Rare but Not Gone: A Relict Population of the Black Sea Ship Sturgeon Acipenser nudiventris Persists in the Rioni River, Georgia

Round 1

Reviewer 1 Report

Beridze et al. worked with local fishers, among others, to collect physical data and tissue samples from a putative, remnant population of ship sturgeon. The authors then used a series of genetics-based assays to validate that the specimens were ship sturgeon and not hybrids. The authors concluded that specimens are in fact ship sturgeon and not hybrids. Generally, I think the paper was well written, the methods are sound, and the conclusions are warranted. Most of the comments below are to improve clarity; however, a bit more focus should be spent considering uncertainty in the inference that these fish are ship sturgeon given the information obtained through this research (see below).

 

General comment:

1) From my reading of the Russian sturgeon-specific assay, the primers chosen worked to identify nearly all individuals tested (175/183) in the paper cited, so the frequency of the null allele (i.e., that doesn’t amplify in Russian Sturgeon) is roughly q = sqrt(q^2) = = sqrt(1-(175/183)) = sqrt(0.0437) = 0.209, which isn’t small. Given a sample size of 22 individuals and the application of this assay, we’d expect that ~1 individual could be missed (i.e., 0.961 = 22 * 0.0437). Of course, that assumes we’re talking about only Russian sturgeon, which we’re not, and so the calculation would get more complicated if we considered different types of hybrids. The general point is that the use of the species-specific assays is great and should be done, but there is still uncertainty in the inference, which should be considered in the Discussion section in an additional paragraph. Potential suggestions would be to consider using an additional Russian sturgeon-specific assay (and potentially others) to decrease uncertainty in the inference.

               

Specific comments:

Line 13 – Replace “missing” with “undetected”

Line 20 – Replace "which haplotype” with “, which the haplotype” (at least, I think)

Line 21 – Please be specific about what is meant by “signs”. Are the authors talking about phenotypes here or the genotypic data they generated?

Line 28-30 – This is a run-on sentence. Please address. One suggestion would be to replace “and is probably close to” with “ – with the species”

Line 42 – Replace “are” with “were”

Line 81 – Was bidirectional Sanger sequencing used? I assume so given the length of the amplicon, but it is never stated.

Line 82 – Please make the “2” in MgCl2 a subscript. Please address this throughout the paper.

Line 84 – Replace “microliter” with “µl”, as it is used throughout the paper.

Line 84 – Add a space between 40ng/µl à “40 ng/µl”.

Line 96 & 104 & 11 – What kind of assay are these? SNP? Presence/absence? I’ve skimmed the citations and they appear to be good choices, but a clearer description of each type of assay used would be helpful.

Line 111 – I think Hauelka et al. 2017 is the proper citation, given where the primers came from (Table S2).

Line 126 – Don’t start sentences with numbers.

Lines 151-152 – Please clarify the language here to state that the commercial haplotypes are associated with the ship sturgeon.

Line 154-155 – Is there a citation for this statement?

Lines 195-197 – Consider evaluating this statement a bit more given what is known about the species in terms of dispersal. How likely is it that they are from the reintroduction efforts? Could they have dispersed to the Rioni?

Author Response

Thank you very much for your time and valuable suggestions and comments.

Section added in the text:

These markers are designed for the species, the protocols are straightforward and easy to implement in the laboratory. However, there can be uncertainties for Russian Sturgeon and Siberian sturgeon-specific markers, these markers differentiate these species from others with 96% and 99% probability respectively [17], while for stellate sturgeon and beluga sturgeon, these assays have shown 100% reliability [16,27].   Based on all these test results, the specimens captured in the Rioni River can be considered pure ship sturgeons, notwithstanding the potential for hybridization with other resident sturgeon species.      

         

Specific comments:

Line 13 – Replace “missing” with “undetected”

Replaced

Line 20 – Replace "which haplotype” with “, which the haplotype” (at least, I think)

Replaced

Line 21 – Please be specific about what is meant by “signs”. Are the authors talking about phenotypes here or the genotypic data they generated?

Added in text, it was based on genetic data.

Line 28-30 – This is a run-on sentence. Please address. One suggestion would be to replace “and is probably close to” with “ – with the species”

Replaced

Line 42 – Replace “are” with “were”

Replaced

Line 81 – Was bidirectional Sanger sequencing used? I assume so given the length of the amplicon, but it is never stated.

Yes, text was edited, we used Sanger sequencing. Samples were sequenced in one direction only, using the reverse primer, and electropherograms visually inspected for base calls; we resequenced any samples that showed ambiguities or poor quality sequence. Per Congiu et al., 2011, Sanger sequencing with the forward primer is contraindicated due to problematic sequence motifs. DNA sequences will be uploaded on Gene Bank.

Line 82 – Please make the “2” in MgCl2 a subscript. Please address this throughout the paper.

Edited as subscript, also through the text

Line 84 – Replace “microliter” with “µl”, as it is used throughout the paper.

Replaced

Line 84 – Add a space between 40ng/µl à “40 ng/µl”.

Edited

Line 96 & 104 & 11 – What kind of assay are these? SNP? Presence/absence? I’ve skimmed the citations and they appear to be good choices, but a clearer description of each type of assay used would be helpful.

Yes, they are SNP, and Presence/absence assays. Text is edited in Detecting Hybrids section

Line 111 – I think Hauelka et al. 2017 is the proper citation, given where the primers came from (Table S2).

Edited, Hauelka et al. 2017 indicated

Line 126 – Don’t start sentences with numbers.

Edited in the text.

Lines 151-152 – Please clarify the language here to state that the commercial haplotypes are associated with the ship sturgeon.

Clarified

Line 154-155 – Is there a citation for this statement?

We have this information from Professor Nikolai Mugue by email communication, he mantioned only two females (one from Volga and one from Ili river) were used in the past to propagate ship sturgeon in aquaculture and those in aquaculture now are decendents of the two females. And HAP01 is Volga haplotype also according to Professor Mugue. Do you think we can indicate personal communication here? Or I can ask Professor Mugue if we can indicate the information?

Lines 195-197 – Consider evaluating this statement a bit more given what is known about the species in terms of dispersal. How likely is it that they are from the reintroduction efforts? Could they have dispersed to the Rioni?

Text added:   We do not know the post-introduction life histories biology of these fish, and how farlong orand where they migrate., Hhowever, there is ~600 km distance between the Krasnodar River and the Rioni River, it is still less unlikely that the reintroduced fish have migrated from the Azov Sea basin and started reproducing in Georgia, in the Rioni River, a conclusion that is reinforced by the observed divergence of haplotypes between the respective populations.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript is interesting because it is one of the few reports about sturgeon fish in Georgia. However, the authors in the introduction should give more complete information about the sturgeon of the country (for example, https://doi.org/10.1007/978-3-642-20611-5_17; Guchmanidze, 2007, etc.).

There are several questions about the research methodology. Why didn't the authors use primers for European sturgeon hybrids? Only 9 individuals were used for molecular analysis. The rest were identified by morphological parameters. Then, in the appendix to this manuscript, I ask you to give a photo of all the individuals that have been identified by morphological parameters.

In the Rioni River, breeding conditions remained only for 9 km of river drainage (so the authors write). However, fish were caught almost 100 km from the mouth. Does this mean that the fish migrated so far? Does the dam on the river affect such migration? What is the age of the fish?

Line 186-197. These are premature conclusions based on only 9 samples.

Line 224-226. These are premature conclusions based on only 9 samples.

Author Response

Dear Sir/Madam,

Thank you very much for the valuable comments and suggestions. We appreciate your time.

Thank you very much for providing the link for the European sturgeon work by Guchmanidze, it was not accessible as long as we tried. However, we used Dr. Guchmanidze’s work Current and Historical Status of Sturgeon (Acipenseridae, Osteichthyes) in Georgia, from 2009.

Regarding European sturgeon primers, there can be diagnostic loci identified but as long as we follow recently published information there are no European sturgeon-specific diagnostic markers designed yet.

The pictures of the records will be provided as a separate file.

Regarding breeding conditions and distribution, the following text is added in the manuscript: The samples of A. nudiventris identified in this study were roughly clustered, with one set of samples from the coastal region (from 0-25 km inland), and the other samples were found ca. 80 km upstream of the Rioni River mouth. Samples from the coastal region ranged in size from 10 cm to 75 cm, while inland samples ranged in size from 30-60 cm. However, we have no sure knowledge of the migratory behavior or history of these individuals, or the precise location of spawning grounds for ship sturgeon in the Rioni River, and it would be premature to draw any geographic or biological inference based upon these data. Rather, since ship sturgeon clearly have survived and persist in the Rioni River, aggressive efforts are needed to monitor ship sturgeon populations in the Rioni, and to identify and protect spawning areas.

Line 186-197. These are premature conclusions based on only 9 samples.

There are only two haplotypes available on GeneBank Caspian Sea (KU321568) and the Ili River (Balkhash Lake basin) (KU321569) haplotypes, and these haplotypes characterize these two populations according to Mugue et al 2016 because Rioni River haplotype is different from these two we presume that the specimens are from remnant Black Sea population.

Line 224-226. These are premature conclusions based on only 9 samples.

All the markers we used are designed and used for sturgeon species and interspecies hybrid identification was tested and validated for different sturgeon species. This is why we really on the results to presume that the specimens are pure species.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Dear authors. Thank you for the answers.