Resettlement of Eurytemora velox (Crustacea: Copepoda) in Europe, the Urals and Western Siberia
Sahar Khodami
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
Introduction: note, E. velox is also found in the UK.
Line 75-75: What is 33 and 34?
Methods: What publication(s) was identification based on?
Results: For more easy interpretation of the results, a tree should be included in addition to the haplotype diagram.
Table 2: Why are Thoracic wings in Table 2 all shown as clubs?; Same applies to elsewhere – what is the significance of the clubs?; Furcall should be Furcal L; righ (multiple times) should be right.
Table 3: Line 1: 1 sequence, not one sequences.
Lines 248-250: Why is this interesting? Make it clear to the reader.
Author Contributions: The initials here don’t match any of the authors.
Comments on the Quality of English LanguageAlthough the logic and ordering appear good, the writing of individual sentences in the Introduction and Discussion needs to be improved greatly.
Author Response
Dear Reviewer 1
The authors would like to thank you for valuable comments that have improved considerably our MS.
Extensive changes were made, and the authors suppose that the text is now more clear and readable. We hope the Editor-in-Chief and reviewers will notice the improvement
Best regards
Coauthors of the paper
Our Response to comments as following:
Reviewer 1
Comments and Suggestions for Authors
Introduction: note, E. velox is also found in the UK.
Unswer: Thanks, we do not list all the finds in Europe, but we make it clear that the species inhabits almost all of Europe
Line 75-75: What is 33 and 34?
Unswer: These are references to literature. Square brackets are added
Methods: What publication(s) was identification based on?
Unswer: We used keys given in the works below. We refer to them in the text. Borutsky et al. 1991 is usually used for European species identification in RF.
- Borutsky, E.S.; Stepanova L.A., Kos M.S. Key to Calanoida fresh waters of the USSR, Leningrad: Nauka, 1991; p. 504. (In Russian)
- Sukhikh, N.M.; Lazareva, V.I.; Alekseev, V.R. Copepod Eurytemora caspica Sukhikh et Alekseev, 2013 (Crustacea, Calanoida) in Volga and Kama River Reservoirs. Inland Water Biology. 2020, 13(2), 198–205. https://doi.org/10.1134/S199508292002014
Results: For more easy interpretation of the results, a tree should be included in addition to the haplotype diagram.
Unswer: We build the CO1 phylogenetic tree displaying also the results of the date calibration analyses.
Table 2: Why are Thoracic wings in Table 2 all shown as clubs?; Same applies to elsewhere – what is the significance of the clubs?; Furcall should be Furcal L; righ (multiple times) should be right.
Unswer: Sorry, we changed these typos
Table 3: Line 1: 1 sequence, not one sequences.
Unswer: Changed
Lines 248-250: Why is this interesting? Make it clear to the reader.
Unswer: We remove this information from Discussion
Author Contributions: The initials here don’t match any of the authors.
Unswer: Changed
Comments on the Quality of English Language
Although the logic and ordering appear good, the writing of individual sentences in the Introduction and Discussion needs to be improved greatly.
Unswer: Changed
Reviewer 2 Report
Comments and Suggestions for AuthorsDear authors,
This study presents new genetic and deep morphological observations of Eurytemora velox, a species of calanoid copepods, from different rivers and water bodies from Caspian Sea region. Additionally, a comprehensive comparison is conducted here between the population of E. velox from European waters (as reported in previous studies) and the specimens collected for this study. Using a multi gene approach and haplotype network analyses, the study explores various hypotheses regarding the origin and distribution patterns of the studied population. Furthermore, A molecular clock analyses, employing different calibration points is used to date the origin of the studied populations. While, the introduction and discussion sections provide sufficient details and offer interesting insights into the species’ origin and gene flow, the results and methods sections require revision to clarify and visualize the genetic findings. I must note that, I am not a taxonomist or an expert in analyzing morphological intraspecific characters for evolutionary studies; therefore, my comments and revisions primarily focused on the genetic data:
1. The introduction provides a comprehensive literature survey that justifies the need for investigations on the unstudied populations of Eurytemora velox in Southern Urals and West Siberia, allowing for comparison with European populations of the species.
2. Considering that only a few species have been genetically analyzed, how can we be certain that the morphotypes examined in this study exhibit the same genotypes as those individuals subjected to genetic analyses? Were the morphological and genetic analyses performed on different individuals?
3. In Figure 1, the map lacks coordinates or names for reference, making it difficult to pinpoint the specific locations mentioned in the study.
4. In line 106, it is mentioned that "both sets of primers" were used. It would be helpful to specify which primers were used for which loci.
5. Lines 116-118 mention the use of the GTR + Gamma model. What criteria were used to select this model? Additionally, why was the Tamura-Nei 93 model chosen for genetic distances instead of a pure genetic distance model?
6. In lines 143-145, it is not clear which gene fragments were sequenced for Eurytemora velox, as the sentence mentions "Crustaceans" when referring to some of the loci.
7. Regarding line 147, Table 1 states that two specimens from the Gulf of Ob and two specimens from Magnitogorsk were genetically analyzed. Does this sentence imply that three different haplotypes were observed out of the four individuals? How different are the haplotypes, and do these two areas share any haplotypes?
8. Despite having COI and ITS sequences from nearly all studied areas, there is no mention of the results of the COI haplotype network or the genetic diversity of the ITS from specimens in other locations. This information should be included.
9. The genetic studies section of the results is quite brief, consisting of only nine lines. It would be beneficial to provide information on the length of the analyzed fragments and details about the studied genotypes for COI and 18S. Additionally, information on the genetic differences between and within sites, as well as the extent of gene flow between rivers, lakes, and gulfs, should be discussed. Furthermore, the results section should include a tree or diagram displaying the results of the date calibration analyses. Consider providing a brief explanation for choosing the calibration points, particularly the outgroups. Additionally, include haplotype networks for each gene fragment.
10. In line 241, it is mentioned that female p5 intraspecific variabilities correlate with human activities. How was this correlation established? Please provide more clarity on this matter.
11. In the discussion section, a significant portion of section 4.2 should be moved to the results section, along with the haplotype network (Figure 6).
12. Line 256 suggests that COI and ITS recovered identical haplotypes between Magnitogorsk and Chelyabinsk. However, the COI haplotype network does not display any haplotypes from Magnitogorsk, and not all shared haplotypes between Magnitogorsk and Chelyabinsk are identical in ITS.
13. In Figure 6, please clarify what doe the size of the circles refers to.
14. Considering the 4% genetic divergence in COI between the Ural's populations and European populations, is it possible that the Ural's populations represent another closely related species rather than E. velox? Additionally, it would be helpful to gain a better understanding of the extent of sequencing of the 18S gene in this study and whether similar patterns were observed when comparing 18S among the specimens.
Comments for author File: 
Comments.pdf
A revision on English language is necessary.
Author Response
Dear Reviewer 2
The authors would like to thank you for valuable comments that have improved considerably our MS.
Extensive changes were made, and the authors suppose that the text is now more clear and readable. We hope the Editor-in-Chief and reviewers will notice the improvement
Best regards
Coauthors of the paper
Our Response to comments as following:
Reviewer 2
- The introduction provides a comprehensive literature survey that justifies the need for investigations on the unstudied populations of Eurytemora velox in Southern Urals and West Siberia, allowing for comparison with European populations of the species.
- Considering that only a few species have been genetically analyzed, how can we be certain that the morphotypes examined in this study exhibit the same genotypes as those individuals subjected to genetic analyses? Were the morphological and genetic analyses performed on different individuals?
Answer: The work was done by specialist in Eurytemora taxonomy and phylogeny. Every specimen was identified by the morphological traits and after part of them was analyzed morphologically and material suitable for genetic analysis was used for genetics. We have DNA sequences of almost all Eurytemora species to understand if this is Eurytemora velox or not. Also we checked our sequences using GenBank database.
- In Figure 1, the map lacks coordinates or names for reference, making it difficult to pinpoint the specific locations mentioned in the study.
Answer: Figure capture includes information (names) for reference. We would not to place a lot of information on the map to avoid information overload
- In line 106, it is mentioned that "both sets of primers" were used. It would be helpful to specify which primers were used for which loci.
Answer: We improve this part in lines 117-119. We do not want to overload the article with information that has already been previously published.
- Lines 116-118 mention the use of the GTR + Gamma model. What criteria were used to select this model? Additionally, why was the Tamura-Nei 93 model chosen for genetic distances instead of a pure genetic distance model?
Answer: We added this information to the text in lines 131-135
- In lines 143-145, it is not clear which gene fragments were sequenced for Eurytemora velox, as the sentence mentions "Crustaceans" when referring to some of the loci.
Answer: we clarify this information. We analyzed CO1, ITS1-2 and 18SrRNA part of genes for Eurytemora velox.
- Regarding line 147, Table 1 states that two specimens from the Gulf of Ob and two specimens from Magnitogorsk were genetically analyzed. Does this sentence imply that three different haplotypes were observed out of the four individuals? How different are the haplotypes, and do these two areas share any haplotypes?
Answer: According to Table 3 and Figure 7B 3 specimens from the Ob Bay and Magnitogorsk are represent three private haplotypes, one of which is shared with one of Chelyabinsk haplotype. These haplotypes are closer to the Volga population [34], which differ from Kyiv one by 0.1% of nucleotide substitutions. (Line 167-170)
- Despite having COI and ITS sequences from nearly all studied areas, there is no mention of the results of the COI haplotype network or the genetic diversity of the ITS from specimens in other locations. This information should be included.
Answer:
The information is added to the genetic results. Haplotype networks (ITS and CO1), including all available E.velox sequences, are given in discussion, as it used a lot of published material.
- The genetic studies section of the results is quite brief, consisting of only nine lines. It would be beneficial to provide information on the length of the analyzed fragments and details about the studied genotypes for COI and 18S. Additionally, information on the genetic differences between and within sites, as well as the extent of gene flow between rivers, lakes, and gulfs, should be discussed. Furthermore, the results section should include a tree or diagram displaying the results of the date calibration analyses. Consider providing a brief explanation for choosing the calibration points, particularly the outgroups. Additionally, include haplotype networks for each gene fragment.
Answer:
We added information on the genetic differences, on the length of the analyzed fragments. Haplotype networks for CO1 and nITS are given in discussion, as it used a lot of published material. A tree displaying the results of the date calibration analyses is given in the text.
- In line 241, it is mentioned that female p5 intraspecific variabilities correlate with human activities. How was this correlation established? Please provide more clarity on this matter.
Answer: This refers to stressful conditions as a result of human activity, leading to mutations. Explanations added in the text
- In the discussion section, a significant portion of section 4.2 should be moved to the results section, along with the haplotype network (Figure 6).
Answer: Since the haplotype network mainly includes previously published material, we believe that this information is most relevant in the Discussion part. By moving this section to the results, we will get repetition of the material in the results and discussion
- Line 256 suggests that COI and ITS recovered identical haplotypes between Magnitogorsk and Chelyabinsk. However, the COI haplotype network does not display any haplotypes from Magnitogorsk, and not all shared haplotypes between Magnitogorsk and Chelyabinsk are identical in ITS.
Answer: we clarified this moment in the text
- In Figure 6, please clarify what does the size of the circles refers to.
Answer: it is done
- Considering the 4% genetic divergence in COI between the Ural's populations and European populations, is it possible that the Ural's populations represent another closely related species rather than E. velox? Additionally, it would be helpful to gain a better understanding of the extent of sequencing of the 18S gene in this study and whether similar patterns were observed when comparing 18S among the specimens.
Answer: 4% genetic divergence in COI corresponds to interspecies divergence in COI among Copepods. The level of difference in nuclear genes, as well as morphological studies also do not support the species status of the Siberian population. In this study, a short 18S gene fragment was sequenced, but it is informative for the genus Eurytemora. Similar picture, when we observe high intraspecies and species divergence in mitochon-drial genes and very low one among nuclear genes was observed among different groups of Copepoda. More information is given in the text in lines 386-393.
