DNA Barcoding and Phylogenetic Relationship of Parabuthus liosoma (Ehrenberg, 1828) (Scorpiones: Buthidae) in Saudi Arabia

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors conducted the first DNA barcoding and phylogenetic study of Parabuthus liosoma in Saudi Arabia. They collected nine specimens from Farasan Island, extracted genomic DNA, sequenced the mitochondrial COI gene, and analyzed genetic variability and phylogenetic relationships.

 

The results are well presented and the analyses are done correctly. They explored phylogenetic reconstruction through multiple methods (Maximum Parsimony, Neighbor-Joining, Bayesian Inference). Barcodes of P. liosoma clearly shows a monophyletic clade and they showed there is strong genetic separation between Arabian and African lineages within the genus.

My main critique relates with two points:

• There are only 9 samples in the analysis which is not really extensive.
• Although COI is standard the authors should have used other markers (at least one more) so that not all conclusions would rely on a single 500 bp sequence.

Overall the discussion could be improved focusing on the integration of more aspects on evolutionary implications (past climatic changes, ecological specialization, etc).

Also, carefully proofread the manuscript to correct minor typographical errors (e.g., "maraphyly" → "monophyly" in the conclusions).

Author Response

 Comments and Suggestions for Authors

Comment 1: The authors conducted the first DNA barcoding and phylogenetic study of Parabuthus liosoma in Saudi Arabia. They collected nine specimens from Farasan Island, extracted genomic DNA, sequenced the mitochondrial COI gene, and analyzed genetic variability and phylogenetic relationships.

Response 1: Thank you.

Comment 2: The results are well presented and the analyses are done correctly. They explored phylogenetic reconstruction through multiple methods (Maximum Parsimony, Neighbor-Joining, Bayesian Inference). Barcodes of P. liosoma clearly shows a monophyletic clade and they showed there is strong genetic separation between Arabian and African lineages within the genus.

Response 2: Thank you.

My main critique relates with two points:

Comment 3: There are only 9 samples in the analysis which is not really extensive.

Response 3: Thank you for your comment regarding the limited sample size in our analysis. We fully acknowledge that only nine scorpion specimens were included, which may constrain the statistical power and generalizability of our findings. However, it is important to note that all samples were collected from the Farasan Protected Area, where strict regulations govern scientific research and biological sampling.

Comment 4: Although COI is standard the authors should have used other markers (at least one more) so that not all conclusions would rely on a single 500 bp sequence.

Response 4: Thank you for your insightful comment regarding the use of additional genetic markers. As noted in the manuscript, we selected the COI gene for DNA barcoding because it is represented by a substantial number of entries in global databases such as BOLD and GenBank, providing robust comparative data for species identification. The widespread use of COI in animal barcoding studies allows for reliable and standardized comparisons across diverse taxa.

Comment 5: the discussion could be improved focusing on the integration of more aspects on evolutionary implications (past climatic changes, ecological specialization, etc).

Response 5: Thank you for the valuable suggestion. We have revised the discussion to better integrate evolutionary implications.

Comment 5: Also, carefully proofread the manuscript to correct minor typographical errors (e.g., "maraphyly" → "monophyly" in the conclusions).

Response 6: Thank you for your attention to detail and for pointing out the typographical error in the conclusions section ("maraphyly" → "monophyly"). We have carefully proofread the entire manuscript and corrected this and other minor typographical errors to improve the overall clarity and accuracy of the text. We appreciate your careful review and helpful suggestions which have contributed to strengthening the quality of our work.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors presented a new DNA barcode for the endemic Buthidae species found in Saudi Arabia. The introduction was nicely written. The method of DNA isolation, PCR and sequencing was performed on standard. Here are a few comments that the authors may need to address to increase the quality of the manuscript:

1. Provide details on how the collected specimen is recognised as Parabuthus liosoma. Is it based on morphological identification? If so, please provide additional details on that.

2. Provide the sex of the collected specimen in Table 1.

3. Line 94 - Indicate the tissues that were used for DNA isolation.

4. Have the isolated DNA being qualified by gel electrophoresis or quantification? 

5. Provide details on how to perform the sequencing. Is it performed by the authors or the samples had been sent to company for sequencing? Please kindly state in the method section.

6. It is highly encouraged that the author to run the phylogenetic tree with ML algorithm, since the authors have detected genetic variations between different scorpion clades. The ML tree may provide additional information on the mutation rate between different entry sequences. 

7. Besides, the authors may also need to consider using a more distant outgroup for the tree construction.

8. It is encouraged to include a nice photo (with scale bar) of Parabuthus liosoma in the manuscript.

Author Response

The authors presented a new DNA barcode for the endemic Buthidae species found in Saudi Arabia. The introduction was nicely written. The method of DNA isolation, PCR and sequencing was performed on standard. Here are a few comments that the authors may need to address to increase the quality of the manuscript:
Thank you
Comment 1. Provide details on how the collected specimen is recognised as Parabuthus liosoma. Is it based on morphological identification? If so, please provide additional details on that.

Response 1:

Comment 2. Provide the sex of the collected specimen in Table 1.

Response2: Thank you. A column indicating the sex of each collected specimen was added to Table 1.

Comment 3. Line 94 - Indicate the tissues that were used for DNA isolation.

Response 3: Thank you for providing that information. Line 94 has been revised to clearly indicate the tissue used for DNA isolation: "Genomic DNA was extracted from leg tissue of preserved specimens using the commercially available DNeasy Blood and Tissue Kit (Qiagen) following the manufacturer's protocol."

Comment 4. Have the isolated DNA being qualified by gel electrophoresis or quantification? 

Response 4: Thank you for providing the refined description. The manuscript's methodology section was updated to accurately reflect the DNA quality assessment procedure as follows: "To assess DNA quality, 5 μL of genomic DNA was mixed with 1 μL of 6× DNA loading buffer (containing 0.25% w/v bromophenol blue and 40% w/v sucrose) and loaded onto a 1% agarose gel stained with 0.5 μg/mL ethidium bromide (Thermo Fisher Scientific)."

Comment  5. Provide details on how to perform the sequencing. Is it performed by the authors or the samples had been sent to company for sequencing? Please kindly state this in the method section.

Response 5: Thank you for providing this information. The methodology section was updated to include the following details about the sequencing process: "The purified COI gene fragments were then sequenced using an ABI 3500 automated sequencer (Applied Biosystems, USA)." This clarifies that the sequencing was performed using a specific instrument.

Comment 6. It is highly encouraged that the author to run the phylogenetic tree with ML algorithm, since the authors have detected genetic variations between different scorpion clades. The ML tree may provide additional information on the mutation rate between different entry sequences. 

Response 6:
Thank you for your suggestion. In this study, we have already employed multiple phylogenetic analysis methods, including Maximum Parsimony and Bayesian Inference, to ensure robust and comprehensive results. While we acknowledge the value of the Maximum Likelihood (ML) approach, the use of these complementary methods has provided consistent and well-supported phylogenetic relationships among the scorpion clades. We believe this multi-method strategy sufficiently addresses the genetic variation and mutation rates observed in our dataset. However, we appreciate your recommendation and will consider incorporating ML analysis in future research to further strengthen our findings.

Comment 7. Besides, the authors may also need to consider using a more distant outgroup for the tree construction.

Thank you for your suggestion. We have updated the Methods section of the manuscript to indicate that Buthus mardochei (ON255580.1) and Microbuthus kristensenorum (ON255621.1) were selected as more distant outgroup taxa for rooting the phylogenetic tree in our analysis.

8. It is encouraged to include a nice photo (with scale bar) of Parabuthus liosomain the manuscript.

Response 8:
Thank you for the valuable suggestion. Unfortunately, we do not currently have a photograph of Parabuthus liosoma with a scale bar. However, we will consider including such an image in future revisions or related studies to enhance the manuscript.

 

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript provides the COI barcode of a scorpion taxon for the first time and presents a limited phylogeny. I recommend publication after moderate revisions.

 

40: "The Parabuthus genus" to "The genus Parabuthus"
42: "features" to "reasons"
44: State where the stridulatory organs are located.
51: Here and elsewhere in the manuscript, place the author's surname (and et al. if relevant) before the brackets, otherwise the reader is confused and must refer to the reference list every time there is a citation.
81: delete scorpion species
88: "hat" to "that"
90: "samples" to "specimens"
Table 1: Spelling error in table "araia"
Figure 2 is too small and needs to be made larger on the page.
198: per year?
198: "the scorpion's" to "scorpion"
202: "of" to "to"
206: "P." to "Parabuthus" (always write out genus name in full at start of a sentence)
207: "the previous" to "previous"
237: species complex is repeated twice in the same sentence, change it.
251: "maraphyly" to "paraphyly"
References: All binomials must be italicised.

Comments on the Quality of English Language

40: "The Parabuthus genus" to "The genus Parabuthus"
42: "features" to "reasons"
44: State where the stridulatory organs are located.
51: Here and elsewhere in the manuscript, place the author's surname (and et al. if relevant) before the brackets, otherwise the reader is confused and must refer to the reference list everytime there is a citation.
81: delete scorpion species
88: "hat" to "that"
90: "samples" to "specimens"
Table 1: Spelling error in table "araia"
Figure 2 is too small and needs to be made larger on the page.
198: per year?
198: "the scorpion's" to "scorpion"
202: "of" to "to"
206: "P." to "Parabuthus" (always write out genus name in full at start of a sentence)
207: "the previous" to "previous"
237: species complex is repeated twice in the same sentence, change it.
251: "maraphyly" to "paraphyly"
References: All binomials must be italicised.

Author Response

Comments and Suggestions for Authors

This manuscript provides the COI barcode of a scorpion taxon for the first time and presents a limited phylogeny. I recommend publication after moderate revisions.

Comment 1:  40: "The Parabuthus genus" to "The genus Parabuthus"

Response 1: Done.
Comment 2: 42: "features" to "reasons"

Response 2: Done.

Comment 3: 44: State where the stridulatory organs are located.
Response 3: Done.

Comment 4:  51: Here and elsewhere in the manuscript, place the author's surname (and et al. if relevant) before the brackets, otherwise the reader is confused and must refer to the reference list every time there is a citation.
Response 4: thank you, I'll definitely make sure to move the author's surname (and "et al." where needed) before the brackets throughout the manuscript. You're right, it will make it much easier for the reader to follow without constantly flipping to the reference list. I appreciate you pointing this out.

Comment 5:   81: delete scorpion species

Response 5: deleted.

Comment 6:   88: "hat" to "that"

Response 6: Thanks for pointing out the typo in line 88. I'll correct "hat" to "that" immediately. Regarding the title of Figure 1, I understand the editor's recommendation and have deleted it as suggested.

Comment 7:   90: "samples" to "specimens"

Response 7: Done.

Comment 8:   Table 1: Spelling error in table "araia"

Response 8: Thank you for the clarification! I have corrected the spelling error in Table 1 to "Arabia".

Comment 9:   Figure 2 is too small and needs to be made larger on the page.

Response 9: Understood. High-resolution figures will be provided for the final version to be edited by the journal.

Comment 10:   198: per year?

Response 10: corrected.
Comment 11: 198: "the scorpion's" to "scorpion"

Response 11: Done.

Comment 12: 202: "of" to "to"
Response 12: Done.

Comment 13: 206: "P." to "Parabuthus" (always write out genus name in full at start of a sentence)
Response 13: Done.

Comment 14: 207: "the previous" to "previous"
Response 14: Done.

Comment 15: 237: species complex is repeated twice in the same sentence, change it.
Response 15: Done.

Comment 16:  251: "maraphyly" to "paraphyly"

Response 16: Done.
Comment 17:  References: All binomials must be italicised.

Response 17: Done.

Comments on the Quality of English Language

Comment 18:  40: "The Parabuthus genus" to "The genus Parabuthus"

Response 18: Done.
Comment 19: 42: "features" to "reasons"

Response 19: Done.

Comment 20: 44: State where the stridulatory organs are located.
Response 20: Done.

Comment 21:  51: Here and elsewhere in the manuscript, place the author's surname (and et al. if relevant) before the brackets, otherwise the reader is confused and must refer to the reference list every time there is a citation.
Response 21: thank you, I'll definitely make sure to move the author's surname (and "et al." where needed) before the brackets throughout the manuscript. You're right, it will make it much easier for the reader to follow without constantly flipping to the reference list. I appreciate you pointing this out.

Comment 22:   81: delete scorpion species

Response 22: deleted.

Comment 23:   88: "hat" to "that"

Response 23: Thanks for pointing out the typo in line 88. I'll correct "hat" to "that" immediately. Regarding the title of Figure 1, I understand the editor's recommendation and have deleted it as suggested.

Comment 24:   90: "samples" to "specimens"

Response 24: Done.

 
Comment 25:   Table 1: Spelling error in table "araia"

Response 25: Thank you for the clarification! I have corrected the spelling error in Table 1 to "Arabia".

Comment 26:   Figure 2 is too small and needs to be made larger on the page.

Response 26: Understood. High-resolution figures will be provided for the final version to be edited by the journal.

Comment 27:   198: per year?

Response 27: corrected.

Comment 28: 198: "the scorpion's" to "scorpion"

Response 28: Done.

Comment 29: 202: "of" to "to"
Response 29: Done.

 

 

Comment 30: 206: "P." to "Parabuthus" (always write out genus name in full at start of a sentence)
Response 30: Done.

Comment 31: 207: "the previous" to "previous"
Response 31: Done.

Comment 32: 237: species complex is repeated twice in the same sentence, change it.
Response 32: Done.

Comment 33:  251: "maraphyly" to "paraphyly"

Response 33: Done.

Comment 34:  References: All binomials must be italicised.

Response 34: Done.