Population Genetic Structure: Where, What, and Why?

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is an extensive review of the 'population genetic structure’ (PGS) concept.  According to the authors, the primary goal of this work was to initiate the process of comprehensive synthesis, as well as to reevaluate the currently available information about PGS. The idea is to present to the interested scientific community what we already know and can expect in the future. At first, I thought it would be interesting to read what the authors had to say about this topic.  But I found it difficult to read the review and difficult to follow the line of reasoning. One of the reasons might be due to English grammar problems. Some of them are mentioned below in this review. But the feeling I had is that the authors were not clear enough in transmitting their message, although some of the text brings very important information. It is a valid initiative. As far as the questions such as where, what, and why, I would have started by answering ‘what’, and would define the main topic right away, that is, I would define what the concept of ‘population genetic structure’ is.  It felt to me that the concept of ‘genetic diversity’ was sometimes confused with ‘population genetic structure’.  To improve the manuscript, I have some suggestions for the authors:

Lines 11-12:  Instead of:  “Yet PGS term is central to population genetics..”,

Please alter to:  “Yet the PGS term is central to population genetics..”,

Lines 12-13:  The authors mention that:  “and it is expected that future research will focus on the evolutionary continuum from populations to species.”

But, from what I understand, researchers have been doing exactly this since population genetic studies started.

Line 57: Please alter the reference citation [19) such as [19].

Line 100:  Instead of: “with comprehensive revision and update”, please change to: “with a comprehensive revision and update”,

Line 127:  Please consider deleting the words “to them” in this sentence.  It is unnecessary.

Line 131: Instead of … “in the following chapter”, I suppose the correct would be “in the following subtitle”.

Line 132:  Please delete the words “to us”. It is unnecessary.

Lines 138-139: Instead of “What is population?”, I suggest modifying to “What is a population?”

Line 148: Please insert a stop sign between “…..therein) and In ecology”

The sentence in lines 148-153 is too long.  I suggest inserting a stop sign after “characteristics” in line 150.  Ex: “….one or more common characteristics. Yet different fields...”.

Line 155: In this line: “…the following paragraphs and chapters, especially after small modification”, I suggest deleting the words “and chapters”. And altering the text to: “especially after a small modification: ….”

Lines 155-156:  Instead of “population is a group of the same species (BSC application) individuals that are more related…”, I suggest the following modifications: “a population is a group of individuals of the same species (BSC application) that are more related…”,

Line 163: Instead of “begins in population”, I suggest modifying to begins in a population”

Line 173: Instead of: “most often, probably, direct or indirect competition”, I imagine it should be altered to: “most often, probably, due to direct or indirect competition”,…to make the sentence meaningful.

Line 178: Please review the sentence in this line: “successions that forming new ecosystems over time”. I suppose it should be “successions that form new ecosystems over time”, or “successions forming new ecosystems over time”

Line 179:  Instead of “Typically, number of reproductively mature individuals in a population…”, please alter to:  “Typically, the number of reproductively mature individuals in a population…”

Lines 180-181:  Instead of: “The infinitely large population have great Nc but exist only in the abstractions and theory,”, please alter to: “An infinitely large population has a great Nc but exists only in the abstractions and theory,”

Line 182:  Please replace “in real world” with “in the real world”

Line 183:  Please replace: “….large, yet grow only to a certain degree…” with: “….large, yet they grow only to a certain degree…”

Lines 193-196:  In this sentence [Because population could have large Nc but only relatively small number of reproducers in it could create a new generation (see [73] and references therein), thus population is defined not only by Nc  but also by effective population size (Ne).],…

I suggest a few modifications, such as: [Because a population could have a large Nc but only a relatively small number of reproducers, it could create a new generation (see [73] and references therein), thus a population is defined not only by the Nc but also by the effective population size (Ne).],…

Lines 199-200:  Please modify this sentence, such as: Both Nc and Ne are marked with decreases over time and are described as examples of the bottleneck effect [74].

Line 211:  “within [67] article”.  This way of writing does not seem correct. 

Line 215:  Instead of: “in lifetime reproductive success among”, please modify it to: “in the lifetime of reproductive success among”

Line 232:  It is better to write such as: “…from the species level perspective,”

Lines 233-234: I would write this part of the sentence as: “panmictic populations [77, 78], two or more populations within a species (see [79] and references therein), and metapopulations [80].”  It is easier to understand that these are the three types of populations meant by the authors.

Line 235:  It is better to write, such as: “Typically, a panmictic population…”

Line 235-236:  It is better to write such as: “of a panmictic species, as it …“

Lines 245-246:  Instead of:  “For instance, famous mysterious European eel (Anguilla anguilla) is considered as panmictic species for a long time, yet”

I suggest the following modifications: “For instance, the famous mysterious European eel (Anguilla anguilla) was considered a panmictic species for a long time, yet”

Line 256:  please replace:  “populations withing species” by “populations within species”

Line 260:  Instead of:  “Typically, in population genetics, metapopulation defined as a group” Please alter to: “Typically, in population genetics, a metapopulation is defined as a group”

Lines 262-263:  Instead of:  “and show central place and role of PGS within proper context.” Please alter to:  “and show the central place and role of PGS within a proper context.”

Line 273:  Replace Sandberg et al [93] by Sandberg et al. [93]

Lines 308-310:  Please review this sentence, as it needs some improvement “For instance, one individual might have genetic program to be large compared to its kind but in environment there are not enough resources, and this led to smaller size of this individual.”                              I would write “might have a genetic program”… “but in a different environment, there may not be enough resources, so that this could lead to a smaller size individual”

Figure 1.  For me, Figure 1a shows genetic variation (different colors) among individuals of a population, and not a population phenetic structure.  To do so, it should show a division among two populations, such as in 1d, where one division could have white color individuals and the other division could have individuals in brown and black colors.  The same reasoning can be applied to Figure 1b. 

Lines 737-739:  This sentence needs reviewing.

Line 834:  Please alter to:  The third is associated with …

Figure 2A is supposed to represent a panmitic species with all patterns of genetic diversity within one population.  But, it is only showing one individual of a population/species, and its DNA.  Shouldn´t it show several individuals, genetically different from one another?

Lines 1326-1329:  “However, instead of this scenario, was opened the window that slowly starting to close only now: both population genetics based on single marker research and population genomics could coexist and provide valuable information about living systems for more then one decade.”

This sentence needs to be reviewed.  A suggestion might be the following:

“However, instead of this scenario, a window has been opened that only now is slowly starting to close: both population genetics based on single marker research and population genomics could coexist and provide valuable information about living systems for more than one decade.”

Lines 1339-1341:  Please review this sentence “Consequently, we advise to start solve this issues by discussions, additional research, synthesis, and minimization of controversy, so future multidisciplinary investigations into ….”

Supplementary material file:

The Glossary S1 file has definitions of the many terms, most of them starting with:  “could be defined as” or “could be understood as”.  Is this necessary? Why don´t the authors just insert the definition of the words to simplify this file?

Ex:  Microevolution: could be understood as a process of small-scale evolutionary changes in populations over a relatively short period of time.

Demographic: could be understood as birth, migration and death relationships within population or species.

Migration: could be understood as organisms moving from one geographical area to another.

I suggest altering to:

Microevolution: a process of small-scale evolutionary changes in populations over a relatively short period of time.

Demographic: birth, migration, and death relationships within a population or species.

Migration: organisms moving from one geographical area to another.

 

Comments on the Quality of English Language

I have pointed out many English language problems in the space above.

Author Response

Comments and Suggestions for Authors

Comment 1: This is an extensive review of the 'population genetic structure’ (PGS) concept.  According to the authors, the primary goal of this work was to initiate the process of comprehensive synthesis, as well as to reevaluate the currently available information about PGS. The idea is to present to the interested scientific community what we already know and can expect in the future. At first, I thought it would be interesting to read what the authors had to say about this topic.  But I found it difficult to read the review and difficult to follow the line of reasoning. One of the reasons might be due to English grammar problems. Some of them are mentioned below in this review. But the feeling I had is that the authors were not clear enough in transmitting their message, although some of the text brings very important information. It is a valid initiative.

Answer 1:

Dear Reviewer,

First of all, we would like to thank you for your time evaluating our manuscript and help to improve it. Thank you for noting that our work is a valid initiative. We are sorry to hear that you found this manuscript not as interesting as you expected it to be mainly due to English grammar problems and difficulties to follow the line of reasoning. We tried to solve these problems by using MDPI Author Services for Rapid and Academic language editing and by adding additional structure within the manuscript major parts. In addition, we expanded glossary and provided abbreviations within Supplementary material.

Comment 2: As far as the questions such as where, what, and why, I would have started by answering ‘what’, and would define the main topic right away, that is, I would define what the concept of ‘population genetic structure’ is.  It felt to me that the concept of ‘genetic diversity’ was sometimes confused with ‘population genetic structure’.  To improve the manuscript, I have some suggestions for the authors:

Answer 2: Thank you. We see your position. However, this review was dedicated not only for the specialists but also for students and scientists that are not related with population genetics field (see Graphical Abstract). Consequently, it was decided that in this case, just like in the case of DNA that is found within chromosomes of nucleus of cell, it is more rational to first mention where ecological and biochemical construction as a background of our research object is located and only then define it as a phenomenon. Once again thanks for constructive comments and suggestions how to improve our manuscript.

 

Comment 3: ‘‘Lines 11-12:  Instead of:  “Yet PGS term is central to population genetics..”,

Please alter to:  “Yet the PGS term is central to population genetics..”,

Answer 3: Corrected according your suggestion.

Comment 4: Lines 12-13:  The authors mention that:  “and it is expected that future research will focus on the evolutionary continuum from populations to species.”

But, from what I understand, researchers have been doing exactly this since population genetic studies started.

Answer 4: In general, for the most part you are right. However, there is a greater need and focus nowadays compared to previous times. In addition, understanding of evolutionary continuum from populations to species should not be limited to genetic diversity but expanded to intertwined relationships of various population structures, especially PGS.

Comment 5: Line 57: Please alter the reference citation [19) such as [19].

Answer 5: Thank you for noting this error. Fixed.

Comment 6: Line 100:  Instead of: “with comprehensive revision and update”, please change to: “with a comprehensive revision and update”,

Answer 6: Corrected.

Comment 7: Line 127:  Please consider deleting the words “to them” in this sentence.  It is unnecessary.

Answer 7: Deleted.

Comment 8: Line 131: Instead of … “in the following chapter”, I suppose the correct would be “in the following subtitle”.

Answer 8: Due to the fact that we added many additional structural units in the manuscript, writing “in the following subtitle” could be misleading, thus we will change this text to: “in the third chapter”.

Comment 9: Line 132:  Please delete the words “to us”. It is unnecessary.

Answer 9: Deleted.

Comment 10: Lines 138-139: Instead of “What is population?”, I suggest modifying to “What is a population?”

Answer 10: Corrected.

Comment 11: Line 148: Please insert a stop sign between “…..therein) and In ecology”

Answer 11: Corrected.

Comment 12: The sentence in lines 148-153 is too long.  I suggest inserting a stop sign after “characteristics” in line 150.  Ex: “….one or more common characteristics. Yet different fields...”.

Answer 12: Done as suggested.

Comment 13: Line 155: In this line: “…the following paragraphs and chapters, especially after small modification”, I suggest deleting the words “and chapters”. And altering the text to: “especially after a small modification: ….”

Answer 13: Suggestion accepted.

Comment 14: Lines 155-156:  Instead of “population is a group of the same species (BSC application) individuals that are more related…”, I suggest the following modifications: “a population is a group of individuals of the same species (BSC application) that are more related…”,

Answer 14: Done as suggested.

Comment 15: Line 163: Instead of “begins in population”, I suggest modifying to begins in a population”

Answer 15: Corrected.

Comment 16: Line 173: Instead of: “most often, probably, direct or indirect competition”, I imagine it should be altered to: “most often, probably, due to direct or indirect competition”,…to make the sentence meaningful.

Answer 16: Fixed.

Comment 17: Line 178: Please review the sentence in this line: “successions that forming new ecosystems over time”. I suppose it should be “successions that form new ecosystems over time”, or “successions forming new ecosystems over time”

Answer 17: Changed to: “successions forming new ecosystems over time”

Comment 18: Line 179:  Instead of “Typically, number of reproductively mature individuals in a population…”, please alter to:  “Typically, the number of reproductively mature individuals in a population…”

Answer 18: Corrected.

Comment 19: Lines 180-181:  Instead of: “The infinitely large population have great Nc but exist only in the abstractions and theory,”, please alter to: “An infinitely large population has a great Nc but exists only in the abstractions and theory,”

Answer 19: Altered.

Comment 20: Line 182:  Please replace “in real world” with “in the real world”

Answer 20: Changed.

Comment 21: Line 183:  Please replace: “….large, yet grow only to a certain degree…” with: “….large, yet they grow only to a certain degree…”

Answer 21: Corrected.

Comment 22: Lines 193-196:  In this sentence [Because population could have large Nc but only relatively small number of reproducers in it could create a new generation (see [73] and references therein), thus population is defined not only by Nc  but also by effective population size (Ne).],…

I suggest a few modifications, such as: [Because a population could have a large Nc but only a relatively small number of reproducers, it could create a new generation (see [73] and references therein), thus a population is defined not only by the Nc but also by the effective population size (Ne).],…

Answer 22: Done as suggested.

Comment 23: Lines 199-200: Please modify this sentence, such as: Both Nc and Ne are marked with decreases over time and are described as examples of the bottleneck effect [74].

Answer 23: Modified.

Comment 24: Line 211:  “within [67] article”.  This way of writing does not seem correct.

Answer 24: Changed to: “within Fedorca et al. [67] article”

Comment 25: Line 215:  Instead of: “in lifetime reproductive success among”, please modify it to: “in the lifetime of reproductive success among”

Answer 25: Corrected.

Comment 26: Line 232:  It is better to write such as: “…from the species level perspective,”

Answer 26: Done as suggested.

Comment 27: Lines 233-234: I would write this part of the sentence as: “panmictic populations [77, 78], two or more populations within a species (see [79] and references therein), and metapopulations [80].”  It is easier to understand that these are the three types of populations meant by the authors.

Answer 27: Suggestions accepted.

Comment 28: Line 235:  It is better to write, such as: “Typically, a panmictic population…”

Answer 28: Corrected.

Comment 29: Line 235-236:  It is better to write such as: “of a panmictic species, as it …“

Answer 29: Changed.

Comment 30: Lines 245-246:  Instead of:  “For instance, famous mysterious European eel (Anguilla anguilla) is considered as panmictic species for a long time, yet”

I suggest the following modifications: “For instance, the famous mysterious European eel (Anguilla anguilla) was considered a panmictic species for a long time, yet”

Answer 30: Modified.

Comment 31: Line 256:  please replace:  “populations withing species” by “populations within species”

Answer 31: Replaced.

Comment 32: Line 260:  Instead of:  “Typically, in population genetics, metapopulation defined as a group” Please alter to: “Typically, in population genetics, a metapopulation is defined as a group”

Answer 32: Altered as suggested.

Comment 33: Lines 262-263:  Instead of:  “and show central place and role of PGS within proper context.” Please alter to:  “and show the central place and role of PGS within a proper context.”

Answer 33: Corrected.

Comment 34: Line 273:  Replace Sandberg et al [93] by Sandberg et al. [93]

Answer 34: Replaced.

Comment 35: Lines 308-310:  Please review this sentence, as it needs some improvement “For instance, one individual might have genetic program to be large compared to its kind but in environment there are not enough resources, and this led to smaller size of this individual.”                              I would write “might have a genetic program”… “but in a different environment, there may not be enough resources, so that this could lead to a smaller size individual”

Answer 35: Improved according your suggestion.

Comment 36: Figure 1.  For me, Figure 1a shows genetic variation (different colors) among individuals of a population, and not a population phenetic structure.  To do so, it should show a division among two populations, such as in 1d, where one division could have white color individuals and the other division could have individuals in brown and black colors.  The same reasoning can be applied to Figure 1b. 

Answer 36: We aimed to represent in Figure 1a different phenotypes found in just one population (it could be distributed in different ratios). Your suggestion to take into account not just one population but all of the same species to represent all different population structures in the same way as PGS is really interesting. However, we are not sure whether it could be correctly done without additional scientific discussions and consensus first. Even if it would be agreed that other population structures indeed should be defined and represented in similar principles like in PGS, we think that current representation of Figure 1a, 1b, 1c is still quite correct, similarly to the case of PGS within panmictic population.

Comment 37: Lines 737-739:  This sentence needs reviewing.

Answer 37: Sentence rewritten: The pangenome concept solves these problems only in part. Pangenomes arise due to gene gain by genomes from other species through horizontal gene transfer (HGT) and differential gene loss among genomes, and have been described in both prokaryotes and eukaryotes, yet our current view of pangenome variation is incomplete [137]. In addition, this concept concerns only genes but not all DNA variants.

Comment 38: Line 834:  Please alter to:  The third is associated with …

Answer 38: Altered.

Comment 39: Figure 2A is supposed to represent a panmitic species with all patterns of genetic diversity within one population.  But, it is only showing one individual of a population/species, and its DNA.  Shouldn´t it show several individuals, genetically different from one another?

Answer 39: Thank you. You are right that this Figure 2A lacks clarity because it wasn‘t clearly stated that this eel represent not one individual but population/species and its DNA represent PGS of this species and not just genome/s of one eel. Additional information now provided in Figure 2 legend. Your solution is also possible, yet it would complicate many things, thus we tend to upgrade not this Figure in this regard but its text in the legend. Text changes: The schematic representation of Population Genetic Structure (PGS)  from simple to complex. (a) One panmictic species contain all patterns of intraspecific genetic diversity within one population; (b) When species comprised from distinct populations, then intraspecific genetic diversity patterns exist not only within each population, but also among them. Black circles together with eel or lions’ pictures and orange circles show species-population level and PGS, respectively. (Image created in BioRender. Maziliauskaitė, E. (2025) https://biorender.com/twuuho4, accessed on 1 August 2025).

Comment 40: Lines 1326-1329:  “However, instead of this scenario, was opened the window that slowly starting to close only now: both population genetics based on single marker research and population genomics could coexist and provide valuable information about living systems for more then one decade.”

This sentence needs to be reviewed.  A suggestion might be the following:

“However, instead of this scenario, a window has been opened that only now is slowly starting to close: both population genetics based on single marker research and population genomics could coexist and provide valuable information about living systems for more than one decade.”

Answer 40: Changed as recommended.

Comment 41: Lines 1339-1341:  Please review this sentence “Consequently, we advise to start solve this issues by discussions, additional research, synthesis, and minimization of controversy, so future multidisciplinary investigations into ….”

Answer 41: Changed to: “Consequently, we advise starting to solve these issues. For instance, by discussions, additional research, synthesis, and minimization of controversy, so future multidisciplinary investigations into ….”.

Comment 42: Supplementary material file:

The Glossary S1 file has definitions of the many terms, most of them starting with:  “could be defined as” or “could be understood as”.  Is this necessary? Why don´t the authors just insert the definition of the words to simplify this file?

Ex:  Microevolution: could be understood as a process of small-scale evolutionary changes in populations over a relatively short period of time.

Demographic: could be understood as birth, migration and death relationships within population or species.

Migration: could be understood as organisms moving from one geographical area to another.

I suggest altering to:

Microevolution: a process of small-scale evolutionary changes in populations over a relatively short period of time.

Demographic: birth, migration, and death relationships within a population or species.

Migration: organisms moving from one geographical area to another.

Answer 42: Thank you. We tried to simplify most of the terms based on your suggestions.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript “Population Genetic Structure: Where, What, Why?” is written about biodiversity on its three main levels: ecosystem, species and intraspecific genetic diversity. Genetic diversity is directly linked with the term of the population genetic structure. Many uncertainties and confusion around  the concept of the population genetic structure of species in scientific studies and literature are discussed. Finding answers and reaching consensus of the scientific community for certain questions regarding population genetic structure is essential for population genetics and related research disciplines. The major areas of interest and research are discussed here: population genetic structure role in microevolution and speciation processes, sustainable use of natural resources and conservation of genetic diversity.  

Other important aspects of this perspective review are proposals of scientific definitions of some terms and concepts, new perspectives and explanations that could be used as basis for future theoretical models and applied research of population genetic structure.

The manuscript is written in a clear and concise manner, making it a valuable resource for anyone working in the field of life sciences, from students to experts.

The only recommendation to be done to the Authors – please, describe your thoughts about concept of core collections (see Brown, 1989) in the field of genetic resources management. It would be a valuable addition to this manuscript.

Brown, A.H.D., 1989. Core collections: a practical approach to genetic resources management. Genome, 31(2), pp.818-824.

Author Response

Comments and Suggestions for Authors

Comment 1: The manuscript “Population Genetic Structure: Where, What, Why?” is written about biodiversity on its three main levels: ecosystem, species and intraspecific genetic diversity. Genetic diversity is directly linked with the term of the population genetic structure. Many uncertainties and confusion around  the concept of the population genetic structure of species in scientific studies and literature are discussed. Finding answers and reaching consensus of the scientific community for certain questions regarding population genetic structure is essential for population genetics and related research disciplines. The major areas of interest and research are discussed here: population genetic structure role in microevolution and speciation processes, sustainable use of natural resources and conservation of genetic diversity.  

Other important aspects of this perspective review are proposals of scientific definitions of some terms and concepts, new perspectives and explanations that could be used as basis for future theoretical models and applied research of population genetic structure.

The manuscript is written in a clear and concise manner, making it a valuable resource for anyone working in the field of life sciences, from students to experts.

The only recommendation to be done to the Authors – please, describe your thoughts about concept of core collections (see Brown, 1989) in the field of genetic resources management. It would be a valuable addition to this manuscript.

Brown, A.H.D., 1989. Core collections: a practical approach to genetic resources management. Genome, 31(2), pp.818-824.

 

Answer 1:

 

Dear Reviewer,

 

Thank you very much for your evaluation of our manuscript. We are glad that you really liked it. Honestly, for the first author this work was a great challenge, as it was the first attempt to write perspective review. For the most part it was quite hard to write this manuscript and from time to time we had to experiment by uniting different terms and concepts and ideas together in various ways in order to produce valuable synthesis about our research object. Consequently, we really appreciate your shared thoughts as it shows that our attempts were not in vain, the creative experimentation was also right thing to do and all this knowledge and evaluation of our manuscript motivates us not only to successfully finish to carry out necessary changes in this work prior acceptance but also to write new perspective reviews in the future.

Regarding your recommendation about core collections, we tried to apply it in the upgraded version of the manuscript. We hope that you will like additional changes in the manuscript. Once again thank you.

Reviewer 3 Report

Comments and Suggestions for Authors

In this work, the authors explore the concepts related to population genetic structure and its importance to various practical areas. It is a large manuscript and a very impressive deep synthesis. However (or maybe partly because of the length and depth), I cannot unfortunately say that it was an easy text to read. Please find below my questions and suggestions. I hope that authors share some of my concerns and find inspiration in the suggestions, and I look forward to reading the updated review.

 

Major content-related concerns

 

1. In my opinion, the manuscript lacks inner structure. With this total length of the text, it’s hard to follow logic within subsections which are up to five pages long (like 3.1.). It might become easier if an additional level of subsubsections was added.

 

2. In addition, the manuscript is obviously long, and sometimes the text could be made more concise without much damage to the conveyed meaning. In my opinion, this text is similar to a lecture transcript: would read fine as a lecture, but a bit long for a review paper.

Please understand me correctly. I’m not trying to just critique your work, and I totally understand that everyone has their own unique style. However, it definitely is longer than average, and I think that finding it long and hard to read could be not only my problem. What I’m trying to say is that if the text becomes more structured, it would be easy find important information, which would lead to more people reading it to the end and more citations finally.

I will provide more particular examples below, and I also tried to illustrate my ideas by rewriting a section of the text in the end of my report. Of course, I do not expect that the authors follow my edits exactly, but I very much hope that they could find some inspiration in it.

 

3. It is strage that the abstract does not contain any conclusions, while in fact the ending of the manuscript does, and they are indeed important. Please consider updating the abstract.

 

4. I find it a bit strange that the problem of species delimitation is totally omitted in the manuscript (mentioned once without any explanation towards the end). In my opinion, it’s extremely hard to speak about intraspecific structure without defining what a species is, exactly, for the purposes of studying PGS.

 

5. L61-62 “by clearly defining used concepts during research.”: i’m curious how the authors suggest calling species in this case. Should something like “phylogenetic species” be used?

 

6. L69 ‘especially birds and mammals” why especially these groups? It’s not obvious.

 

7. I feel that the species concepts in the Introduction section could be explained in more detail, maybe as a table / schematic? It’s a nice review the authors are citing but the concept quite central to this manuscript, so it could be important to provide more background here..

 

8. Glossary in Supplement it a very interesting idea, but I feel its implementation could be improved.

First, I cannot quite catch the intended audience. At the one hand, it should be quite inexperienced specialists, as such basic terms as chromosome, allele or locus are defined... but at the same time, some much more complex and niche terms, like census size, carrying capacity, genetic load, acclimatization potential or Fst do not have a definition here.

Second, I do not understand the logic behind ordering. It is designed to follow the order of the terms’ appearance in the manuscript? I believe, in this case alphabetic would be better, as most people will look up some terms at random rather than read it along switching back and forth between the article and the supplement.

Third, speaking about the ease of reading, I would greatly suggest incroprorating the idea of the glossary into the manuscript itself by highlighting the term (in bold and/or italics) when it is used for the first time and followed by the definition. It will provide more structure. Please see the very end of my report for a couple of examples.

If the authors feel that a separate glossary could also be useful in some situations, as for me, it is okay to provide this information once more in the Supplement. Actually, going through the text like this will also help identify some definitions missing from the glossary in the supplement. Moreover, the supplement would be a nice place to provide literature references to key definitions.

 

9. It would be very helpful to make a list of non-standard abbreviations like PGS, PSS, EP etc. (for those readers who did not read the whole manuscript thoroughly but instead wanted to look into a particular section or, for example, have been reading this review over a long time and forgot some of the abbreviations).

 

10. L232 “only three main types of populations”: are these types of populations indeed? Looks more like types of PGS within a species. This would really be nicely (and easily!) illustrated by a figure, as this is an important concept and much more complex than PPS / PGS / PSS / PAS illustrated in Figure 1.

update: This comment was written before I encontered Fig. 2, which partly encompasses this idea. Maybe it would make sense to add the third structure (metapopulation) to the figure and moving it above.

 

11. L233 “, as distinct most diverse structural units of species:”: is this another definition of the same term population?

 

12. “The research showed that insects feeding on genetically modified plants (those unable to send electrical signals) gained weight more rapidly than those feeding on wild-type plants, indicating that insects found it more difficult to predate plants with systems for sending electrical signals [43] (and references therein).”: this is a direct quote from this review, which creates two problems. First, if this a direct quote this long, it should be either quoted or paraphrased. Second, in this context it is unclear how these plants were genetically modified, while it is important to the

 

13. Speaking about the structure of the text: e.g. L357-397 could be a subsection “Population ethology structure” or something along these lines.

 

14. “[134] and references therein”: I have nothing against this review cited multiple times, but it was published in 2004, >20 years ago, and references therein are thus even older. Maybe cite something newer in addition or instead? For example, there is a 2024 book “DNA Barcoding: Methods and Protocols” (https://link.springer.com/book/10.1007/978-1-0716-3581-0), and citing particular chapters from it seems a good idea.

 

15. “genetic diversity could be defined as DNA variations found in organisms” <...> “Due to the fact that all species are genetically unique, this definition of genetic diversity is also quite abstract as it encompasses both interspecific and intraspecific genetic diversity. Consequently, if we want to exclude interspecific level, then intraspecific genetic diversity could be defined as DNA variations found within the same species.”: this explanation is very long. Why not define intraspecific genetic diversity at the beginning? It’s quite clear that if genetic diversity could be defined as DNA variations found in organisms, then intraspecific genetic diversity could be defined as DNA variations found in organisms of one species and interspecific genetic diversity could be defined as DNA variations found in organisms of different species.

 

16. It is also strange that modified nucleotides (epigenetic modification of DNA, if you will) are not mentioned either here or later when speaking about nucleotides.

 

17. L451 prions : there is no reference to a review of prions as hereditary entitites.

 

18. “during Ice Age [138]”: it is cited as an example of using ancient DNA, but I cannot see such information here. Please check.

 

19. L490-511 “The double-helix DNA structure was discovered in 1953 [148] <...>will concentrate more about DNA genetic properties and its use within population genetics framework.”: I honestly cannot see clear relevance of this text fragment to the major topic of this research and think that this paragraph can be safely omitted.

 

20. L519-520 “quantitative genetic parameters, such as haplotype diversity (Hd) and nucleotide diversity (π), are calculated.”: needs a reference to what these parameters are and how they are calculated. This being said, I need to add that this thesis is also misleading, as Hd and pi can be calculated taking into account gaps in alignment.

 

21. L524-525 “yet only direct sequencing [74] allows us to study each nucleotide position in the obtained short and long DNA sequences [156]”: the term “direct sequencing” could be highly misleading in this particualr case. I would use just “sequencing”. One the one hand, “direct sequencing” had been used to describe Sanger sequencing as opposed to Maxam-Gilbert degradation-based technique. On the other hand, now “direct sequencing” can be used to mean TGS techniques that do not include DNA amplification as opposed to the methods exploiting PCR-based library preparation.

 

22. L607-608 “Genomic repetitive DNA sequences (Repeatomes, REPs)”: I feel that this wording could lead to a misunderstanding, as “repetitive sequences” does not equal “repeatomes”. Repeatome is already a sum of repetitive sequences in a species. I would just use “repetitive DNA sequences (REPs)”.

 

23. L616 “A genome sequence is a snapshot of a strain in time [137].”: without some context it’s impossible to guess that now are speaking about prokaryotes; otherwise, a concept of strain sounds very strange.

 

24. L633-636 “For instance, based on selected method the estimated number of protein-coding genes in Eurasian perch (Perca fluviatilis) could range from 23,397 to 24,326, yet total scaffold size (bp) ranged from 630,662,671 to 958,225,764 bp (see [186] and references therein).” : while I totally share and support the thesis illustrated by this example and actually think this would be true for basically any genome of a free-living eukaryote, giving the estimate of gene number and genome size does not clearly illustrate it. Otherwise, one could argue that perch has very large genes, for example. It would be better to provide information about the percent of the genome assembly actually occupied by genes.

 

25. L639 “Over 50% of animal genomes belong to the phylum Chordata”: I would add an introductory sentence that states the problem of unequal representation of different taxonomic groups in genome projects (I guess, this was the motivation). Otherwise, the examples below are unclear.

 

26. L699 “Aneuploidy (see [189]) is the simplest system for us to measure genetic variability,”: I don’t understand this thesis. First, the term “genetic variability” appears only in the paragraph above and is not defined there; neither is it defined in the glossary. What is the difference between genetic diversity and genetic variability? From what was described above, I would talk about aneuploid systems as a tool to link cytogenetics and genome assembly (even though it does not completely apply to the yeast model described in [189]). So, please consider revising this logical connection.

 

27. What is the function of Box 1? Does it provide questions to which the authors try to find some answers below or open questions? I could not find proof for any of these hypothesis and was lost here. Please provide some background.

I’m inclined towards the first hypothesis. In this case, it would be a great help to the reader if the authors revised the next subsections to reflect these questions.

 

28. It’s a pity that this manuscript does not include any overview of how the population structure can be estimated and compared. However, I completely understand the motivation. Well, we could only hope for a future review about this topic, which could be titled “Population Genetic Structure: How”.

 

An example on how this text could be structured and shortened:

 

Original text:

What is the PGS of the species? The answer to this main question is directly linked to understanding genetic diversity of species. There are many reviews dedicated to intraspecific genetic diversity (see [14, 21, 54]), its different aspects [3, 132, 133], and how to study it using major methods [7] related to application of various molecular markers (see [134] and references therein), thus in this paper most of this information will not be presented but interested readers will be guided to important literature sources. In this review we will use a commonly accepted, yet oversimplified, definition of genetic diversity. That is, genetic diversity could be defined as DNA variations found in organisms. Major oversimplification: there are other known biological molecules that could be genetic material for certain organisms. For instance, viruses are considered as quasispecies (see [135]) and are not included as part of life, yet they still are biological-genetic systems, and in some of their group’s RNA and not DNA is the essential genetic material [136] (and references therein). That said, based on current comprehensive scientific knowledge, we could rationally limit the term and concept of genetic diversity to just DNA within the biological molecules, which could code genetic information, context encompassing most, if not all, eukaryotes. This leaves decoding by RNA molecules (see [55] and references therein) and functioning of some proteins, such as prions, to major areas of research for transcriptomics and proteomics, respectively, and outside the scope of the mentioned term and concept of genetic diversity of eukaryotes. Due to the fact that all species are genetically unique, this definition of genetic diversity is also quite abstract as it encompasses both interspecific and intraspecific genetic diversity. Consequently, if we want to exclude interspecific level, then intraspecific genetic diversity could be defined as DNA variations found within the same species. To sum up, DNA variations could and should be treated as the major key to understanding the intraspecific genetic diversity, and this leads us to the necessity to mention DNA and its variations in more detail. However, on a broader scale, genetic diversity includes all heritable variation at the molecular level.

 

Suggested changes:

In order to understand what PGS is, we need to understand the genetic diversity of a species. There are many reviews dedicated to intraspecific genetic diversity (see [14, 21, 54]), its different aspects [3, 132, 133], and how to study it [7] with molecular markers (see [134] and references therein), thus interested readers will be referred to relevant literature sources. In this review we define genetic diversity as DNA variations found in organisms. This definition has some disadvantages.

First, it is oversimplified, as some other biological molecules are known to be genetic material. For instance, viruses are considered as quasispecies (see [135]) and are not included as part of life, yet they still are biological-genetic systems, and in some viruses RNA is the essential genetic material [136,55] (and references therein). Moreover, some proteins, such as prions [reference missing!], act as hereditary factors. These particular examples are outside the scope of this review. However, on a broader scale, genetic diversity includes all heritable variation at the molecular level.

Second, due to the fact that all species are genetically distinct, this definition encompasses both interspecific and intraspecific genetic diversity. Consequently, intraspecific genetic diversity could be defined as DNA variations found within a species. To sum up, DNA variations could and should be treated as the key to understanding the intraspecific genetic diversity, and this leads us to the necessity to consider DNA and its variations in more detail.

 

Comments on the Quality of English Language

In my opinion, the text could be edited for brevity and also proofread, but this is a minor concern. Please find below some awkward phrases I noticed while reading but not this is by no means an exhaustive list.

 

1. “Species are comprised of populations that encompasses intraspecific genetic variability in the form of DNA”: the sentence is grammatically awkward

 

2. “Most widely accepted” => the most

 

3. L148 “references therein)” missing dot

 

4. L176 “predator-victim dynamics” it’s normally predator–prey

 

5. L243-245 “Even species that are considered panmictic, could surprise us with deviations from our theoretical assumptions.”: excessive comma

 

6. Fig 1 : is it designed to be available in BioRender to the readers? It is not.

Fig 2: the same problem.

 

7. L256 “withing” should be “within”

 

8. L327 “phenotype plasticity” => “phenotypic plasticity”?

 

9. (multiple occurences) “code genetic information”, “code genes” etc => “code for genetic information” or “encode genetic information”

 

10. L588-589 “Both selectively neutral genetic variations and genes [162], as well as DNA parts near them, included in the concept of genetic diversity.”: should be something like “Both substitutions in genes and selectively neutral genetic variations [162], as well as adjacent DNA regions, are included in the concept of genetic diversity.”

 

11. L603 “and unique DNA sequences, which functions are not clear”: better “and unique DNA sequences, functions of which are unclear” or just “and unique DNA sequences with unclear functions”

 

12. L730 “It could be concluded that within an eukaryotic cell there is not one mtDNA copy”: it’s not “concluded”, it’s rather “It should be kept in mind” or “It is worth noting”

 

13. L737 “Complications not ending here,”: “These are not the only complications” or “Complications are not ending here”

 

14. L773 “environmentally, responsive epigenetic mechanisms”: excessive comma

 

15. L773 “can allow acclimatization” : “can allow for acclimatization”

 

16. L778 “According Gargiulo et al [75],”: “According to Gargiulo et al. [75],”

 

17. L824 “gene drift (or random genetic drift) that defined”: “gene drift (or random genetic drift) that is defined”

 

18. L869 “defined prior C. Darwin”: “defined prior to Charles Darwin”

 

19. L870 “biologic evolution”: “biological evolution”

 

20. L900 “For more information see Charlesworth & Charlesworth [209], Balkenhol et al [210].”: Previously, only reference numbers were provided, and this was quite enough.

 

21. L1104 “genetics have”: “genetics has” or “geneticists have”

 

22. L1190 “while previously only non-economically important species has been largely neglected”: “only” seems excessive

 

23. L1263 “It is worth to mention that scientific works that dedicated”: “It is worth to mention that scientific works dedicated”

 

24. L1326-1327 “However, instead of this scenario, was opened the window that slowly starting to close only now:”: “However, instead of this scenario, a window opened that is only slowly starting to close now:”

 

25. L1345 “within and among populations”: “within and between populations”?

Author Response

Comments and Suggestions for Authors

Comment 1: In this work, the authors explore the concepts related to population genetic structure and its importance to various practical areas. It is a large manuscript and a very impressive deep synthesis. However (or maybe partly because of the length and depth), I cannot unfortunately say that it was an easy text to read. Please find below my questions and suggestions. I hope that authors share some of my concerns and find inspiration in the suggestions, and I look forward to reading the updated review.

Answer 1:

Dear Reviewer,

Thank you very much for everything, especially for noting that our work was able to reach impressive deep synthesis level and your constructive comments, questions and suggestions that we found extremely helpful for improving our manuscript. For the most part your suggestions were accepted without any modifications. We expect that English of our manuscript will be greatly improved after necessary corrections will be carried out by the experts associated with MDPI Authors service for Academic English editing. We also hope that you will get to evaluate our updated version of the manuscript and that you will like it even more than the current version.

Comment 2: Major content-related concerns

 

1. In my opinion, the manuscript lacks inner structure. With this total length of the text, it’s hard to follow logic within subsections which are up to five pages long (like 3.1.). It might become easier if an additional level of subsubsections was added.

Answer 2: We agree with your suggestion, thus we tried to add additional inner structure within the updated version of the manuscript.

Comment 3: 2. In addition, the manuscript is obviously long, and sometimes the text could be made more concise without much damage to the conveyed meaning. In my opinion, this text is similar to a lecture transcript: would read fine as a lecture, but a bit long for a review paper.

Answer 3: Thank you for sharing your thoughts. We expect that text would be more concice after English correction that was mentioned before by the specialists.

Comment 4: Please understand me correctly. I’m not trying to just critique your work, and I totally understand that everyone has their own unique style. However, it definitely is longer than average, and I think that finding it long and hard to read could be not only my problem. What I’m trying to say is that if the text becomes more structured, it would be easy find important information, which would lead to more people reading it to the end and more citations finally.

Answer 4: We agree with you that inner structure could greatly improve the overall quality of the manuscript and be useful to the readers. We are aware that on one hand this additional inner structure will a little bit increase the overall length of the manuscript but on the other hand it would be a valuable improvement to the readers that avoid reading the whole manuscripts from start to finish but using manuscripts with the “take what you want/need and leave the rest” approach. Consequently, we think that your suggested inner structure is the best solution for the current case and we added it.

Comment 5: I will provide more particular examples below, and I also tried to illustrate my ideas by rewriting a section of the text in the end of my report. Of course, I do not expect that the authors follow my edits exactly, but I very much hope that they could find some inspiration in it.

Answer 5: Thank you very much. Your efforts and ideas are greatly appreciated. We used most of your suggestions prior sending the revised manuscript for English correction to the the specialists in MDPI.

Comment 6: 3. It is strage that the abstract does not contain any conclusions, while in fact the ending of the manuscript does, and they are indeed important. Please consider updating the abstract.

Answer 6: Thank you. Based on your observation and suggestion we added additional sentence at the end of the Abstract: “It could be concluded that the PGS should be viewed as fragile genetic mosaic encompassing at least three spatial and one temporal dimensions.”.

Comment 7: 4. I find it a bit strange that the problem of species delimitation is totally omitted in the manuscript (mentioned once without any explanation towards the end). In my opinion, it’s extremely hard to speak about intraspecific structure without defining what a species is, exactly, for the purposes of studying PGS.

Answer 7: Thank you very much for your remark regarding lack of information considering species delimitation in the manuscript. We added an additional chapter 2.1 “Species Concepts” including Table 1. Entitled” Comparison of commonly used species concepts by key features, temporal orientation, applicability to asexual organisms and ontological status” and included new text starting from Line 144 till Line 196 defining species conception.

Comment 8: 5. L61-62 “by clearly defining used concepts during research.”: i’m curious how the authors suggest calling species in this case. Should something like “phylogenetic species” be used?

Answer 8: Previously we used Biological Species Concept (BSC), mainly defined as: a species as a group of actually or potentially interbreeding natural populations that are reproductively isolated from other such groups. Later in the text now we still use abbreviation BSC. Its conception now is mentioned in more detail, and we hope that this concept and term is now better defined in the manuscript, as well as in the Supplementary Material. Additional species concepts now also mentioned in more detail.

Comment 9: 6. L69 ‘especially birds and mammals” why especially these groups? It’s not obvious.

Answer 9: This statement was taken directly from Shaw et al. [13] reference. In order to avoid any confusion, sentence part “especially birds and mammals” was deleted.

Comment 10: 7. I feel that the species concepts in the Introduction section could be explained in more detail, maybe as a table / schematic? It’s a nice review the authors are citing but the concept quite central to this manuscript, so it could be important to provide more background here..

Answer 10: Based on your suggestion, additional Table 1 was created in a separate “2.1. Species Concepts” subchapter. We hope that you will like it.

Comment 11: 8. Glossary in Supplement it a very interesting idea, but I feel its implementation could be improved.

First, I cannot quite catch the intended audience. At the one hand, it should be quite inexperienced specialists, as such basic terms as chromosome, allele or locus are defined... but at the same time, some much more complex and niche terms, like census size, carrying capacity, genetic load, acclimatization potential or Fst do not have a definition here.

Second, I do not understand the logic behind ordering. It is designed to follow the order of the terms’ appearance in the manuscript? I believe, in this case alphabetic would be better, as most people will look up some terms at random rather than read it along switching back and forth between the article and the supplement.

Third, speaking about the ease of reading, I would greatly suggest incroprorating the idea of the glossary into the manuscript itself by highlighting the term (in bold and/or italics) when it is used for the first time and followed by the definition. It will provide more structure. Please see the very end of my report for a couple of examples.

If the authors feel that a separate glossary could also be useful in some situations, as for me, it is okay to provide this information once more in the Supplement. Actually, going through the text like this will also help identify some definitions missing from the glossary in the supplement. Moreover, the supplement would be a nice place to provide literature references to key definitions.

Answer 11: We agree that Glossary could and should be upgraded, thus we done it now. Previously we used order based on terms (that are not explained later in more detail) at first mention in the manuscript text but now based on your advise we order everything based on alphabet. We agree that it would be good if Glossary would be not  in the Supplementary Material but in the manuscript text, yet decided to not do it, as then the length of the manuscript would increase drastically. In addition, then there would be a necessity renumber references in the text that is important but very tiring technical work. If you think that Glossary within the manuscript itself is still necessary after the next revision, then we will consider it once again. Even so, your idea regarding bold text application to all necessary terms at first mention is greatly appreciated, accepted and used.

The intended audience is not one specific (unless, it is defined by broad “scientists“ category) and this information is represented in new Graphical Abstract.

Comment 12: 9. It would be very helpful to make a list of non-standard abbreviations like PGS, PSS, EP etc. (for those readers who did not read the whole manuscript thoroughly but instead wanted to look into a particular section or, for example, have been reading this review over a long time and forgot some of the abbreviations).

Answer 12: Your suggestion is constructive indeed and accepted, thus we created additional abbreviation list in the Supplementary Material.

Comment 13: 10. L232 “only three main types of populations”: are these types of populations indeed? Looks more like types of PGS within a species. This would really be nicely (and easily!) illustrated by a figure, as this is an important concept and much more complex than PPS / PGS / PSS / PAS illustrated in Figure 1.

update: This comment was written before I encontered Fig. 2, which partly encompasses this idea. Maybe it would make sense to add the third structure (metapopulation) to the figure and moving it above.

Answer 13: Thank you. Despite of great increasing of the manuscript space each time when new Figures are added, we agree that metapopulation case requires additional representation as Figure. Consequently, we created additional Figure 3 (it could not be just Figure 2C because then it would be hard to see all necessary images in Figure 2) for this purpose.

Comment 14: 11. L233 “, as distinct most diverse structural units of species:”: is this another definition of the same term population?

Answer 14: In general, yes. We tried to avoid too many repetitions of word population and also show quite different perspective using descriptions like structural units.

Comment 15: 12. “The research showed that insects feeding on genetically modified plants (those unable to send electrical signals) gained weight more rapidly than those feeding on wild-type plants, indicating that insects found it more difficult to predate plants with systems for sending electrical signals [43] (and references therein).”: this is a direct quote from this review, which creates two problems. First, if this a direct quote this long, it should be either quoted or paraphrased. Second, in this context it is unclear how these plants were genetically modified, while it is important to the 

Answer 15: Text was modified to: “The research showed that insects feeding on genetically modified plants (after modification plants lost ability to send electrical signals) gained weight more rapidly than those feeding on wild-type plants, indicating that insects found it more difficult to predate plants with active systems for sending electrical signals [43] (and references therein).”.

Comment 16: 13. Speaking about the structure of the text: e.g. L357-397 could be a subsection “Population ethology structure” or something along these lines.

Answer 16: Yes. Done as you suggested.

Comment 17: 14. “[134] and references therein”: I have nothing against this review cited multiple times, but it was published in 2004, >20 years ago, and references therein are thus even older. Maybe cite something newer in addition or instead? For example, there is a 2024 book “DNA Barcoding: Methods and Protocols” (https://link.springer.com/book/10.1007/978-1-0716-3581-0), and citing particular chapters from it seems a good idea.

Answer 17: Yes, we agree that additional reference with recent knowledge should be added as well. Added your suggested reference as book.

Comment 18: 15. “genetic diversity could be defined as DNA variations found in organisms” <...> “Due to the fact that all species are genetically unique, this definition of genetic diversity is also quite abstract as it encompasses both interspecific and intraspecific genetic diversity. Consequently, if we want to exclude interspecific level, then intraspecific genetic diversity could be defined as DNA variations found within the same species.”: this explanation is very long. Why not define intraspecific genetic diversity at the beginning? It’s quite clear that if genetic diversity could be defined as DNA variations found in organisms, then intraspecific genetic diversity could be defined as DNA variations found in organisms of one species and interspecific genetic diversity could be defined as DNA variations found in organisms of different species.

Answer 18: Thank you. Instead of our old paragraph now we using your suggested revised text.

Comment 19: 16. It is also strange that modified nucleotides (epigenetic modification of DNA, if you will) are not mentioned either here or later when speaking about nucleotides.

Answer 19: Sentence was modified accordingly: In general, all DNA variations, including epigenetic modifications of nucleotides, could be reduced to and understood as all possible variants of nucleotide positions within ssDNA sequence, and observed by aligning and comparing two or more sequences.

Comment 20: 17. L451 prions : there is no reference to a review of prions as hereditary entitites.

Answer 20: New reference was added to the manuscript:

Shorter, J.; Lindquist, S. Prions as adaptive conduits of memory and inheritance. Nat. Rev. Genet. 2005, 6, 435–450. https://doi.org/10.1038/nrg1616

Comment 21: 18. “during Ice Age [138]”: it is cited as an example of using ancient DNA, but I cannot see such information here. Please check.

Answer 21: Thank you. You are right that this reference is not clear enough, thus we add additional two references that are clear regarding Ice Age. References:

Seifertová, M.; Bryja, J.; Vyskočilová, M.; Martínková, N.; Šimková, A. Multiple Pleistocene refugia and post-glacial colonization in the European chub (Squalius cephalus) revealed by combined use of nuclear and mitochondrial markers. J. Biogeogr. 2012, 39, 1024–1040. https://doi.org/10.1111/j.1365-2699.2011.02661.x

Hadly, E.A.; Ramakrishnan, U.; Chan, Y.L.; van Tuinen, M.; O'Keefe, K.; Spaeth, P.A.; Conroy, C.J. Genetic response to climatic change: Insights from ancient DNA and phylochronology. PLoS Biol. 2004, 2, e290. https://doi.org/10.1371/journal.pbio.0020290

Comment 22: 19. L490-511 “The double-helix DNA structure was discovered in 1953 [148] <...>will concentrate more about DNA genetic properties and its use within population genetics framework.”: I honestly cannot see clear relevance of this text fragment to the major topic of this research and think that this paragraph can be safely omitted.

Answer 22: Actually, this paragraph purpose was to expand information about the DNA diversity, mainly DNA conformation nuances. It was logical step after DNA strands diversity. Even so, we decided to remove this paragraph as you suggested.

Comment 23: 20. L519-520 “quantitative genetic parameters, such as haplotype diversity (Hd) and nucleotide diversity (π), are calculated.”: needs a reference to what these parameters are and how they are calculated. This being said, I need to add that this thesis is also misleading, as Hd and pi can be calculated taking into account gaps in alignment.

Answer 23: Based on your observation, haplotype diversity (Hd) and nucleotide diversity (π) now are defined in Supplementary Material. You are also right that unfortunately previous text indeed could mislead readers, thus we modified this text: Quite often indels, especially those that represent one or few nucleotide deletions or insertions, are still ignored in practice of DNA sequence analysis when quantitative genetic parameters, such as haplotype diversity (H_d) [170] and nucleotide diversity (π) [171], are calculated. That is not to say that the mentioned parameters could not be calculated both including gaps as 5^th nucleotide position in DNA and excluding them. Examples and evidence that indels could provide an additional layer of important information, and in turn should not be discarded, keeps growing [172, 173].

Comment 24: 21. L524-525 “yet only direct sequencing [74] allows us to study each nucleotide position in the obtained short and long DNA sequences [156]”: the term “direct sequencing” could be highly misleading in this particualr case. I would use just “sequencing”. One the one hand, “direct sequencing” had been used to describe Sanger sequencing as opposed to Maxam-Gilbert degradation-based technique. On the other hand, now “direct sequencing” can be used to mean TGS techniques that do not include DNA amplification as opposed to the methods exploiting PCR-based library preparation.

Answer 24: Done as you suggested. Thank you.

Comment 25: 22. L607-608 “Genomic repetitive DNA sequences (Repeatomes, REPs)”: I feel that this wording could lead to a misunderstanding, as “repetitive sequences” does not equal “repeatomes”. Repeatome is already a sum of repetitive sequences in a species. I would just use “repetitive DNA sequences (REPs)”.

Answer 25: Thank you. Changed based on your suggestion.

Comment 26: 23. L616 “A genome sequence is a snapshot of a strain in time [137].”: without some context it’s impossible to guess that now are speaking about prokaryotes; otherwise, a concept of strain sounds very strange.

Answer 26: The sentence was modified accordingly: A genome sequence is a snapshot of an individual (in case of procaryotes and certain parasitic eucaryotes, a strain) at a given point in time [157].

Comment 27: 24. L633-636 “For instance, based on selected method the estimated number of protein-coding genes in Eurasian perch (Perca fluviatilis) could range from 23,397 to 24,326, yet total scaffold size (bp) ranged from 630,662,671 to 958,225,764 bp (see [186] and references therein).” : while I totally share and support the thesis illustrated by this example and actually think this would be true for basically any genome of a free-living eukaryote, giving the estimate of gene number and genome size does not clearly illustrate it. Otherwise, one could argue that perch has very large genes, for example. It would be better to provide information about the percent of the genome assembly actually occupied by genes.

Answer 27: Thank you very much. Additional text (and reference) was added based on your suggestion:  Similarly, majority of human nuclear genome is made up of non‑coding sequences like introns (almost 26%) and TEs (nearly 45%) and only less than 1.5% of it consists of the suspected 20,000–25,000 protein‑coding sequences [205].

Comment 28: 25. L639 “Over 50% of animal genomes belong to the phylum Chordata”: I would add an introductory sentence that states the problem of unequal representation of different taxonomic groups in genome projects (I guess, this was the motivation). Otherwise, the examples below are unclear.

Answer 28: Additional new text added: Nowadays comparative genomics potential is reduced due to unequal research and representation of genomes of different organisms encompassing distinct evolutionary and taxonomic groups.

Comment 29: 26. L699 “Aneuploidy (see [189]) is the simplest system for us to measure genetic variability,”: I don’t understand this thesis. First, the term “genetic variability” appears only in the paragraph above and is not defined there; neither is it defined in the glossary. What is the difference between genetic diversity and genetic variability? From what was described above, I would talk about aneuploid systems as a tool to link cytogenetics and genome assembly (even though it does not completely apply to the yeast model described in [189]). So, please consider revising this logical connection.

Answer 29: Thank you. We used genetic variability here as synonym of genetic diversity. Modified sentence: Aneuploidy (see [208]) is the simplest system for us to research genetic diversity at chromosomes level, as alleles could be detected only in certain loci of chromosomes and directly translate to traits revealed in phenotypes.

Comment 30: 27. What is the function of Box 1? Does it provide questions to which the authors try to find some answers below or open questions? I could not find proof for any of these hypothesis and was lost here. Please provide some background.

I’m inclined towards the first hypothesis. In this case, it would be a great help to the reader if the authors revised the next subsections to reflect these questions.

Answer 30: In order to remove additional confusion and reduce the length of the manuscript, Box 1 was removed. In addition, most questions in Box 1 could be found throughout the manuscript and alternative option to expand it with more questions is not viable for this manuscript anymore.

Comment 31: 28. It’s a pity that this manuscript does not include any overview of how the population structure can be estimated and compared. However, I completely understand the motivation. Well, we could only hope for a future review about this topic, which could be titled “Population Genetic Structure: How”.

Answer 31: Yes, you are absolutely right that “Population Genetic Structure: How?” could and should be written as separate perspective review by our research group or other scientists in the future.

Comment 32: An example on how this text could be structured and shortened:

 

Original text:

What is the PGS of the species? The answer to this main question is directly linked to understanding genetic diversity of species. There are many reviews dedicated to intraspecific genetic diversity (see [14, 21, 54]), its different aspects [3, 132, 133], and how to study it using major methods [7] related to application of various molecular markers (see [134] and references therein), thus in this paper most of this information will not be presented but interested readers will be guided to important literature sources. In this review we will use a commonly accepted, yet oversimplified, definition of genetic diversity. That is, genetic diversity could be defined as DNA variations found in organisms. Major oversimplification: there are other known biological molecules that could be genetic material for certain organisms. For instance, viruses are considered as quasispecies (see [135]) and are not included as part of life, yet they still are biological-genetic systems, and in some of their group’s RNA and not DNA is the essential genetic material [136] (and references therein). That said, based on current comprehensive scientific knowledge, we could rationally limit the term and concept of genetic diversity to just DNA within the biological molecules, which could code genetic information, context encompassing most, if not all, eukaryotes. This leaves decoding by RNA molecules (see [55] and references therein) and functioning of some proteins, such as prions, to major areas of research for transcriptomics and proteomics, respectively, and outside the scope of the mentioned term and concept of genetic diversity of eukaryotes. Due to the fact that all species are genetically unique, this definition of genetic diversity is also quite abstract as it encompasses both interspecific and intraspecific genetic diversity. Consequently, if we want to exclude interspecific level, then intraspecific genetic diversity could be defined as DNA variations found within the same species. To sum up, DNA variations could and should be treated as the major key to understanding the intraspecific genetic diversity, and this leads us to the necessity to mention DNA and its variations in more detail. However, on a broader scale, genetic diversity includes all heritable variation at the molecular level.

 

Suggested changes:

In order to understand what PGS is, we need to understand the genetic diversity of a species. There are many reviews dedicated to intraspecific genetic diversity (see [14, 21, 54]), its different aspects [3, 132, 133], and how to study it [7] with molecular markers (see [134] and references therein), thus interested readers will be referred to relevant literature sources. In this review we define genetic diversity as DNA variations found in organisms. This definition has some disadvantages.

First, it is oversimplified, as some other biological molecules are known to be genetic material. For instance, viruses are considered as quasispecies (see [135]) and are not included as part of life, yet they still are biological-genetic systems, and in some viruses RNA is the essential genetic material [136,55] (and references therein). Moreover, some proteins, such as prions [reference missing!], act as hereditary factors. These particular examples are outside the scope of this review. However, on a broader scale, genetic diversity includes all heritable variation at the molecular level.

Second, due to the fact that all species are genetically distinct, this definition encompasses both interspecific and intraspecific genetic diversity. Consequently, intraspecific genetic diversity could be defined as DNA variations found within a species. To sum up, DNA variations could and should be treated as the key to understanding the intraspecific genetic diversity, and this leads us to the necessity to consider DNA and its variations in more detail.

Answer 32: Thank you so much. This is really helpful and greatly increasing the overall quality of the manuscript.

Comment 33: Comments on the Quality of English Language

In my opinion, the text could be edited for brevity and also proofread, but this is a minor concern. Please find below some awkward phrases I noticed while reading but not this is by no means an exhaustive list.

1. “Species are comprised of populations that encompasses intraspecific genetic variability in the form of DNA”: the sentence is grammatically awkward
2. “Most widely accepted” => the most
3. L148 “references therein)” missing dot
4. L176 “predator-victim dynamics” it’s normally predator–prey
5. L243-245 “Even species that are considered panmictic, could surprise us with deviations from our theoretical assumptions.”: excessive comma
6. Fig 1 : is it designed to be available in BioRender to the readers? It is not.

Fig 2: the same problem.

7. L256 “withing” should be “within”
8. L327 “phenotype plasticity” => “phenotypic plasticity”?
9. (multiple occurences) “code genetic information”, “code genes” etc => “code for genetic information” or “encode genetic information”
10. L588-589 “Both selectively neutral genetic variations and genes [162], as well as DNA parts near them, included in the concept of genetic diversity.”: should be something like “Both substitutions in genes and selectively neutral genetic variations [162], as well as adjacent DNA regions, are included in the concept of genetic diversity.”
11. L603 “and unique DNA sequences, which functions are not clear”: better “and unique DNA sequences, functions of which are unclear” or just “and unique DNA sequences with unclear functions”
12. L730 “It could be concluded that within an eukaryotic cell there is not one mtDNA copy”: it’s not “concluded”, it’s rather “It should be kept in mind” or “It is worth noting”
13. L737 “Complications not ending here,”: “These are not the only complications” or “Complications are not ending here”
14. L773 “environmentally, responsive epigenetic mechanisms”: excessive comma
15. L773 “can allow acclimatization” : “can allow for acclimatization”
16. L778 “According Gargiulo et al [75],”: “According to Gargiulo et al. [75],”
17. L824 “gene drift (or random genetic drift) that defined”: “gene drift (or random genetic drift) that is defined”
18. L869 “defined prior C. Darwin”: “defined prior to Charles Darwin”
19. L870 “biologic evolution”: “biological evolution”
20. L900 “For more information see Charlesworth & Charlesworth [209], Balkenhol et al [210].”: Previously, only reference numbers were provided, and this was quite enough.
21. L1104 “genetics have”: “genetics has” or “geneticists have”
22. L1190 “while previously only non-economically important species has been largely neglected”: “only” seems excessive
23. L1263 “It is worth to mention that scientific works that dedicated”: “It is worth to mention that scientific works dedicated”
24. L1326-1327 “However, instead of this scenario, was opened the window that slowly starting to close only now:”: “However, instead of this scenario, a window opened that is only slowly starting to close now:”
25. L1345 “within and among populations”: “within and between populations”?

Answer 33: Thank you very much. For the most part we done as you suggested. We tried to remove these awkward phrases and where we failed to do it we hope and expect that everything will be ok after English correction by the specialists in MDPI. Regarding BioRender, Figures were not available because it was set for August month. Now everything should work fine.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript was submitted to MDPI Author Services for Rapid and Academic language editing, and has been significantly improved.  The authors also added additional structure within the major parts of the manuscript. I noticed that a table was included (Table 1), comparing the commonly used species concepts, and I found that very interesting and useful for a better understanding of the text.  The authors have also accepted most of my suggestions and justified the ones that could not be accepted. Therefore, I agree now with its publication in this important Journal.

Author Response

Comment1: The manuscript was submitted to MDPI Author Services for Rapid and Academic language editing, and has been significantly improved.  The authors also added additional structure within the major parts of the manuscript. I noticed that a table was included (Table 1), comparing the commonly used species concepts, and I found that very interesting and useful for a better understanding of the text.  The authors have also accepted most of my suggestions and justified the ones that could not be accepted. Therefore, I agree now with its publication in this important Journal.

 

Answer1: Dear Reviewer,

 

Thank you so much for your time, energy, positive objective evaluation of our work and previous suggestions that helped to improve the manuscript and Supplementary Materials and think more about certain natural phenomena and how to interpret it. We really appreciate that and are glad that you liked carried out changes in the manuscript compared to its previous version.

Reviewer 3 Report

Comments and Suggestions for Authors

I very much like the updated version of the manuscript and would like to sincerely thank the authors for investing such effort in the revision! It seems much easier to read and find necessary information in it. I totally agree with the authors’ decisions and am proud if my comments helped trigger some of these positive changes.

I have a few very minor comments.

 

- I like the abbreviation list, but I don’t see a reference to it in the main text, so it is not easy to find. Please check and consider adding a note into the main text.

 

- The abbreviation list might benefit from a critical re-reading, maybe by a colleague.

Here are examples of very small but still a bit confusing details, please take a look:

 

“Biparental inheritance: DNA inherited by both parents.” should probably be “Biparental inheritance: DNA inherited from both parents”

 

“Predator-prey dynamics: dynamic ecological relationship among organisms when one of them (predator) hunt and eat another organism (prey).” should probably be “Predator-prey dynamics: dynamic ecological relationship between organisms when one of them (predator) hunts and consumes another organism (prey).”

Author Response

Comment1: I very much like the updated version of the manuscript and would like to sincerely thank the authors for investing such effort in the revision! It seems much easier to read and find necessary information in it. I totally agree with the authors’ decisions and am proud if my comments helped trigger some of these positive changes.

 

Answer1: Dear Reviewer,

 

To be honest, our work was evaluated by a few Reviewers and all of them provided good and important suggestions how to improve the manuscript, yet your constructive, objective and realistic comments and suggestions were extremely valuable and the most helpful for us during this comprehensive revision process, thus we really appreciate that, and would like to sincerely thank you. Be sure that your comments and suggestions not only triggered many positive changes in the manuscript but also helped us to gain great experience and to think more about certain aspects when preparing future manuscripts, especially associated with PGS phenomena. In addition, it is indeed nice to hear from you that you liked our updated version of the manuscript.

Comment2: I have a few very minor comments.

 

- I like the abbreviation list, but I don’t see a reference to it in the main text, so it is not easy to find. Please check and consider adding a note into the main text.

Answer2: Yes, you are right. In the beginning of the manuscript, we added the direction to the Supplementary Materials regarding the abbreviation list, so readers could find it, as well as Glossary of terms, more easily.

Comment3: - The abbreviation list might benefit from a critical re-reading, maybe by a colleague.

Here are examples of very small but still a bit confusing details, please take a look:

 

“Biparental inheritance: DNA inherited by both parents.” should probably be “Biparental inheritance: DNA inherited from both parents”

 

“Predator-prey dynamics: dynamic ecological relationship among organisms when one of them (predator) hunt and eat another organism (prey).” should probably be “Predator-prey dynamics: dynamic ecological relationship between organisms when one of them (predator) hunts and consumes another organism (prey).”

Answer3: Thank you. First, we corrected everything based on your suggestions. After that there was an attempt to re-read Supplementary Materials and correct possible errors.