Genetic and Phenotypic Characteristics of the Salmo trutta Complex in Italy

Round 1

Reviewer 1 Report

This paper is an extensive review of the genomes of the Salmo truth complex in Italy. It gives an interesting overview of the evolution and genetic diversity of the species, primarily in Italy, but also to some extent in the rest of the Mediterranean and Central European regions. 

The paper gives an - as far as I have been able to judge - complete overview of the genomic investigations carried out on the Salmo trutta complex, and discuss the findings in the context of what is known on the evolution of the species. 

The review provides good background knowledge to improve management of the IUCB Red list of threatened species, as som species in the S. trutta complex are threatened according to the IUCB list. 

The literature list is extensive, as it should be, also including other Salmo species, particularly S salar.

Author Response

This paper is an extensive review of the genomes of the Salmo truth complex in Italy. It gives an interesting overview of the evolution and genetic diversity of the species, primarily in Italy, but also to some extent in the rest of the Mediterranean and Central European regions.

The paper gives an - as far as I have been able to judge - complete overview of the genomic investigations carried out on the Salmo trutta complex, and discuss the findings in the context of what is known on the evolution of the species.

The review provides good background knowledge to improve management of the IUCB Red list of threatened species, as som species in the S. trutta complex are threatened according to the IUCB list.

The literature list is extensive, as it should be, also including other Salmo species, particularly S salar.

Reply: more than 20 bibliographic entries have been added to the list (with methods also used on other Salmo Specie as the Atlantic salmon).

Reviewer 2 Report

Dear authors,

I have thoroughly read your manuscript and unfortunately, I have not been satisfied with its structure and the extent of the content. I would propose a major revision before any publication. Please see my following comments and if the Editors agree I would suggest a second round of reviews if not rejected.

Title. Genomic --> Genetic. The word "genetic" is more appropriate based on the content of the article.
Title. analysis --> analyses. The article reviews the relevant analyses, not just one of them.

Affiliation's citation numbering is missing from some authors.

Correspondence asterisk is on the last author but the details are of the first author.

The abstract ends insufficiently with no conclusions.

The scope of the review is not clear. How does this review fit the scope of the Journal of Applied Sciences?

Sections' titles are too general. Please try to be more specific.

Also, this article as a review it should be more comprehensive and include all the significant relevant articles such as DOI 10.1007/s10750-012-1321-8, DOI 10.1007/s10750-006-0479-3, DOI 10.1111/j.0022-1112.2004.00458.x, DOI 10.1023/B:EBFI.0000005762.81631.fa, DOI 10.1111/bij.12279, DOI 10.1080/11250000600679751, DOI 10.1006/mpev.1994.1008 and others.

Part between lines 77-120 lacks references.

Lines 129-131. How can the multispecies coalescent method be very useful in the phylogenetic analysis of the S.trutta complex? Please explain.

Lines 138-144. Please add references.

Line 146. Please add references for such cases.

Lines 149-154. The reference is for Araneae. What about fish species?

Line 173. What about EST-SSRs? Microsatellites are not always neutral. Please add references.

Line 187-188. Please add references.

Section 3. This section has just theoretical information about microsatellites and no data for S.trutta complex. It could be merged with Section 2.

Section 4. this Section is proportionally small. It could be merged with the previous ones.

L257. Please add references for STACKs and other programs.

L320-324. Please add references.

L353-363. Reference "30" is cited 4 times in the same paragraph and it is the only reference of the paragraph.

Section 8. This section lacks references.

Figures 5, 6 and 8 are mentioned but I cannot locate the actual Figures.
Figures 3, 4 and 7 are not even mentioned. Am I missing something?

Author Response

First of all, all authors of paper would like to thank the three referees for their positive appreciation (interesting, well organized, well written, etc.) of the work and for their valuable comments. In the revised version, more than 20 bibliographic entries have been added to the list. This topic is much debated at international level and in Italy in relation to the importance of the subject (impressive number of articles) in the context of the conservation of freshwater fish species and the European policies for the protection of native species. The aim of the review is to provide an original work and an update of the researches carried out in the last years in particular in the field of genetics and genomics. Some of the topics covered (for example description of the phenotipic traits in section 8) are the result of two Life+ projects and one research project and are based on our personal experience (more than twenty years) in the field of freshwater fish conservation and laboratory genetic analysis.  We have accepted all requests from the referee 2.

• Genomic --> Genetic. The word "genetic" is more appropriate based on the content of the article.

 Replay: we accept the request and we have modify the text accordingly.

• analysis --> analyses. The article reviews the relevant analyses, not just one of them.

Replay:  we accept the request and we have modify the text accordingly

• Affiliation's citation numbering is missing from some authors.

Replay: we accept the request and we have modify the text accordingly.

• Correspondence asterisk is on the last author but the details are of the first author.

Replay: we accept the request and modify the text accordingly.

• The abstract ends insufficiently with no conclusions.

Replay:  we accept the request and we have modify the text accordingly.

• Sections' titles are too general.

Replay:  we accept the request and modify the text accordingly.

• The following references have been included in the text:

DOI 10.1007/s10750-012-1321-8,

Meraner, A.; Gratton, P.; Baraldi, F.; Gandolfi, A. (2013). Nothing but a trace left? Autochthony and conservation status of Northern Adriatic Salmo trutta inferred from PCR multiplexing, mtDNA control region sequencing and microsatellite analysis. Hydrobiologia  2013, 702 (1), 201-213.

DOI 10.1007/s10750-006-0479-3,

Meraner, A., Baric, S., Pelster, B., Dalla Via J.  Trout (Salmo trutta) mitochondrial DNA polymorphism in the centre of the marble trout distribution area. Hydrobiologia  2007, 579, 337–349.

DOI 10.1111/j.0022-1112.2004.00458.x,

Caputo, V , Giovannotti, M , Nisi Cerioni, P , Caniglia, M. L , Splendiani, A. Genetic diversity of brown trout in central Italy. J. fish biol. 2004, 65 (2), 403-418.

DOI 10.1023/B:EBFI.0000005762.81631.fa,

Marzano, F.N.; Corradi, N.; Papa, R.; Tagliavini, J.; Gandolfi, G. Molecular Evidence for Introgression and Loss of Genetic Variability in Salmo (trutta) macrostigma as a Result of Massive Restocking of Apennine Populations (Northern and Central Italy). Environ. Biol. Fishes 2003, 68, 349–356.

DOI 10.1111/bij.12279,

Fruciano, C., Pappalardo, A.M., Tigano, C., Ferrito, V., Phylogeographical relationships of Sicilian brown trout and the effects of genetic introgression on morphospace occupation, Biol. J. Linn. Soc. 2014, 112, 3, 387–398.

DOI 10.1080/11250000600679751,

Splendian,i A., Giovannotti, M.,  Nisi Cerioni, P., Caniglia  M.L., Caputo, V.(2006) Phylogeographic inferences on the native brown trout mtDNA variation in central Italy. Ital. J. Zool. 2006, 73:2, 179-189.

DOI 10.1006/mpev.1994.1008

Patarnello, T., Bargelloni, L., Caldara, F., Colombo, L. Cytochrome b and 16S rRNA sequence variation in the Salmo trutta (Salmonidae, Teleostei) species complex. Mol. Phylogenet Evol. 1994, 3(1):69-74.

• Part between lines 77-120 lacks references.

The following references have been included in the text:

Hewitt, H.G.M. Some genetic consequences of ice ages, and their role in divergence and speciation. Biol. J. Linn. Soc. 1996, 58, 247–276.

Bernatchez L. 2001. The evolutionary history of brown trout (Salmo trutta L.) inferred from phylogeographic, nested clade, and mismatch analyses of mitochondrial DNA variation. Evolution 2001, 55, 351–379.

Hamilton, K.E., Fergusson, A., Taggart, J.B., Tómasson ,T. Post-glacial colonization of brown trout, Salmo trutta L.: Ldh-5 as a phylogeographic marker locus. J. Fish Biol. 1989, 35, 651–664.

Cortey, M., Vera, M., Pla C., Garcìa-marìn, J. Northern and Southern expansions of Atlantic brown trout (Salmo trutta) populations during the Pleistocene. Biol. J. Linn. Soc. 2009, 97, 904–917.

• Lines 129-131. How can the multispecies coalescent method be very useful in the phylogenetic analysis of the S.trutta complex? Please explain.

Replay: this sentence has been removed from the text

• Lines 138-144. Please add references.

Replay: the following references have been included in the text:

Rubinoff, D., Holland, B.S. Between Two Extremes: Mitochondrial DNA is neither the Panacea nor the Nemesis of Phylogenetic and Taxonomic Inference. Syst. Biol. 2005, 54, 6, 952–961.

Tobe, S.S., Kitchener, A.C., Linacre, A.M.T. Reconstructing Mammalian Phylogenies: A Detailed Comparison of the Cytochrome b and Cytochrome Oxidase Subunit I Mitochondrial Genes. PLoS ONE 2010, 5(11), e14156.

• Line 146. Please add references for such cases.

Replay: this sentence has been removed from the text

• Lines 149-154. The reference is for Araneae. What about fish species?

Replay: the reference has been remouved.

mtDNA

The following references have been included in the text

Snoj, A., Melkic, E., Susnik ,S., Muhamedagic, S., Dovc, P. DNA phylogeny supports revised classification of Salmothymus obtusirostris. Biol. J. Linn. Soc. 2002, 77, 399–411.

Berrebi, P., Caputo Barucchi, V., Splendiani, A., Muracciole, S., Sabatini, A., Palmas, F., Tougard, C., Arculeo, M., Maric S. Brown trout (Salmo trutta L.) high genetic diversity around the Tyrrhenian Sea as revealed by nuclear and mitochondrial markers. Hydrobiologia 2019, 826, 209–231.

Cortey, M., Garcìa-Marìn, J.L. Evidence for phylogeographically informative sequence variation in the mitochondrial control region of Atlantic brown trout. J. Fish Biol. 2002, 60, 1058–1063.

Cortey, M., Pla, C. Garcìa-Marìn J.L.  Historical biogeography of Mediterranean trout. Mol. Phylogenetics Evol. 2004,  33, 831–844.

Giuffra 1994

Microsatellites

The following references have been included in the text

Araguas, R.M., Vera, M., Aparicio, E., Sanz, N., Fernández-Cebrián, R., Marchante, C., García-Marín, J.L. Current status of the brown trout (Salmo trutta) populations within eastern Pyrenees genetic refuges. Ecol. Freshw. Fish. 2017, 26, 120-132.

Patrick Berrebi, P., Tougard, C., Dubois, S., Shao ,Z., Koutseri, I., Petkovski, S., Crivelli A.J. Genetic Diversity and Conservation of the Prespa Trout in  the Balkans. Int. J. Mol. Sci. 2013, 14, 23454-23470.

Meraner, A., Gratton, P., Baraldi, F.; Gandolfi, A. Nothing but a trace left? Autochthony and conservation status of Northern Adriatic Salmo trutta inferred from PCR multiplexing, mtDNA control region sequencing and microsatellite analysis. Hydrobiologia 2013, 702 (1), 201-213.

Line 173. What about EST-SSRs? Microsatellites are not always neutral. Please add references.

We accept the request and we have modify the text accordingly.

The following references have been included in the text

Lemopoulos, A., Prokkola, J.M., Uusi-Heikkilä, S., Anti Vasemägi,A.,  Ari Huusko, A., Hyvärinen, P., Koljonen, M.L.,  Koskiniemi, J., Vainikka, A. Comparing RADseq and microsatellites for estimating genetic diversity and relatedness — Implications for brown trout conservation. Ecol. Evol. 2019, 9, 2106– 2120.

• Line 187-188. Please add references.

Replay: the following references have been included in the text.

Grohme, M.A., Soler, R.F., Wink, M., Frohme, M. Microsatellite marker discovery using single molecule real-time circular consensus sequencing on the Pacific Biosciences RS. Biotechniques 2013, 55(5), 253-6.

• Please add references for STACKs and other programs.

Replay: the following references have been included in the text

Catchen, J.M., Amores, A., Hohenlohe, P., Cresko, W., Postlethwait, J.H. Stacks: building and genotyping Loci de novo from short-read sequences. G3 (Bethesda) 2011, 1, 171–182.

Minh, B.Q., Schmidt, H.A., Chernomor, O., Schrempf, D., Woodhams, M.D., von Haeseler, A., Lanfear, R. IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Mol. Biol.Evol. 2020, 37, 5, 1530–1534.

Bradbury, P.J., Zhang, Z., Kroon, D.E., Casstevens, T.M., Ramdoss, Y., Buckler, E.S. TASSEL: Software for association mapping of complex traits in diverse samples. Bioinformatics 2007, 23, 2633-2635.

Eaton, D.A.R. PyRAD: assembly of de novo RADseq loci for phylogenetic analyses , Bioinformatics 2014, 30, 13, 1844–1849.

Puritz, J.B., Hollenbeck, C.M., Gold, J.R. dDocent: a RADseq, variant-calling pipeline designed for population genomics of non-model organisms. PeerJ. 2014, 2, e431.

Sovic, M.G., Fries, A.C., Gibbs, H.L. AftrRAD: a pipeline for accurate and efficient de novo assembly of RADseq data. Mol. Ecol. Resour. 2015, 15(5), 1163-71.

• L320-324. Please add references

Replay: the following references have been included in the text

Splendiani, A., Palmas, F., Sabatini, A., Caputo Barucchi, V. 2019. The name of the trout: considerations on the taxonomic status of the Salmo trutta L., 1758 complex (Osteichthyes: Salmonidae) in Italy. Europ. Zool. J. 2019, 86, 432–442.

Cortey, M., C. Pla & J. L. Garcı´a-Marı´n, 2004.

• L353-363. Reference "30" is cited 4 times in the same paragraph and it is the only reference of the paragraph.

Replay:  we accept the request and we have modify the text accordingly

• Section 8. This section lacks references.

Lorenzoni, M., Carosi, A., Giovannotti M., La Porta G., Splendiani A.  Caputo Barucchi V. Morphological survey as powerful detection tool of pure and local phenotypes in Salmo trutta complex. Knowl. Manag. Aquat. Ecosyst. 2019, 420, 48.

Aparicio, E., García-Berthou, E., Araguas, R.M., Martínez, P., García-Marin, J.L. Body pigmentation pattern to assess introgression by hatchery stocks in native Salmo trutta from Mediterranean streams. J. Fish Biol. 2005, 67, 931–949.

Kottelat and Freyhof (2007)

Zaccara et al. (2015)

Delling et al. (2019)

• Figures 5, 6 and 8 are mentioned but I cannot locate the actual Figures.

Replay: this sentence has been removed from the text

• Figures 3, 4 and 7 are not even mentioned. Am I missing something?

Replay: this sentence has been removed from the text

Reviewer 3 Report

In a well written text, the authors present an analysis of the distribution of Salmo trutta in Italy and in surrounding areas.

In general, it is not clear why it is interesting a review article on the genetic and phenotypic characteristics of Salmo trutta specifically in Italy and why it is interesting to focus on different parts of Italy in separate paragraphs. The authors should stress this point explicitly. I would found more interesting a more general review covering the whole distribution of the species complex.

The title is misleading because genomic analysis is only a small part of the review. The main comments regard the distinction of Salmo trutta populations based on the mtDNA control region and not on genomic data. In addition, the review does not only refer to genetic analysis but it is extended to the phenotypic analysis as well.

In the abstract it is not obvious that the paper is a review article. Also, it is not obvious why this review is written for and what are the main conclusions that we would expect if we overview the literature.

Line 44 and line 325: Salmo trutta should be in italics.

Fig 1. What is the meaning of the colors? Also what is the meaning of the drawings in the up-right corner?  they look like three snowflakes but I don’t see the point of being there.

Fig 2. This figure is not found in the text. The thermometer presenting in the up-right corner indicates that the colors represent temperatures, but it is not shown the temperature scale.

Line 382: I cannot find figures 5 and 6

Line 458: I cannot find figure 8

In general, I find the two figures of the review irrelevant with the main topic of the paper. I would prefer a map showing the potential migrations of the Salmo species (as described in lines 110 and below) rather than two figures for the temperatures of the seas. Also, it would be useful a map with the current distribution of the species, with the different genetic lineages annotated. Plus, I would find interesting if the figures 5, 6 and 8 which are described in the text were actually presented.

I find the description of the mtDNA (lines 133-167) and microsatellites (171-188) unnecessary. Nobody expects to learn how mtDNA or microsats are used as molecular markers. Paragraph 4 (lines 190-197) gives all needed information in relation to Salmo trutta.

The conclusions paragraph are vague.

Author Response

First of all, all authors of paper would like to thank the three referees for their positive appreciation (interesting, well organized, well written, etc.) of the work and for their valuable comments. In the revised version, more than 20 bibliographic entries have been added to the list. This topic is much debated at international level and in Italy in relation to the importance of the subject (impressive number of articles) in the context of the conservation of freshwater fish species and of the European policies for the protection of native species. The aim of the review is to provide an original work and an update of the researches carried out in the last years in particular in the field of genetics and genomics. Some of the topics covered (for example description of the phenotipic traits in section 8) are the results of two Life+ projects and one research project and they are based on our personal experience (more than twenty years) in the field of freshwater fish conservation and laboratory genetic analysis.  

We have accepted all requests from the referee 3.

1)Line 44 and line 325: Salmo trutta should be in italics.

Replay: we accept the request and we have modify the text accordingly.

2) Fig 1 and Fig. 2

Replay: we accept the request and we have modify the text accordingly.

The following references have been included in the text:

Hayes, A., Kucera, M., Kallel, N., Sbaffi, L., & Rohling, E. J. (2005). Glacial Mediterranean sea surface temperatures based on planktonic foraminiferal assemblages. Quaternary Science Reviews, 24(7-9), 999-1016.

3) Line 382: I cannot find figures 5 and 6

Replay: this sentence has been removed from the text

4) Line 458: I cannot find figure 8

Replay: this sentence has been removed from the text

5) Introduction, genetic analysis, conclusions

Replay: we accept the request and we have modify the text accordingly.

Round 2

Reviewer 2 Report

Title. Phenotipic -->Phenotypic