Using ISSR Genomic Fingerprinting to Study the Genetic Differentiation of Artemia Leach, 1819 (Crustacea: Anostraca) from Iran and Neighbor Regions with the Focus on the Invasive American Artemia franciscana

Round 1

Reviewer 1 Report

This manuscript assesses the genetic variability of different Artemia populations from Western Asia (Iran, Iraq and Turkey), including both autochthonous and invasive populations. ISSR genomic fingerprint has been the chosen technique to study it. The authors hypothesize that the establishment of exotic species in new geographical habitats should be accompanied by intra-specific genetic divergence to better adapt to the new environmental conditions.

The study is described properly and clearly with an adequate explanation of the materials and methods utilized, an appropriate presentation of the results obtained, a precise discussion, a reasonable use of references, and realistic conclusions drawn. The molecular population genetics approach that the authors have chosen is appropriate to ascertain the contribution of the genetic variability to the success of the invasion. This kind of approaches has been scarcely used in the case of the A. franciscana invasion of the Eurasian area. Consequently, it presents a novelty in this sense.

The interest in the presented data lays in the gained knowledge on the mechanisms that make an allochthonous species to behave as exotic invasive. In this case, the initial hypothesis is well supported by the data obtained. Probably, it would be necessary some additional research to expand the hypothesis to some other areas where invasion has succeeded, but this study perfectly serves as a “proof of concept”.

For all these reasons, I suggest this manuscript to be published after some language editing and a minor revision of some few details that I list below:

• Line 14. The introduction of franciscana was not always intentional. In most of cases it was just the logic consequence of its extensive use in larviculture plants. Please, tune down this statement.
• Line 24. ‘…variability compared…’ instead of ‘…variability compare…’
• Line 33. Change ‘… has the ...’ to ‘… that has a …’
• Lines 86-87. Change ‘… in relationship with native populations and colonized American franciscana…’ to ‘… in native and colonizing populations of American A. franciscana…’
• Line 152. Use provide, not provides.
• Line 153. Use the same structure: locality name (abbreviation) for all cited places.
• Lines 155-158. Rephrase these lines. They are difficult to understand. Maybe beginning with ‘… that shared a common ancestor with urmiana. Although most of the parthenogenetic populations were located in the clade P1, the majority of CAM specimens …’
• Line 153. Difficult to understand. Change ‘… were attributed among native and invasive populations (69 % vs 31 %).’ to ‘… were attributed among native and invasive populations more that within populations (69 % vs 31 %).’
• Line 200. Change ‘consisted’ to ‘explained’.
• Line 205. Change ‘single individual’ to ‘a single individual’.
• Lines 219-220. Change ‘… triploids, which these have been also confirmed by phylogenetic tree.’ to ‘… triploids. This observation has been also confirmed by the phylogenetic tree.’
• Line 228. Change ‘… urmiana has represented an unexpectedly …’ to ‘… it has presented an unexpectedly …’
• Line 229. Change ‘have also’ to ‘have been also’.
• Lines 235-236. Change ‘as well among’ to ‘as well as among’.
• Lines 240-251. This paragraph is only presenting results. Nothing is discussed and nothing is added to what it has been already said in the results section. Move to results or delete.
• Lines 252-259. Some comments here on the genetic variation of the invasive populations reported in the manuscript would be of interest, not only those reported in other studies.
• Line 260. Delete ‘of’.
• Line 274. Point missing before ‘We’.
• 7 and 8. Headings are swapped.
• Table 8. Invasive population from Iraq is missing.

Author Response

The English isn’t awful, but it does need the attention of a native English speaker

Response： The manuscript has been revised by a native English speaker. The revised draft is highlighted in blue colors.

I don’t suppose it’s entirely necessary to explain why radioactive marking of the PCR products was chosen instead of the other less hazardous options, but I am most curious

Response: All laboratory security regulations have been followed by the University of Heidelberg. In addition, using radioactive-labeled PCR products loading on the 6% polyacryl amid gel markers can potentially generate large number of bands with high resolutions.

Methods state that “only unequivocally reproducible bands were scored” – how many replicates were performed before a band was considered unequivocally reproducible?

Response: this method has been optimized by the first author before. Please, check the following articles:

1) Eimanifar, A.; Wink. M. Fine-scale population genetic structure in Artemia urmiana (Günther, 1890) based on mtDNA sequences and ISSR genomic fingerprinting. Org Divers Evol 2013, 13, 531–543.

2) Eimanifar, A.; Marden, B.; Braun, M.S.; Wink, M. Analysis of the genetic variability of Artemia franciscana Kellogg, 1906 from the Great Salt Lake (USA) based on mtDNA sequences, ISSR genomic fingerprinting and biometry. Marine Biodiversity 2015, 45: 311–319.

The observation that invasive populations have lower genetic variability than origin populations is unsurprising given the necessary founder effect; the opposite effect is most interesting. It would be useful to include a discussion of the relationship between time-since-population-founding and genetic diversity in these invasive populations

Response: There is a lake of information about time of introducing of invasive population in studied localities.  Due to that, we cannot discuss about the relationship between time-since-population-founding and genetic diversity in these invasive populations.

A note on language, see, for example line 261-262 “….than our parthenogenic populations” – the populations are not yours. They might belong to someone. But they don’t belong to you. (Much less offensive in the context of brine shrimp than in the context of humans, where I’ve also seen this construction used, but claiming ownership of that which is not yours is a good thing to avoid).

Response: Revised

Reviewer 2 Report

• The English isn’t awful, but it does need the attention of a native English speaker
• I don’t suppose it’s entirely necessary to explain why radioactive marking of the PCR products was chosen instead of the other less hazardous options, but I am most curious
• Methods state that “only unequivocally reproducible bands were scored” – how many replicates were performed before a band was considered unequivocally reproducible?
• The observation that invasive populations have lower genetic variability than origin populations is unsurprising given the necessary founder effect; the opposite effect is most interesting. It would be useful to include a discussion of the relationship between time-since-population-founding and genetic diversity in these invasive populations
• A note on language, see, for example line 261-262 “….than our parthenogenic populations” – the populations are not yours. They might belong to someone. But they don’t belong to you. (Much less offensive in the context of brine shrimp than in the context of humans, where I’ve also seen this construction used, but claiming ownership of that which is not yours is a good thing to avoid).

Author Response

Line 14. The introduction of franciscana was not always intentional. In most of cases it was just the logic consequence of its extensive use in larviculture plants. Please, tune down this statement.

Response: Revised

Line 24. ‘…variability compared…’ instead of ‘…variability compare…’

Response: Revised

Line 33. Change ‘… has the ...’ to ‘… that has a …’

Response: Revised

Lines 86-87. Change ‘… in relationship with native populations and colonized Americanfranciscana…’ to ‘… in native and colonizing populations of American A. franciscana…’

Response: Revised

Line 152. Use provide, not provides.

Response: Revised

Line 153. Use the same structure: locality name (abbreviation) for all cited places.

Response: Revised

Lines 155-158. Rephrase these lines. They are difficult to understand. Maybe beginning with ‘… that shared a common ancestor with urmiana. Although most of the parthenogenetic populations were located in the clade P1, the majority of CAM specimens …’

Response: Revised

Line 183. Difficult to understand. Change ‘… were attributed among native and invasive populations (69 % vs 31 %).’ to ‘… were attributed among native and invasive populations more that within populations (69 % vs 31 %).’

Response: Revised

Line 200. Change ‘consisted’ to ‘explained’.

Response: Revised

Line 205. Change ‘single individual’ to ‘a single individual’.

Response: Revised

Lines 219-220. Change ‘… triploids, which these have been also confirmed by phylogenetic tree.’ to ‘… triploids. This observation has been also confirmed by the phylogenetic tree.’

Response: Revised

Line 228. Change ‘… urmiana has represented an unexpectedly …’ to ‘… it has presented an unexpectedly …’

Response: Revised

Line 229. Change ‘have also’ to ‘have been also’.

Response: Revised

Lines 235-236. Change ‘as well among’ to ‘as well as among’.

Response: Revised

Lines 240-251. This paragraph is only presenting results. Nothing is discussed and nothing is added to what it has been already said in the results section. Move to results or delete.

Response: We moved that paragraph to the result section.

Lines 252-259. Some comments here on the genetic variation of the invasive populations reported in the manuscript would be of interest, not only those reported in other studies.

Response: the position of Lines 252-259. Changed to 285-265, before discuss about our result

Line 260. Delete ‘of’.

Response: Revised

Line 274. Point missing before ‘We’.

Response: Revised

Table 7 and 8. Headings are swapped.

Response: Revised

Table 8. Invasive population from Iraq is missing.

Response: GAA is population of Iraq (last row, it is marked in the red color)