Genetic Variability, Population Structure, and Relatedness Analysis of Meagre Stocks as an Informative Basis for New Breeding Schemes
, Konstantinos Tzokas 3, Leonidas Papaharisis 3, Katerina Ekonomaki 1, Dimitrios Chatziplis 4, Costas Batargias 5 and Costas S. Tsigenopoulos 1
Round 1
Reviewer 1 Report
fishes-1413735
Nousias et al
In this work, the genetic diversity and differentiation between eleven meagre stocks is evaluated.
The goals of this study (as stated by the authors, lines 78-82) were “to investigate the genetic diversity among stocks of breeders and their offspring, to describe their putative relatedness, inbreeding and population structure, and last to propose an implementation of population genetics analytical tools based on microsatellite markers as a cost-effective solution to set the basis for the design and establishment of breeding programs.”
The justification of the work is adequate. The MS is well written (needs an English revision) and interesting, but I have some concerns that need to be addressed before recommending its publication.
General comments
Regarding the third objective (lines 80-82), since the authors do not propose any specific mating scheme for their case study (11 stocks) or a simulation of the impact of the choice of breeders of one or another population is not shown, in my opinion, this is not an objective of the work, but rather a conclusion or recommendation. Please move this sentence to the discussion o conclusion.
References in the text must be numbered.
Species names must be in italics.
In the references section, it is necessary to review the format and adjust it to that of the journal.
The numbers of tables and/or figures in the MS must be correlative. The supplementary material must be indicated with an S and be correlative also (with respect to the rest of the supplementary material).
Please, review the order in which they appear in the text.
The article, especially both the introduction and discussion section, needs to be enriched with more references.
The Introduction section is general and short. In lines 43 to 50, the relationship between fitness and effective population size is discussed, I recommend adding some recent reference on this topic (see for instance https://doi.org/10.1098/rspb.2019.1989). Also, I recommend enriching the introduction with some recent reference
Line 61 Wright et al, 2004 is not in the references list
Line 70 tonnes (FAO, 2016. Please update this information
Line 78. Please change was by were
Line 86 Sampling. The “old breeders”, are all of them from the wild? There is any information about the sampling location? About age? Please clarify.
DNA techniques/PCR... line 95 The multiplex design is based on heterologous primers developed, the optimization refers to the amplification conditions, isn’t it? Please explain. This is important because the use of microsatellites from a different species explains the low numbers of alleles detected.
Line 97, Is the concentration of primers 10µmol/l? or 10µM (=10pmol/L)
Line 100 please change steps by step
2.3.1. section. There is nothing to argue with in this section, but those results are presented as supplementary material, and they are, in my opinion, central to this study. I could not find the significance of the Fis values. The frequency of null alleles in the rest of the MS is not mentioned or discussed. The same occurs with Shannon’s diversity Index. Please, explain better on the text.
Lines 117-126. Please, explain why you choose Structure to infer the structure of the population in this case. This program tends to inadequately solve situations in which there is no equilibrium or the difference in population sizes is large (as in this case)
Line 146, explain better and reference Pew et al, adequately
Table 1. It is recommended to use the same number of decimal places in all numbers. Line 163 Please use “is” instead of “are” and “first” instead of “last”.
Line 172 Figure 2 is cited, where is figure 1?
Lines 179-184. Try to summarize this information. My advice is to add HW concept to make it easier and because the term is introduce afterwards.
Lines 185-187. Please rephrase. With all due respect it is not well expressed. It cannot be said that negative values of Fis in a population with random mating indicate a certain degree of mating between unrelated individuals, because then it would not be random.
Lines 188-190 Authors said that as stocks A-H are breeders, the heterozygous excess detected is understandable (it makes sense). Regarding the number of individuals and the number of alleles I will expect more stocks in equilibrium. In low polymorphic markers is easy to recover all the alleles and that for the frequencies to be in equilibrium. Please elaborate on that.
Authors mention the “statistically significant limits of the HW equilibrium” I could not find the significance values. Could you please add them to the table?
Regarding Structure results, as I interpret figure 3, there is no a single individual from cluster 2 without any introgression from another cluster. In fact, all of them are hybrid with individuals from Cluster 3 (old) or 5 (recent). Various levels of introgression can be detected on population K. It would be interesting to explain those results based on the location of origin for those stocks.
In the next section references to the figures are not in order and it is difficult to follow. Results are interesting but an explanation in light of the situation is missing. An example of the usefulness of this analysis in deciding a mating scheme would be ok.
Line 264, instead of “the least variance” use “the smallest” and in line 266 instead of more variance use high
The interpretation of figure 10 on page 10 agrees with the scenario retrieved by Structure. As I already mentioned a “next step section” will be needed, with a proposal for stocks management or how these methods can be used to establish breeding nucleuses (line 343).
Author Response
Reviewer 1:
Comments and Suggestions for Authors
In this work, the genetic diversity and differentiation between eleven meagre stocks is evaluated.
The goals of this study (as stated by the authors, lines 78-82) were “to investigate the genetic diversity among stocks of breeders and their offspring, to describe their putative relatedness, inbreeding and population structure, and last to propose an implementation of population genetics analytical tools based on microsatellite markers as a cost-effective solution to set the basis for the design and establishment of breeding programs.”
The justification of the work is adequate. The MS is well written (needs an English revision) and interesting, but I have some concerns that need to be addressed before recommending its publication.
General comments
Regarding the third objective (lines 80-82), since the authors do not propose any specific mating scheme for their case study (11 stocks) or a simulation of the impact of the choice of breeders of one or another population is not shown, in my opinion, this is not an objective of the work, but rather a conclusion or recommendation. Please move this sentence to the discussion o conclusion.
-Reply : The sentence was removed from the manuscript as it was already mentioned in the Discussion section 341-342
References in the text must be numbered.
-Reply : All references in the text were numbered
Species names must be in italics.
-Reply : Species names now are correctly written in italics
In the references section, it is necessary to review the format and adjust it to that of the journal.
-Reply : We corrected the references’ format according to the journal guidelines
The numbers of tables and/or figures in the MS must be correlative. The supplementary material must be indicated with an S and be correlative also (with respect to the rest of the supplementary material).
Please, review the order in which they appear in the text.
-Reply : We followed the reviewer’s suggestion and corrected the ordering of the tables/figures and the supplementary material accordingly.
The article, especially both the introduction and discussion section, needs to be enriched with more references.
- Reply: The introduction was enriched with numerous references and the following part was added : (line 37) “Highly fecund species or r-strategists, like fish, exhibit higher polymorphism than species that produce low numbers of eggs and/or offspring of bigger body size, also termed K-strategists, with the propagule size (the size of the stage that leaves its parents and disperses, egg or juvenile) being highly predictive of a species genetic diversity (Romiguier et al., 2014; Ellegren & Galtier 2016). ”
(The references are just kept in this form for review purposes, in the text the have been replaced by their corresponding numbers accordingly)
The Introduction section is general and short. In lines 43 to 50, the relationship between fitness and effective population size is discussed, I recommend adding some recent reference on this topic (see for instance https://doi.org/10.1098/rspb.2019.1989). Also, I recommend enriching the introduction with some recent reference
- Reply: Following the recommendation, we now added the following sentence:
“However, genetic purging is evident in wild populations of fish species with low population sizes or low genetic diversity estimates, as well as but for populations that have persisted in low sizes and in an environmental niche since their formation (Yates et., 2019). Moreover, purging in wild populations of low size may have assuaged the effects deleterious alleles in such populations have on fitness, thus preventing a decline in fitness (Danzmann et al., 1998), considering that the population sizes are not extremely low, below critical thresholds (Jamieson & Allendorf, 2012), making these populations highly specialized and at the same time vulnerable to new selective pressures”
(The references are just kept in this form for review purposes, in the text the have been replaced by their corresponding numbers accordingly)
Line 61 Wright et al, 2004 is not in the references list
-Reply : Corrected
Line 70 tonnes (FAO, 2016. Please update this information
-Reply: Thank you for this, we updated the information (APROMAR,2019)
Line 78. Please change was by were
- Reply: It was corrected accordingly
Line 86 Sampling. The “old breeders”, are all of them from the wild? There is any information about the sampling location? About age? Please clarify.
- Reply: We replaced the section with the following:
“In September 2017, 302 meagre samples were collected from eight breeders stocks (A-H) aged more than 6 years old that have been purchased from commercial companies except stock G which was composed of wild fish. The last three stocks (I-K) were sampled in June 2020 and consisted of 643 offspring (younger than 4 years old) of the above eight stocks.”
DNA techniques/PCR... line 95 The multiplex design is based on heterologous primers developed, the optimization refers to the amplification conditions, isn’t it? Please explain. This is important because the use of microsatellites from a different species explains the low numbers of alleles detected.
Reply: Indeed, the cross-species amplification generally results in a lower number of alleles. Here the optimization refers to the use of the Qiagen kit and the choice of the specific 12 from a greater repertoire of already developed microsatellite loci in phylogenetically-close species like yellow meagre (Cynoscion acoupa) [Casmic-14, Farias et al. (2006)], Japanese meagre (Argyrosomus japonicus) [UBA, Archangi et al. (2009)] and red drum (Sciaenops ocellatus) [Soc, O’Malley et al. (2003) & Saillant et al. (2004)]. But all these are already reported in the paper cited, i.e. Nousias et al. (2020)
Line 97, Is the concentration of primers 10µmol/l? or 10µM (=10pmol/L)
Reply: Corrected
Line 100 please change steps by step
Reply: Corrected
2.3.1. section. There is nothing to argue with in this section, but those results are presented as supplementary material, and they are, in my opinion, central to this study. I could not find the significance of the Fis values. The frequency of null alleles in the rest of the MS is not mentioned or discussed. The same occurs with Shannon’s diversity Index. Please, explain better on the text.
Reply: We have moved the Fis table into the main MS and report the significant values.
We also commented the micro-checker as well as the linkage disequilibrium results at the start of the discussion and results section. (reviewer 3 requested it as well)
Concerning Shannon’s information index: this index is used because of its higher sensitivity, it takes values out of the restrictions of 0-1 that He takes. So it has increased in popularity in population genetics studies in the last few years. It actually follows the same pattern as He, increasing and decreasing accordingly. That is why it is not mentioned in the text
Lines 117-126. Please, explain why you choose Structure to infer the structure of the population in this case. This program tends to inadequately solve situations in which there is no equilibrium or the difference in population sizes is large (as in this case)
Reply: This is correct! But our purpose was not to infer population genetics structure but rather to just delineate stocks and we now explain the importance of using the non-admixture model in our analyses which in a way counterbalance the use of variable alpha prior option.
Added : “While STRUCTURE has been reported to provide inaccurate estimates when a uniform alpha prior is used to infer ancestry between populations of unequal sizes (Wang, 2016), this applies to the admixture model analysis and can be corrected with the use of variable alpha prior option. However, in the non-admixture approach, the unequeal sample sizes do not influence the result”
Line 146, explain better and reference Pew et al, adequately
Reply: reference corrected
Table 1. It is recommended to use the same number of decimal places in all numbers. Line 163 Please use “is” instead of “are” and “first” instead of “last”.
Reply: Corrected
Line 172 Figure 2 is cited, where is figure 1?
Reply: Corrected
Lines 179-184. Try to summarize this information. My advice is to add HW concept to make it easier and because the term is introduce afterwards.
Lines 185-187. Please rephrase. With all due respect it is not well expressed. It cannot be said that negative values of Fis in a population with random mating indicate a certain degree of mating between unrelated individuals, because then it would not be random.
Reply: corrected
“Negative FIS values in a panmictic population, with the sample size being representative, indicate an increased degree of mating between individuals less related to each other, while positive values indicate the opposite. “
Lines 188-190 Authors said that as stocks A-H are breeders, the heterozygous excess detected is understandable (it makes sense). Regarding the number of individuals and the number of alleles I will expect more stocks in equilibrium. In low polymorphic markers is easy to recover all the alleles and that for the frequencies to be in equilibrium. Please elaborate on that.
Reply: clarified in the paper, in materials and methods section as well as the discussion
Authors mention the “statistically significant limits of the HW equilibrium” I could not find the significance values. Could you please add them to the table?
Reply: added the limits in the section where Fis is discussed.
Regarding Structure results, as I interpret figure 3, there is no a single individual from cluster 2 without any introgression from another cluster. In fact, all of them are hybrid with individuals from Cluster 3 (old) or 5 (recent). Various levels of introgression can be detected on population K. It would be interesting to explain those results based on the location of origin for those stocks.
-Reply : Most of the stocks were purchased from other companies and only one was the “wild” one. These are mostly from France and Spain and therefore we expect a relatively low differentiation per se since they normally belong to the “Atlantic-W.Med” stock (see Haffray et al., 2012) which is theoretically very distinguishable from the East Mediterranean one.
In the next section references to the figures are not in order and it is difficult to follow. Results are interesting but an explanation in light of the situation is missing. An example of the usefulness of this analysis in deciding a mating scheme would be ok.
Reply: corrected
Line 264, instead of “the least variance” use “the smallest” and in line 266 instead of more variance use high
Reply: Corrected
The interpretation of figure 10 on page 10 agrees with the scenario retrieved by Structure. As I already mentioned a “next step section” will be needed, with a proposal for stocks management or how these methods can be used to establish breeding nucleuses (line 343).
Reply: we elaborated on this a little more on the last part of the discussion
Reviewer 2 Report
The present study carried out analyzes of genetic diversity and structure of 11 Meagre stocks cultivated in Crete, Greece. The general interest of the manuscript is very specific, however, the authors have conducted several analyzes to address questions about genetic distance and relatedness between stocks, which are relevant to the management of aquaculture species in general, therefore, it is worth publishing.
Although the total number of individuals sampled from each stock is very discrepant, the authors discuss issues related to this. However, there are some issues that still need to be improved and/or better clarified.
In "Introduction" there are not enough arguments to show the relevance of the study indeed. Furthermore, there is no information about the study focal stocks in "Materials and Methods"; and, most importantly, there are no conclusions about the relevance of the results in the final session.
Thus, I strongly suggest restructuring the text to highlight the main issues addressed at the beginning of the article (Introduction), and a summary of the implications of the findings for the field of study at the end of the article (last paragraph). See my suggestions and considerations in the attached file.
Comments for author File: 
Comments.pdf
Author Response
Reviewer 2:
Comments and Suggestions for Authors (but also see attached revision on the pdf itself!)
Reply: All corrections were done according to the suggestions in the pdf
The present study carried out analyzes of genetic diversity and structure of 11 Meagre stocks cultivated in Crete, Greece. The general interest of the manuscript is very specific, however, the authors have conducted several analyzes to address questions about genetic distance and relatedness between stocks, which are relevant to the management of aquaculture species in general, therefore, it is worth publishing.
Although the total number of individuals sampled from each stock is very discrepant, the authors discuss issues related to this. However, there are some issues that still need to be improved and/or better clarified.
In "Introduction" there are not enough arguments to show the relevance of the study indeed. Furthermore, there is no information about the study focal stocks in "Materials and Methods"; and, most importantly, there are no conclusions about the relevance of the results in the final session.
Thus, I strongly suggest restructuring the text to highlight the main issues addressed at the beginning of the article (Introduction), and a summary of the implications of the findings for the field of study at the end of the article (last paragraph). See my suggestions and considerations in the attached file.
Reply: The stocks are not cultured in Crete but refer to commercial stocks of companies based in continental Greece and Spain and produce a large number of eggs and juveniles in the Mediterranean region.
In the M&M section, we now give more information on the origin of stocks and we hope that some points are better clarified.
We added studies that are relevant and elaborated on the findings of this study
Reviewer 3 Report
This manuscript reports the genetic diversity and genetic population structure of farmed populations of meagre that belong to commercial hatcheries in Greece, one of the main producers of Mediterranean marine species, using microsatellite markers. In my opinion, obtained results are very important for fish stocks management in farms. However, I think that some major issues should be addressed before manuscript publication as it is described below:
- Introduction
- LINE 52, 53, 63. Suggestion: The scientific names are not italicized (check the entire manuscript)
- LINE 70. There are more actualized production statistics for the species in the Mediterranean (2020), according to Spanish Aquaculture Business Association (APROMAR)
- LINE 71. What are the bottlenecks in the production of the species? How important is the work? Such information must be provided based on the scarcity of scientific studies, compared to other important species cultivated in the region. In addition, the species needs genetic studies due to the particular problems (See https://www.diversifyfish.eu/) (Comment these informations also in the Discussion Section)
- Material and Methods
-
- LINE 104. Why effective population size on farmed meagre stocks were not estimated? If possible, estimate these parameters.
- Results and Discussion
-
- The results need to be better discussed, and compared to previous genetic studies, relating them with the formation of broodstocks. The discussion is poor regarding the development of the production of the species based on the results
- Improve quality Figure 2
- The author discussed the methods of analysis, but should further discuss how his results of diversity, genetic structure, and relatedness could contribute to the formation of base populations of the species, and how genetic diversity can be improved. The discussion, in my opinion, is poor.
Author Response
Reviewer 3:
Comments and Suggestions for Authors
This manuscript reports the genetic diversity and genetic population structure of farmed populations of meagre that belong to commercial hatcheries in Greece, one of the main producers of Mediterranean marine species, using microsatellite markers. In my opinion, obtained results are very important for fish stocks management in farms. However, I think that some major issues should be addressed before manuscript publication as it is described below:
- Introduction
- LINE 52, 53, 63. Suggestion: The scientific names are not italicized (check the entire manuscript)
- LINE 70. There are more actualized production statistics for the species in the Mediterranean (2020), according to Spanish Aquaculture Business Association (APROMAR)
Reply: The following is now inserted:
The aquaculture production of meagre (Argyrosomus regius) in the Mediterranean area exceeded 41 thousand tonnes in 2019 showing a growth of 10.5% compared to the previous year while an additional growth of 2.8% is expected actually (APROMAR, 2020 http://apromar.es/) with Egypt producing the greatest part (approx. 75%) of this production followed by far from Spain (9%), Turkey (6%) and Greece (4%).
- LINE 71. What are the bottlenecks in the production of the species? How important is the work? Such information must be provided based on the scarcity of scientific studies, compared to other important species cultivated in the region. In addition, the species needs genetic studies due to the particular problems (See https://www.diversifyfish.eu/) (Comment these informations also in the Discussion Section)
Reply: The following is now inserted using information from the Aquaculture Europe magazine, 2017, vol. 42, issue 2, in which some Diversify results were mentioned (https://www.diversifyfish.eu/uploads/1/4/2/0/14206280/diversify_featured_article_aes_42_sept_2017.pdf)
“ Despite some attractive attributes of meagre like the large size, good processing yield, low fat content, excellent taste and firm texture (Monfort, 2010), there are few major drawbacks identified like the limited genetic variation of the available broodstocks, variable growth rates and the wide occurrence of systemic granulomatosis which constitute bottlenecks to the expansion of the industry” , and this study is coming to meet the first one (i.e. the genetic study)
- Material and Methods
- LINE 104. Why effective population size on farmed meagre stocks were not estimated? If possible, estimate these parameters.
Reply: With all due respect to the reviewer suggestion, the estimation of Ne for the 11 stocks is not very informative in this scenario. This is because, the study delineates the most probable genetic groups, of these 11 stocks of breeders and offspring, which do not constitute “populations” sensu stricto. In Figure 1, the effective number of alleles, Ne is calculated, which represents the number of alleles with equal frequencies that would take, to give the observed He in each stock. Because alleles with low frequencies contribute very little to the effective number of alleles, a large difference between the number of alleles and the effective alleles shows that the low frequency alleles are low in the stock, so we consider that this aspect of the genetic diversity is more in context with the current study.
- Results and Discussion
- The results need to be better discussed, and compared to previous genetic studies, relating them with the formation of broodstocks. The discussion is poor regarding the development of the production of the species based on the results
- Improve quality Figure 2
- The author discussed the methods of analysis, but should further discuss how his results of diversity, genetic structure, and relatedness could contribute to the formation of base populations of the species, and how genetic diversity can be improved. The discussion, in my opinion, is poor.
Reply: Discussion of the results was enriched, relevant studies were referenced and not so precise parts were removed from the discussion part
Round 2
Reviewer 1 Report
The manuscript has been greatly improved by the changes made by the authors. The authors have responded to all my concerns. In my opinion it is a paper of sufficient quality to be published in Fishes and I am sure it will be of interest to the journal's readers.
Author Response
We would like to thank the reviewer for the feedback in the first revision
Reviewer 2 Report
Accept in present form.
Author Response
We would like to thank the reviewer for the feedback in the first revision
Reviewer 3 Report
Author Affiliation:
Improve author affiliations following “Instructions for Authors”: The PubMed/MEDLINE standard format is used for affiliations: complete address information including city, zip code, state/province, and country
Abstract
LINE 22 -Insert comma “,and outline an efficient methodology for establishing the basis of new breeding schemes.”
.
Introduction
LINE 28 – Include FAO information in the reference list
LINE 30 – Replace “Greater” by “Higher”
LINE 43 – Delete “(e.g. Atlantic salmon Salmo salar L. 1758 [10], while other factors are also in play”
LINE 95 – Insert comma “Conversion Ratio (FCR) and the fast growth rate [28], and has emerged as a promising “
LINE 98 – Insert comma “compared to the previous year, while an additional growth of 2.8% is expected actually 98”
LINE 100 – Insert comma “of this production, followed by Spain (9%), Turkey (6%) and Greece (4%).”
LINE 102-105 – Reference
LINE 106-112 – Improve
Materials and Methods
I suggest a not detailed map (or location reference) of sampling sites in any genetic diversity and structure research approaching aquaculture species
Author Response
Reviewer 3 (2nd revision):
Comments and Suggestions for Authors
Improve author affiliations following “Instructions for Authors”: The PubMed/MEDLINE standard format is used for affiliations: complete address information including city, zip code,state/province, and country
Reply: Corrected
Abstract
LINE 22 -Insert comma “,and outline an efficient methodology for establishing the basis of new breeding schemes.”
Reply: Corrected
Introduction
LINE 28 – Include FAO information in the reference list -
Reply: The following link was included
(www.fao.org/fishery/statistics/global-aquaculture-production/en)
LINE 30 – Replace “Greater” by “Higher”
Reply: Corrected
LINE 43 – Delete “(e.g. Atlantic salmon Salmo salar L. 1758 [10], while other factors are also inplay”
Reply: OK, changed the reference to its right place in the middle of the sentence
“The success of genetic improvement is attributed partly to capturing the broad range of genetic diversity at the start of a breeding program [10], while other factors are also in play”
LINE 95 – Insert comma “Conversion Ratio (FCR) and the fast growth rate [28], and has emerged as a promising “
Reply: Corrected
LINE 98 – Insert comma “compared to the previous year, while an additional growth of 2.8% isexpected actually 98”
Reply: Corrected
LINE 100 – Insert comma “of this production, followed by Spain (9%), Turkey (6%) and Greece(4%).”
Reply: Corrected
LINE 102-105 – Reference
Reply:
The following reference was added : [32]
Aquaculture Europe magazine, 2017, vol. 42, issue 2 (in which some Diversify results are mentioned) and the following link was added at line 104,105 https://www.diversifyfish.eu/uploads/1/4/2/0/14206280/diversify_featured_article_aes_42_sept_2017.pdf)
LINE 106-112 – Improve
Reply: Rephrased as :
“The objectives of the current study were to assess the genetic diversity in different meagre breeders and offspring stocks based on microsatellite markers and then using this information to illustrate their putative relatedness, inbreeding and population structure. The results outline the importance of implementing cost-effective population genetics analytical tools for the genetic evaluation of breeding stocks as the starting point for the future design and fine-tuning of breeding programs.”
Materials and Methods
I suggest a not detailed map (or location reference) of sampling sites in any genetic diversity and structure research approaching aquaculture species
Reply: -We agree that for any study of genetic study and structure report a map or a figure with the sampling points is essential. But we would like to remind the reviewer that all these stocks are kept in land-based tanks and were collected from eight breeder stocks purchased from other commercial companies except one stock which was composed of wild fish from Southern Spain (see also lines 101-105). Therefore, we consider that the addition of a detailed map of samples is not possible and will not be of value since most of the stocks are captive-bred and not wild.
