Molecular Analysis of Two Endemic Squalius Species: Evidence for Intergeneric Introgression among Cyprinids and Conservation Issues
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsMembers of the cyprinid genus Squalius include 28 species distributed in Europe. Some species are of conservation concern. To promote effective conservation action, we must know which species occur where and whether they hybridize. Valic et al. made collections of S. illyricus and S. zyrmanjae at eight sites in Croatia and subjected them to analyses of mitochondrial cytochrome b and nuclear Cyfun P DNA sequence variation. While the results showed the collections to indeed belong to those species, results for S. zrmajae from the Krka River suggest a hybridization event with another species, S. dergle. The methods are appropriate, and the results are of interest to the conservation community. The presentation will have to be sharpened and the prose strengthened. I’ve marked the text to assist with the latter. A better integration of the findings with the literature on hybridization in cyprinids – which the authors briefly mention at lines 71-72 – would really strengthen the Discussion. Context-dependent comments follow.
Abstract. – The sentence at lines 27-28 is redundant with the abstract just above, and can be deleted.
Introduction. – The sentence at line 77 would be clearer if the authors were to write of segregation of species-specific alleles. “Recombination”, suggesting generation of new genetic variation at meiosis, misleads the reader in this context.
At line 94, the authors are not “determining” the species, but rather identifying, characterizing or distinguishing them.
At line 97, to avoid the appearance of bias, the authors might write of “assessing the possibility of hybridization”.
Methods. – The contents of Tables 2 and 3 can be presented as well-crafted sentences within the text.
At lines 153 and 154, we need supporting citations for MrBayes and Modeltest 3.7.
Results. – Figure 3 is too small to read. I suggest that the authors present this figure on a page oriented in landscape mode.
Similarly, Figure 4 is too small. If the authors were to use the full width of the page (not just the width of the text column), then it would prove readable. In the caption for Figure 4, is PP posterior probability? Please write that out.
Discussion. – Line 212 needs a call-out for Figure 3.
The paragraph starting at line 217 has important interpretation, yet I cannot assess its defensibility because Figure 3 is just too small for me to read. At line 230, it is not the rivers, but rather DNA sequences from fish from these two rivers that comprise two species.
At lines 243, 245, and 249, the authors might present S. zyrmanjae and S. derge in quotation marks to indicate the uncertainty of the taxonomic affiliations.
At line 255, the authors write that no clear explanation is known for why cyprinids hybridize so frequently. More is known than is acknowledged here. Cyprinid fishes often spawn in groups in the water column, with distinct species often spawning in proximity, such that sperm drift and interspecific fertilization is reasonably frequent. Many of the respective species are tetraploid (are Squalius species diploid or tetraploid?), and hence more amenable to hybridization. Indeed, some cyprinid species are allotetraploid, i.e., they resulted from historic hybridization events. This passage should include mention of other known hybridization events in cyprinids and discuss the sorts of issues that I mention here. The authors have a good start on developing this passage with references 22-27, some discussing cyprinids (there are other such citations to be found in the literature) and some more general on mechanism.
At line 268, does lowering of water masses denote sea level decline?
At line 291, it is knowledge of systematic relationships that is still incomplete.
At line 305, hybridization is a POTENTIAL problem that needs to be REsolved. Some hybrids have conservation value. Fred Allendorf and colleagues have written of the conservation value of some introgressed cutthroat trout populations, for example, a body of writing that might be cited in this context.
The sentence now appearing at lines 309-310 should be moved up to appear at line 307; microsatellites are too variable to be used for the purpose at hand, and mention should be deleted.
Data availability statement. – The GenBank accession numbers should be presented here.
References. – The stylistic for capitalizing or not capitalizing the key words of titles of journal papers should be consistent ad in conformance with journal stylistics. Species’ Latin names should be italicized at all usages.
Comments for author File: Comments.zip
Comments on the Quality of English LanguageThe prose will have to be strengthened. I’ve marked the text to assist with that.
Author Response
Please see the attachment
Author Response File: 
Author Response.docx
Reviewer 2 Report
Comments and Suggestions for Authors
The manuscript examines the problematic issue of the phylogeny of cyprinids, indicating possible intergeneric hybridization and introgression of genes. As evidence, data on the discordance of phylogenies obtained using mitochondrial and nuclear markers and morphologic data are presented. Other reasons may be invoked to explain the discordance, such as incorrect identification of species, including for accessions whose sequences are deposited in the GeneBank; imperfection of clustering algorithms; poor resolution of the applied markers; manifestation of ancient polymorphism, etc.
The originality of the text of the manuscript is more than 55%.
Although I am not a native speaker, the English was clear and easy to read.
Some minor comments that could improve the manuscript:
1) It is not clear which genera are referred to in the Key contribution section (line 32) and Conclusion section (line 305).
2) There is over-citation: [3-17] (lines 62-63). This may be halved.
3) Figure 3 is not clearly visible.
4) Comparison of morphological characteristics (lines 62-63) would be better presented in a table.
5) A sentence in the Conclusion section: “Furthermore, many non-native species such as the northern pike (Esox lucius) and the perch (Perca fluviatilis) have been introduced into this river basin, devastating the local ichthyofauna.” (lines 307-308) is not relevant to the problem discussed in the manuscript and should be deleted.
The manuscript can be accepted for publication after minor revision.
Comments for author File: 
Comments.pdf
Author Response
Please see the attachment
Author Response File: Author Response.docx
Reviewer 3 Report
Comments and Suggestions for AuthorsThere are many grammatical mistakes in the paper, some of which are noted in the attached pdf. There should be a table of the morphometric and meristic data. In the discussion morphological data are compared, but no table summarizing data from the fish you captured. The possibility of your results being sympleisiomorphic character states is not discusses. You have to at least consider this and attempt to refute it to make the paper more sound.
Comments for author File: Comments.pdf
Please do not use nouns (e.g., fish) as adjectives.
Author Response
Reviewer 3
There are many grammatical mistakes in the paper, some of which are noted in the attached pdf. There should be a table of the morphometric and meristic data. In the discussion morphological data are compared, but no table summarizing data from the fish you captured. The possibility of your results being sympleisiomorphic character states is not discusses. You have to at least consider this and attempt to refute it to make the paper more sound.
Our response:
We are very thankful to the Reviewer 3 for his suggestion.
We made numerous grammatical corrections in the text. Suggested corrections by the Reviewer 3, but also by the Reviewer 1 were included.
A table of the morphometric and meristic data is attached as supplement, because there are 30 pages in PDF document. This was made from our original field tables that are in Croatian, for all fishes examined, and for other investigated parameters. Because of this, table had to be translated and several other parameters deleted. We do not think that this would be a problem, but have to emphasize that this data is not of crucial importance for our paper, because the accent is on molecular methods. If it would be necessary to additionally edit the tables or display their results in a different way we kindly ask the Reviewer 3. for further instructions. In the discussion, morphological data are compared, but only to give already known morphological characters of the genus Squalius and Scardinius, which are used to differentiate these two genera (citations provided). In our original paper this was not presented, but previous reviewer wanted this to be in the paper.
About the possibility of our results being symplesiomorphic character states, it is true that we did not discuss that. In the case of plesiomorphy, cytochrome b sequence of S. zrmanjae would be located much lower in the phylogenetic tree, actually it would be at a node that is sister to a large number of other species. But this is not the case in our phylogenetic tree, S. zrmanjae is located far from the main nodes, and the branch length itself is relatively short, which indicates either hybridization or apomorphic properties. The same thing is in the case of the Cyfun phylogenetic tree, where S. zrmanjae is always shown as a derived taxon. In our paper we discussed hybridisation in the light of molecular results, specifically mitochondrial and nuclear markers, and implied that we found resemblance in the nuclear region between two genera.
Remarks from Reviewer 3, which have been given in the attached pdf, have been answered in the same PDF that is also attached to this paper. If the additional clarification should be needed, we will be happy to answer it.
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
Comments and Suggestions for AuthorsComments for author File: Comments.pdf
Author Response
We are very thankful to the Reviewer 3 for his suggestions, and we have made all the changes, according to the Reviewer 3 comments. All revisions to the manuscript are marked up in the PDF provided from the Reviewer 3 so they can be easily viewed, and accordingly, changes in the manuscript have been made.
Author Response File: Author Response.pdf
