brief description is given.

 

References.  I did not check to see if the references corresponded to the text.  No time.  I do note that the journal doesn't require DOI numbers but that they are highly recommended.  I too recommend that they be included since it seems to be the norm now and it helps readers track the citations and they show up in citation tracking databases, which helps all authors.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

This is contained in the Comments and Suggestions for Authors section.

Much improvement needed.  A scientific writing service is suggested.

Author Response

REVIEW REPORT 1:

 

Review of "The mosquitoes of Morelos, Mexico: DNA-Barcodes, distribution, ecology and the discovery of Culiseta vulcanica n. sp. (Diptera: Culicidae)" by Ortega-Morales et al.

 

This paper is a result of a long-term survey in a small Mexican state near Mexico City. All due diligence was paid to researching background information on species that were historically reported in the area. The new state records are important additions as well as the records of the species collected during the survey. Barcode sequence of a subset of species is also an important addition to knowledge of mosquitoes of the area, and all of Mexico. The addition of a species new to science is a big plus. The descriptions of ecology for a number of species was good and the species chosen well. I liked that more information was provided for species newly found in the state (Table 3). Likewise, I liked that species not collected were discussed. Also good are discussions of species that could be found in the state but haven't been yet, and the names, with references, of species removed from previous lists.

 

In general, the paper is understandable, except for a few instances where I couldn't quite tell what was meant. I have marked these instances on the manuscript. However understandable the language is it contains much awkward or non-standard wording. These also are marked on the manuscript throughout. It is not to be assumed that I have always chosen the best alternative wording and I left many questions for the authors to clarify. Neither is it to be assumed that I found all instances of needed edits. In other words, I didn't set out to re-write the paper, that will be up to the authors to be satisfied with their work.

ANSWER: Authors greatly appreciate the editing work on the mansucript. All changes and suggestions were incorporated into our original manuscript

 

A few things stood out for me.

 

The system of physiographic regions was confusing, and the reference given (number 41) did not lead me to an explanation, or anything approaching the subject. My impression is that perhaps translation of the regions to English was not always the best. I did try to find this online but the various "hits" were not consistent among themselves. Please clarify.

ANSWER: This reference was corrected

 

Figure 1 needs a reference and if it is original or after some source (related to the above).

ANSWER: The legend “Map originally elaborated for this article” is added

 

Table 1 also needs a reference or statement that it is a construct of the authors. Note that Table 2 is quite well-referenced.

ANSWER: The reference (41) from INEGI is corrected

 

Figure 2. Please clarify that this is a species accumulation curve for samples taken in this study, not all mosquitoes reported from the area.

ANSWER: “Figure 2. Species accumulation curve for the 43 mosquito species (200 collections) collected in Morelos during 2009–2018”, indicating that only the species that were found during our collections (n=43) were included

 

Starting with line 312. COI is most often used for discovering clusters of similar species and pointing to cryptic taxa. It is not usually intended for phylogenetic analysis. It's a similarity measure. I'm sure I'm not telling the authors anything they don't know but the suggestion here is that this is a phylogenetic tree.

ANSWER: Autohrs appreciate this comment, yes authors are aware of the limitations of COI

 

Photos. Are the review photos to the same resolution as will be used for publication? If so, they need to be improved. I leave this to journal standards, however.

ANSWER: Yes, all photos were taken with the same resolution (≥400 dpi)

 

Table 4 information needs a reference of references.

ANSWER: The reference is added in this section

 

Description. It is up to the authors, but I would have designated the larvae as paratypes since they are associated in the minds of the authors with the holotype and were before them when they did the description. Obviously, they were since a brief description is given.

ANSWER: 10 larvae were designed as paratypes

 

References. I did not check to see if the references corresponded to the text. No time. I do note that the journal doesn't require DOI numbers but that they are highly recommended. I too recommend that they be included since it seems to be the norm now and it helps readers track the citations and they show up in citation tracking databases, which helps all authors.

ANSWER: Not all DOI are available for all references, since the journal dosent requiere DOI numbers, we prefer to leave the references as they are

 

 

 

 

 

 

 

 

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This study reported the species diversity, DNA barcoding, ecology and a novel species of mosquito from Mexico. Overall, the information reported in this manuscript is interesting and will be useful for further investigation relating to the disease epidemiology involving these mosquito species. Following are my comments and suggestions that I think could help to improve the manuscript.

- Abstract, there is no information related to the DNA barcoding results despite it was included in the title of the manuscript.

- Subheading 2.4, I think it is better to separate the genetic data analysis from the molecular work, i.e. adding another subheading e.g. Genetic data analysis.

- Table 2, what is meaning of the bold characters? Please specify.

- Line 188 – 189 and Fig. 2, please add some discussion on this topic. Based on Fig. 2, more species are expected to discover with increasing sampling effort?

- Page 9, subheading 3.3.1 – 3.3.4, name of the genus must be italic.

- line 312 – 354, this section needs additional information regarding the DNA barcoding analyses such as intraspecific and interspecific genetic divergences, percentage of successful identification based on COI sequences.

- Figure 3, the figure seems to be incomplete, some parts are missing. The posterior probability of Bayesian analysis lower than 0.5 should not present because it indicates low support. Figure caption needs more information e.g. number of COI sequences, number of species, the details of specimen label.

- Figure 6 and 7, it will be nice and useful to indicate important diagnostic morphological characteristics (e.g. in lines 388 – 389) of the novel species in these figures.

- Line 482, I suggest that “Conclusion” should be separated into different subheading.

- Table 4, this information should be present in “Results” section.

- Section 4.2 – 4.4 and 4.6 can be merged.

- There is no discussion on the DNA barcode. Please add this discussion. 

Author Response

REVIEW REPORT 2:

This study reported the species diversity, DNA barcoding, ecology and a novel species of mosquito from Mexico. Overall, the information reported in this manuscript is interesting and will be useful for further investigation relating to the disease epidemiology involving these mosquito species. Following are my comments and suggestions that I think could help to improve the manuscript.

Abstract, there is no information related to the DNA barcoding results despite it was included in the title of the manuscript.

ANSWER: This legend is included in the abstract: “Phylogenetic trees were constructed for 32 species, including Ae. shannoni which is new record for genbank databeses. Additionally, we report COI barcode sequences of one undescribed species, Culiseta vulcanica Ortega n. sp., which is described and deposited in CC-UL collection”.

Subheading 2.4, I think it is better to separate the genetic data analysis from the molecular work, i.e. adding another subheading e.g. Genetic data analysis.

ANSWER: The subheading “2.5 Genetic Data Analysis” is added

 

Table 2, what is meaning of the bold characters? Please specify.

ANSWER: The legend “New records for Morelos are indicated in bold” is added in the Table 2

 

Line 188 – 189 and Fig. 2, please add some discussion on this topic. Based on Fig. 2, more species are expected to discover with increasing sampling effort?

ANSWER: Yes, according Fig. 2 more species are expected to be discovered in Morelos. In this curve, we show only the 43 spp that we found. We improve the legend to “Figure 2. Species accumulation curve for the 43 mosquito species (200 collections) collected in Morelos during 2009–2018”, indicating that only the species that were found during our collections (n=43) were included

 

Page 9, subheading 3.3.1 – 3.3.4, name of the genus must be italic.

ANSWER: All genera were italized

 

line 312 – 354, this section needs additional information regarding the DNA barcoding analyses such as intraspecific and interspecific genetic divergences, percentage of successful identification based on COI sequences.

ANSWER: This paragraph was divided in two sections for improve understanding: 2.4. Polymerase Chain Reaction and DNA Sequencing and 2.5. Genetic Data Analysis

 

Figure 3, the figure seems to be incomplete, some parts are missing. The posterior probability of Bayesian analysis lower than 0.5 should not present because it indicates low support. Figure caption needs more information e.g. number of COI sequences, number of species, the details of specimen label.

ANSWER: We have reviewed the figure and corrected it as requested. Thank you very for the recommendations.

Figure 6 and 7, it will be nice and useful to indicate important diagnostic morphological characteristics (e.g. in lines 388 – 389) of the novel species in these figures.

ANSWER: Figures 6 and 7 were improved adding arrows pointing the diagnostic caracteres of the new species

 

Line 482, I suggest that “Conclusion” should be separated into different subheading.

ANSWER: Thank you for the suggestions, most of authors prefer to leave this section in its current state

 

Table 4, this information should be present in “Results” section.

ANSWER: Table 4 is presented in the Results section now

 

Section 4.2 – 4.4 and 4.6 can be merged.

ANSWER: Thank you for the suggestions, most of authors prefer to leave this sections in its current state

 

There is no discussion on the DNA barcode. Please add this discussion. 

ANSWER: We have added a paragraph and discussed our findings with regards the COI DNA barcodes as suggested.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript concerns mosquitoes inhabiting two regions of Mexico, including conserved forest regions, that have not been studied so far. The authors examined a relatively large number of individuals of all life stages collected over ca. 10 years (2008-2018). In the province studied, the authors found 8 more species compared to previous studies covering mostly urban areas. Morphological analyses supported by DNA analysis revealed the presence of a mosquito species new to knowledge, a description of which the authors included in the manuscript. An additional novelty as the work contributes to knowledge is the DNA-barcode sequence for Aedes shannoni.

A closer look at the results shows that the study has its shortcomings. Actually, the study contributes only two new DNA-barcodes to knowledge, despite the impressive study material: 26 species have not been sequenced. The authors explain that they could not obtain DNA for some species because most of them were collected as immature stages and all mounted on microscope slides. This is hardly convincing because it is possible to isolate DNA from most small invertebrates without damaging the individual (so-called “non-destructive methods”). Unfortunately, this material is no longer recoverable. However, even without it, the results obtained are worth publishing, but unfortunately not in their present form. The manuscript needs to be heavily redrafted, correctly written in English and resubmitted.

Major issues:

Fig. 1: The current map does not contribute relevant data for understanding the study; it should include sampling sites with a graphic representation of the size of the collected sample, the largest localities marked, the colors should be checked, as those used in the legend are not informative (lakes and rivers, for example, are not visible).

Table 1 is unnecessary, the authors do not correlate species with environmental factors, so it can be included in the supplements.

L119-121: "Hotshot technique" is known only to selected laboratories, so it should be described in detail; if the samples were sonicated, the device and settings used should be given, and the composition of the alkaline lysis buffer should be given.

The entire paragraph 2.4 should be rewritten. There are mistakes (e.g. ) and redundant information (e.g. ).

There are errors (e.g. L130: PCR "was carried out (...) forward primers developed by Folmer et al. [45]" - what about a reverse primer?) and redundant information (e.g. “which amplify long target region of the COI gene” – is there any shorter one used in this study?), but these are not all examples.

Fig. 2 does not contribute relevant data for understanding the study; much more needed here would be phenological data.

Table 2 in this form is needless, because it contributes little information. There should be a material table in the main text, with less data than Table 3, only the most important data: species, environment, location, GenBank accession number to the DNA sequence that was obtained in the study. All other information from tab. 2 and 3 should be moved to the supplementary material (ideally, it should be an Excel sheet with the ability to sort the data).

Paragraph 3.2 should be rewritten. It is descriptive and contains elements of discussion.

Paragraph 3.4: results of the phylogenetic analysis should be rewritten. What does is mean “ three strong groups of monophyletic species”? and where they are in the tree? what supports the clades have?  

L343-354: why has not DNA analysis been used to solve this problem?

I am not a morphologist, but in my opinion the description of the new species does not meet the standards: there are no drawings of morphological features distinguishing the new species, also there is a lack of DNA results in the differential diagnosis (incorrectly named “Systematics”).

Fig. 3 - the tree is illegible; should be edited, long unnecessary sequence names shortened, and preferably converted to GenBank accession numbers; species names should be marked with braces, etc.

The last three lines in tab. 4 should be included in the main table, e.g. marked with an asterisk or in bold “Human biting /Approaching to humans” species.

Minor issues (but also important):

Correctly, the new species is described by the abbreviation sp. nov. (species nova/novum)

Correct names: GenBank (without databases), GeneDoc, a stereomicroscope Discovery V8 (Zeiss), etc.

Amplicons (instead of “PCR products”)

L177: collection sites? or collected samples?

L179: “6 dissected male genitalia” are not the specimens, but their fragments (what about the specimens they belonged to?

English is not acceptable in many places.

Comments on the Quality of English Language

English is not acceptable in many places.

Author Response

REVIEW REPORT 3:

The manuscript concerns mosquitoes inhabiting two regions of Mexico, including conserved forest regions, that have not been studied so far. The authors examined a relatively large number of individuals of all life stages collected over ca. 10 years (2008-2018). In the province studied, the authors found 8 more species compared to previous studies covering mostly urban areas. Morphological analyses supported by DNA analysis revealed the presence of a mosquito species new to knowledge, a description of which the authors included in the manuscript. An additional novelty as the work contributes to knowledge is the DNA-barcode sequence for Aedes shannoni.

A closer look at the results shows that the study has its shortcomings. Actually, the study contributes only two new DNA-barcodes to knowledge, despite the impressive study material: 26 species have not been sequenced. The authors explain that they could not obtain DNA for some species because most of them were collected as immature stages and all mounted on microscope slides. This is hardly convincing because it is possible to isolate DNA from most small invertebrates without damaging the individual (so-called “non-destructive methods”). Unfortunately, this material is no longer recoverable. However, even without it, the results obtained are worth publishing, but unfortunately not in their present form. The manuscript needs to be heavily redrafted, correctly written in English and resubmitted.

Major issues:

Fig. 1: The current map does not contribute relevant data for understanding the study; it should include sampling sites with a graphic representation of the size of the collected sample, the largest localities marked, the colors should be checked, as those used in the legend are not informative (lakes and rivers, for example, are not visible).

ANSWER: The colors on the map in Fig. 1 were only used to differentiate between the different regions and physiographic sub regions of Morelos. We only used the dense vegetation shape file to indicate the conserved areas in Morelos, where the mosquito diversity was greater

 

Table 1 is unnecessary, the authors do not correlate species with environmental factors, so it can be included in the supplements.

ANSWER: This table was prepared to indicate the municipalities sampled, as well as to describe the biomes of the different physiographic regions in Morelos

 

L119-121: "Hotshot technique" is known only to selected laboratories, so it should be described in detail; if the samples were sonicated, the device and settings used should be given, and the composition of the alkaline lysis buffer should be given.

ANSWER: Thank you very for the comments, We have added the technique in full and added the device and setting as requested.

The entire paragraph 2.4 should be rewritten. There are mistakes (e.g. ) and redundant information (e.g. ). There are errors (e.g. L130: PCR "was carried out (...) forward primers developed by Folmer et al. [45]" - what about a reverse primer?) and redundant information (e.g. “which amplify long target region of the COI gene” – is there any shorter one used in this study?), but these are not all examples.

ANSWER: Thank you very much for spotting the aforementioned errors. They have now been corrected and the information provided accordingly. No shorter region of the COI gene was employed. We have corrected the text accordingly.

Fig. 2 does not contribute relevant data for understanding the study; much more needed here would be phenological data.

ANSWER: Thank you very much for your suggestion about Fig. 2. However, all co-authors think that this figure is necessary and it provides the reader with the information regarding how many species are being found in the Morelos, which will be of relevant importance for colleagues carrying out vector control in the region.

 

Table 2 in this form is needless, because it contributes little information. There should be a material table in the main text, with less data than Table 3, only the most important data: species, environment, location, GenBank accession number to the DNA sequence that was obtained in the study. All other information from tab. 2 and 3 should be moved to the supplementary material (ideally, it should be an Excel sheet with the ability to sort the data).

ANSWER: Thank you very much for your suggestion about Table 2. However, all co-authors think that this table is of relevant importance as it details the list of species and if they are new record for Morelos State.

Paragraph 3.2 should be rewritten. It is descriptive and contains elements of discussion.

ANSWER: We looked over the paragraph 3.2. and made amendments accordingly.

Paragraph 3.4: results of the phylogenetic analysis should be rewritten. What does is mean “ three strong groups of monophyletic species”? and where they are in the tree? what supports the clades have?  

ANSWER: Thank you very much for the comments. We have revised paragraph 3.4 and changed the text accordingly, and made change to make it more clearly.

 

L343-354: why has not DNA analysis been used to solve this problem?

ANSWER: This would be a second step of our study. Currently we are conducted field collections to obtain additional and fresh material of these species

 

I am not a morphologist, but in my opinion the description of the new species does not meet the standards: there are no drawings of morphological features distinguishing the new species, also there is a lack of DNA results in the differential diagnosis (incorrectly named “Systematics”).

ANSWER: We follow the description model of new species of mosquitoes published in various articles. We do not include drawings because we prefer include photographs, which were modified, adding arrows that indicate some of the diagnostic characteristics for identify the new species

 

Fig. 3 - the tree is illegible; should be edited, long unnecessary sequence names shortened, and preferably converted to GenBank accession numbers; species names should be marked with braces, etc.

ANSWER: Tree have been modified accordingly.

The last three lines in tab. 4 should be included in the main table, e.g. marked with an asterisk or in bold “Human biting /Approaching to humans” species.

ANSWER: The table 4 was modified.

 

Minor issues (but also important):

Correctly, the new species is described by the abbreviation sp. nov. (species nova/novum)

ANSWER: The International Code of Zoological Nomenclature accepts “sp. nov.” for abbreviate “new species”

 

Correct names: GenBank (without databases), GeneDoc, a stereomicroscope Discovery V8 (Zeiss), etc.

ANSWER: These words were corrected

 

Amplicons (instead of “PCR products”)

ANSWER: These words were corrected

 

L177: collection sites? or collected samples?

ANSWER: collection samples, this was corrected in the manuscript

 

L179: “6 dissected male genitalia” are not the specimens, but their fragments (what about the specimens they belonged to?

ANSWER: We only dissected six male genitalia, since these samples were mounted on microscope slides and were identified in parallel with the male mosquitoes of origin, they were considered specimens

 

English is not acceptable in many places.

ANSWER: English was reviewed, and the language in the text was improved

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The revised manuscript is much improved over the first version.  A few futher comments are included with the pdf.  Recognition that the presumed new species was actually a synonym is a big plus, thus saving the literature from containing another synonym.  The evidence of this that is presented in quite convincing.

The only problem I had, which has already been discussed with the editor, was the addition of an author who turns out to have photographed the lectotype and labels.  I recommended that it would be more appropriate for that person to be acknowledged, along with the instituional source for the lectotype.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The English for the most part only requires some minor edits, which I included on the draft.  There may be other small issues I missed so further copy editing should be done  by another reviewer or a journal copy editor.

Author Response

Review Report 1

The revised manuscript is much improved over the first version. A few futher comments are included with the pdf. Recognition that the presumed new species was actually a synonym is a big plus, thus saving the literature from containing another synonym. The evidence of this that is presented in quite convincing.

The only problem I had, which has already been discussed with the editor, was the addition of an author who turns out to have photographed the lectotype and labels. I recommended that it would be more appropriate for that person to be acknowledged, along with the instituional source for the lectotype.

The English for the most part only requires some minor edits, which I included on the draft. There may be other small issues I missed so further copy editing should be done by another reviewer or a journal copy editor.

 

Author’s answers to reviewer 1: Thank you very for your comments. These have been addressed and now the added author has now been placed in the acknowledgements sections.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for your modifications of the manuscript according to my comments and suggestions. All are adequately responded.

Author Response

Dear Authors,

 

Thank you for your modifications of the manuscript according to my comments and suggestions. All are adequately responded.

 

Author’s answers to reviewer 2: Your welcome.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

 

1. I maintain my point that Fig. 1 in its present version does not contribute relevant data for understanding the study.

2. Newly introduced text contains typos, including erroneous spacing between numbers and units, etc.

3. Chapter 2.4. Polymerase Chain Reaction and DNA Sequencing continues to be poorly written: details of the PCR reaction are missing, while some erroneous information is still left, e.g. "using forward primers developed by Folmer" - "forward" should be removed, because two primers are needed for PCR and probably both were from Folmer's paper. Also, information about amplicon length ("658 bp region") appears three times. 

4. The text was divided into laboratory methods and DNA analysis incorrectly. DNA data analysis does not begin until "The dataset was analyzed in MEGA v.6(...)”.

5. I can't evaluate how fig.3 was changed because I don't have access to the new version.

6. I can't evaluate how tab. 4 was changed, because I don't have access to the new version.

7. My main comments were rejected by the authors, arguing that “all co-authors think that this figure/table/text is necessary/correct/etc.”. This, by the way, also applies to the comments of the two other reviewers; actually, it concerns all recommendations that required more work from the authors.

Comments on the Quality of English Language

The English language has not been corrected; only some changes have been made, moreover, there are typos in the new text too.

Author Response

Review Report 3

 

1. I maintain my point that Fig. 1 in its present version does not contribute relevant data for understanding the study.

Author’s answers to reviewer 3: Thank you very much for your comments. The figure does indeed contribute to data for this manuscript. It shows to the reader, and it explains, the different ecological regions of Morelos’ state, we have done similar studies in different state of Mexico, and map has always been welcomed. We agreed that this map is of paramount importance for the papers, and decided to leave it.

 

2. Newly introduced text contains typos, including erroneous spacing between numbers and units, etc.

Author’s answers to reviewer 3: Thank you very much for your comments, we have reviewed the manuscript again and corrected the typos.

3. Chapter 2.4. Polymerase Chain Reaction and DNA Sequencing continues to be poorly written: details of the PCR reaction are missing, while some erroneous information is still left, e.g. "using forward primers developed by Folmer" - "forward" should be removed, because two primers are needed for PCR and probably both were from Folmer's paper. Also, information about amplicon length ("658 bp region") appears three times.

 

Author’s answers to reviewer 3: Thank you very much for your comments. We have corrected the sentence and have deleted the…forward primers…Also the previous sentence have been changed following your comments.

 

 

4. The text was divided into laboratory methods and DNA analysis incorrectly. DNA data analysis does not begin until "The dataset was analyzed in MEGA v.6(...)”.

Author’s answers to reviewer 3: Thank you very much for your comments. We have corrected the sentences and relevant paragraph, adding the suggested information.

 

5. I can't evaluate how fig.3 was changed because I don't have access to the new version.

Author’s answers to reviewer 3: Thank you very much for your comments. We have corrected the figure and the figure caption.

 

 

6. I can't evaluate how tab. 4 was changed, because I don't have access to the new version.

Author’s answers to reviewer 3: Thank you very much for your comments. The Table 4 was corrected and added in landscape layout so that columns are clearer.

 

7. My main comments were rejected by the authors, arguing that “all co-authors think that this figure/table/text is necessary/correct/etc.”. This, by the way, also applies to the comments of the two other reviewers; actually, it concerns all recommendations that required more work from the authors.

Author’s answers to reviewer 3: Thank you very much for your comments. We have all corrected all your comments and followed your suggestions, except we do think the Figure 1 is rather important for the paper and for the readers, and we have decided to keep it.

 

The English language has not been corrected; only some changes have been made, moreover, there are typos in the new text too.

Author’s answers to reviewer 3: Thank you very much for your comments. We have all corrected the text and followed your comments and suggestions.

Author Response File: Author Response.pdf