Association of Morphological, Ecological, and Genetic Diversity of Aerva javanica Populations Growing in the Eastern Desert of Egypt

Round 1

Reviewer 1 Report

I was asked to review a manuscript entitled „ Association of morphological, ecological and genetic diversity of Aerva javanica populations growing in the Eastern Desert of Egypt”.

The authors deserve appreciation for their attempt to compile the results of genetic analysis, morphological characteristics and information from the analysis of soil samples. Relatively few authors take such a comprehensive approach to assessing the diversity of medicinal plants. Such studies are important especially in the context of rapid and progressive climate change.

Here are all my comments on the manuscript.

1) All Latin names of species should be written in italics.

2) Abbreviations should not be used in the abstract.

3) In the introduction, the description of the species should be extended and the addition of photographs of the plant would also be welcome.

The materials and methods section is the one that has been the most concerning to me.

4) In the description of the performance of genetic analysis, there is no information on how many plants DNA was isolated from.

5) There is also no information whether the reproducibility of band patterns obtained after PCR was verified. I know from experience that there are problems with the repeatability of thinner and less intense bands when separating fragments in agarose gel.

6) The analysis of the data is definitely insufficient. We cannot consider genetic diversity analysis if only hierarchical grouping was performed.

7) There is a lack of information about which distance coefficient was used and which grouping method was performed. Information about the use of specific software is far too little.

8) I also need to confirm the results of grouping using principal coordinate analysis (PCoA).

9) Distance matrices (genotype and phenotype) should be compared with each other and with the geographical distance using the Mantel test to check if there is a link between genetic/morphological variation and the site of the population.

10) A variance analysis for morphological traits is missing.

11) In order to understand how morphological, genetic and environmental data should be analysed, I suggest to read the paper:  https://link.springer.com/article/10.1007/s10722-017-0555-8

12) It is also worthwhile to include climatic data for collection sites. Such data can be obtained from the WorldClim database - Global Climate Data

13) Maps depicting the collection sites are not very legible - they should be enlarged and presented graphically in a different way.

14) Some tables are redundant - Table 4, Table 5,

15) The discussion should be extended in the context of the results obtained after the extension of statistical analysis.

I encourage the authors to thoroughly improve the computational side of the work and re-submit the manuscript, because despite many shortcomings, I consider these studies to be valuable.

Author Response

We thankfully acknowledge your comments regarding our manuscript.

1) All Latin names of species should be written in italics.

Response:

            Done accordingly.

2) Abbreviations should not be used in the abstract.

Response

            Done accordingly.

3) In the introduction, the description of the species should be extended and the addition of photographs of the plant would also be welcome.

Response

            The plant description extended according to the suggestion and photograph of the plant already inserted in the main text (Fig.2).

4) In the description of the performance of genetic analysis, there is no information on how many plants DNA was isolated from.

Response

            Already denoted in the main text as DNA was extracted from young leaves.

5) There is also no information whether the reproducibility of band patterns obtained after PCR was verified. I know from experience that there are problems with the repeatability of thinner and less intense bands when separating fragments in agarose gel.

Response

            We have taken the PCR banding patterns into consideration while scoring the ISSR fingerprinting, by running the PCR and gels at the same conditions, and repeated unconvincing fingerprinting profiles. Moreover we followed the same standard regarding the thickness of bands, while scoring the bands for all primers. All scorable bands had been estimated using gel documentation system and counted manually through all authors very thoroughly.

6) The analysis of the data is definitely insufficient. We cannot consider genetic diversity analysis if only hierarchical grouping was performed.

Response

            In our MS genetic diversity was clarified using both morphological and genetic information’s using cluster and Biplot analysis to confirm such diversity showing the relationship among different populations.

7) There is a lack of information about which distance coefficient was used and which grouping method was performed. Information about the use of specific software is far too little.

Response

            Cluster analysis showing the relationships between the studied populations of Aerva javanica using Pearson correlation coefficient Ward minimum variance method and this added to the text.

8) I also need to confirm the results of grouping using principal coordinate analysis (PCoA) as confirmed previously by many researchers.

Response

          Done accordingly

9) Distance matrices (genotype and phenotype) should be compared with each other and with the geographical distance using the Mantel test to check if there is a link between genetic/morphological variation and the site of the population.

Response

            The objectives of the study focus mainly on the association of morphological, ecological and genetic diversity of Aerva javanica in different populations growing in the Eastern Desert of Egypt and not within populations so we did not applied Mantle test.

10) A variance analysis for morphological traits is missing.

Response

            Done accordingly

11) In order to understand how morphological, genetic and environmental data should be analysed, I suggest to read the paper:  https://link.springer.com/article/10.1007/s10722-017-0555-8

Response

            Thanks a lot. We used such link as suggested.

12) It is also worthwhile to include climatic data for collection sites. Such data can be obtained from the WorldClim database - Global Climate Data

Response

            Aridity in the Eastern Desert in Egypt is prevailing all the year and no so much fluctuation so we did not include effect the temperature and we concerned about soil chemical and physical properties as it have a prominent effect on genetic diversity as indicated.  

13) Maps depicting the collection sites are not very legible - they should be enlarged and presented graphically in a different way.

Response

            It is corrected accordingly.

14) Some tables are redundant - Table 4, Table 5,

Response

            Each table has an important data for results and discussion section and cannot be deleted.

15) The discussion should be extended in the context of the results obtained after the extension of statistical analysis.

Response

            It was done accordingly

I encourage the authors to thoroughly improve the computational side of the work and re-submit the manuscript, because despite many shortcomings, I consider these studies to be valuable.

Response

            Thanks so much for your valuable comments that make the MS more sound.

Reviewer 2 Report

The manuscript "Association of morphological, ecological and genetic diversity of Aerva javanica populations growing in the Eastern Desert of Egypt" reported about ten populations of A. javanica and usefulness ISSR molecular markers and morphological characters in relation to ecological features of the plant growing site. The idea of this work is interesting, but I have some questions:

Some editorial errors in the text (e.g. see the References)

 

Materials and Methods:

 

Explain, please, how did you measured DNA quantity? Did the Authors use the spectrophotometric measurements? Explain, please, how did you choose the primers. There is no information about primer design. Explain, please, what was the temperature profile of the PCR reactions for ISSR markers? What was the annealing temperature for used primers?

 

Overall, I think that the article has potential to be published in Agronomy.

Author Response

The idea of this work is interesting, but I have some questions:

Response

Thanks for your comments and encouraging us

-Some editorial errors in the text (e.g. see the References)

Response

            It has been corrected.

-Explain, please, how did you measured DNA quantity? Did the Authors use the spectrophotometric measurements? Explain, please, how did you choose the primers. There is no information about primer design. Explain, please, what was the temperature profile of the PCR reactions for ISSR markers? What was the annealing temperature for used primers?

Response

1- how did you measured DNA quantity? Did the Authors use the spectrophotometric measurements?

We applied 2 µl of the extracted DNA from various populations having a final conc. four ng/L, using (spectrophotometric). This already clarified very well in the main text.

2- Explain, please, how did you choose the primers. There is no information about primer design.

Response

            We used universal primers as they applied previously to estimate the genetic diversity among plants at various level some gave scorable bands and other did not.

3- Explain, please, what was the temperature profile of the PCR reactions for ISSR markers? What was the annealing temperature for used primers?

Response

            Already clarified in the main text.  

Reviewer 3 Report

This article, Association of morphological, ecological…..Eastern Desert of Egypt” by El-Tayeh et al. has reported results of the genetic variation of several populations of Aerva javanica, a plant species used for its medicinal value in that region. Additionally, the authors have examined the morphological variations of the selected plant populations and several soil characteristics respective to those plant populations. Finally, the authors have tried to explore the associations of the variations in soil characteristics with the genetic variations and morphological variations to suggest the possible role of abiotic factors in shaping the genetic variations observed in the selected plant populations. Further, the authors suggested that understanding the underlying factors for observed genetic variations on this important plant species could lead to a fruitful conservation effort.

Overall, this is an interesting study and lots of good information related to the plant species, Aerva javanica, grown for medicinal purposes in Egypt. This article has been drafted well in most parts, but still needs many corrections for both English language and typographical errors. I am sure, one or two rounds of reading and intensive corrections could bring this article to a publishable level. I would strongly suggest the authors go through the manuscript thoroughly to identify errors. Meanwhile, I will point out a few obvious mistakes that should be taken care of in a revised version of this manuscript.

First, I would like to make some general comments on the manuscript that are of primary concern.

I understand that the soil type and soil chemistry play an important role in the natural occurrence of certain plant species. However, I do not have much background knowledge in this particular system. So, my questions are: are these plant populations being found in wild habitat or under cultivated/man managed conditions? If they are in their wild habitat, has there been any effort to grow a certain populations in different growing conditions/soil types purposefully? Do you think soil type is an ecological barrier that has led to the genetic diversity in the plant populations being studied? Do you think if one would try to do some type of transfer experiment (growing some populations in different environment/soil type), the plants will survive or behave differently? If you conduct a transfer experiment, do you think the morphological differences observed in plants will change? Is the morphological characteristics observed related to soil type or genetically inherited?

Second, I would like you to improve on the method sections as there are some important information missing.

If you could present a figure of the plant and the morphological characters of plants considered in this study, that would help the readers like me to understand the article better. Currently, there are no information on the “materials and methods” section about the plant morphological characters. Were the plants considered of the same age? Did you control for plant height, leaf position etc. Please give more details. How many samples were collected per population for genetic analysis? I think there are other marker systems you should have used in today’s time instead of using ISSR marker. ISSR marker system is several decade-old tool and rarely used. Use of co-dominant markers could provide your more resolution and a better understanding of the genetic diversity.

There are many editorial mistakes and here some of them:

Scientific names should be in italics throughout the manuscript. Delete ISSR before polymorphism in line 19 Replace “affecting” by “influencing” in line 25 In Egypt, (line 36) Proved not “proofed” (line 56) Delete “DNA markers” (line 65) In line 68, complete the sentence at “use”. Then In ISSR is a new sentence. Line 73, “population genetic studies”, not “population studies” Line 92, delete “used and” Line 102, give name of city and county of the university DNA extraction and purification, not “refining” “Lamdas” line 134, check how to write correctly Just ISSR, not ISSR-PCR in line 141 and elsewhere in the manuscript Lowest leaf length, not leaf length lowest (line 163) No need of 3, reader is going to read your gel pictures. Restructure your sentence (line 283-286). This is long and partly not correct in language usage Not use what you mean by “”on the main of morphological properties” in line 315. Please correct. Add “of” in front of A. javanica (line 344).

 

 

 

Author Response

Overall, this is an interesting study and lots of good information related to the plant species, Aerva javanica, grown for medicinal purposes in Egypt. This article has been drafted well in most parts, but still needs many corrections for both English language and typographical errors. I am sure, one or two rounds of reading and intensive corrections could bring this article to a publishable level. I would strongly suggest the authors go through the manuscript thoroughly to identify errors. Meanwhile, I will point out a few obvious mistakes that should be taken care of in a revised version of this manuscript.

Response

            Thanks for your comments and encouraging us

-First, I would like to make some general comments on the manuscript that are of primary concern.

I understand that the soil type and soil chemistry play an important role in the natural occurrence of certain plant species. However, I do not have much background knowledge in this particular system. So, my questions are: are these plant populations being found in wild habitat or under cultivated/man managed conditions? If they are in their wild habitat, has there been any effort to grow a certain populations in different growing conditions/soil types purposefully? Do you think soil type is an ecological barrier that has led to the genetic diversity in the plant populations being studied? Do you think if one would try to do some type of transfer experiment (growing some populations in different environment/soil type), the plants will survive or behave differently? If you conduct a transfer experiment, do you think the morphological differences observed in plants will change? Is the morphological characteristics observed related to soil type or genetically inherited?

Response

            It is totally a weeds plant, we do not know if it could be cultivated by human (some weeds very difficult to be managed by human)- Our results confirmed that soil type is an ecological barrier that has led to the genetic diversity in the plant populations. The plant populations in our MS found in wild habitat.

 

-Do you think if one would try to do some type of transfer experiment (growing some populations in different environment/soil type), the plants will survive or behave differently? We will try this in the future thanks for the suggestion and see what will be? If you conduct a transfer experiment, do you think the morphological differences observed in plants will change?

Response

             We will do this in the future and see what will be?

-Is the morphological characteristics observed related to soil type or genetically inherited?

Response

            Related to both and confirmed this from our results.

-Second, I would like you to improve on the method sections as there are some important information missing.

If you could present a figure of the plant and the morphological characters of plants considered in this study, that would help the readers like me to understand the article better. Currently, there are no information on the “materials and methods” section about the plant morphological characters. Were the plants considered of the same age? Did you control for plant height, leaf position etc. Please give more details. How many samples were collected per population for genetic analysis? I think there are other marker systems you should have used in today’s time instead of using ISSR marker. ISSR marker system is several decade-old tool and rarely used. Use of co-dominant markers could provide your more resolution and a better understanding of the genetic diversity.

Response

1-If you could present a figure of the plant and the morphological characters of plants considered in this study, that would help the readers like me to understand the article better.

            We attached photographs for the plant according to the suggestion.

2-Currently, there are no information on the “materials and methods” section about the plant morphological characters.

Response

            We add information accordingly.

-I think there are other marker systems you should have used in today’s time instead of using ISSR marker. ISSR marker system is several decade-old tool and rarely used. Use of co-dominant markers could provide your more resolution and a better understanding of the genetic diversity

Response

            Either RAPD or ISSR-PCR can quickly reveal the difference between individuals with a high degree of similarity and include multiple polymorphic loci. Update to date be several researchers like, Guilengue et al. Agronomy 2020, 10, 21; Abdel Latef et al., Plants 2019, 8, 303 and Badr et al., Feddes Repertorium 2018, 129, 173–184 used such primers. Next works we will use more specific primers for plant barcoding and study the genetic diversity among them thanks for suggestion.

There are many editorial mistakes and here some of them:

Scientific names should be in italics throughout the manuscript. Delete ISSR before polymorphism in line 19 Replace “affecting” by “influencing” in line 25 In Egypt, (line 36) Proved not “proofed” (line 56) Delete “DNA markers” (line 65) In line 68, complete the sentence at “use”. Then In ISSR is a new sentence. Line 73, “population genetic studies”, not “population studies” Line 92, delete “used and” Line 102, give name of city and county of the university DNA extraction and purification, not “refining” “Lamdas” line 134, check how to write correctly Just ISSR, not ISSR-PCR in line 141 and elsewhere in the manuscript Lowest leaf length, not leaf length lowest (line 163) No need of 3, reader is going to read your gel pictures. Restructure your sentence (line 283-286). This is long and partly not correct in language usage Not use what you mean by “”on the main of morphological properties” in line 315. Please correct. Add “of” in front of A. javanica (line 344).

Response

            Everything is corrected appropriately in the main text.

Reviewer 4 Report

Note that all latin names should be written using italic style (Aerva javanica, Citrullys colocynthis etc.).

Rows 15-16: In this study, variations...were analyzed (not was analyzed).

Row 19: The ISSR polymorphism ...was 79.4% (not were).

Row 20: delete „of” before „the results”.

Row 23: „The soil analysis revealed that it had crucial impact on genetic variation”. So, please detail this impact on genetic variation, becausereading the next sentence it seems that all the „genetic” impact is related to the frequency of the plants, which is, in my opinion, a fake conclusion.

 

Too many keywords. They have to be different of those included in title.

 

Row 36: put comma after „Egypt”.

Row 82: „eastern desert of...”  are written with lowercase letters but in the next paragraph with uppercase letters.

Row 84: after „Egypt” and comma put a space.

Row 93: delete the space before „ten”.

Row 105: „were” instead of „was”, because is about „the average value and standard deviation”.

Row 150: delete „;” before 33 and put a bracked „)”.

Row 159: put a space among „pop” and „7”.

Row 160: „in populations 7 and 9” not „in population...”

The results presented in rows 157-160 and most of those presented below as comparations, have to be supported by a statistical test of differences significance (T test, ANOVA etc.).

Fig. 2: I do not know how relevant is to perform a cluster including phenotypical traits but it seems a good ideea when comparing to that based on genetic markers.

Row 191: „which illustrates” instead of „illustrate”.

Rows 193, 194: „contains” instead of „contain”.

Table 3: from the statistical point of view, is not right to note min and max values as means and standard deviations. Therefore, min and max are represented by individual values.

Row 285: „utilization” instead of „utilized”.

Row 332: „reflects”.

Row 356: delete one of the two full stops.

Author Response

We would like to give you a special thanks for your careful reading of the whole MS and also for your meaningful corrections that helped us a lot to improve our manuscript significantly.

Note that all latin names should be written using italic style (Aerva javanica, Citrullys colocynthis etc.).

Response

            Done accordingly.

Rows 15-16: In this study, variations...were analyzed (not was analyzed).

Response

            Corrected.

Row 19: The ISSR polymorphism ...was 79.4% (not were).

Response

            Corrected.

Row 19: The ISSR polymorphism ...was 79.4% (not were).

Response

            Corrected.

Row 19: The ISSR polymorphism ...was 79.4% (not were).

Response

            Corrected.

Row 20: delete „of” before „the results”.

Response

            Corrected.

Row 23: „The soil analysis revealed that it had crucial impact on genetic variation”. So, please detail this impact on genetic variation, because reading the next sentence it seems that all the „genetic” impact is related to the frequency of the plants, which is, in my opinion, a fake conclusion.

Response

            Already rephrased.

Too many keywords. They have to be different of those included in title.

Response

            Corrected.

Row 36: put comma after „Egypt”.

Response

            Corrected.

Row 82: „eastern desert of...”  are written with lowercase letters but in the next paragraph with uppercase letters.

Response

            Corrected.

Row 84: after „Egypt” and comma put a space.

Response

            Corrected.

Row 93: delete the space before „ten”.

Response

            Corrected.

Row 105: „were” instead of „was”, because is about „the average value and standard deviation”.

Response

            Corrected.

Row 150: delete „;” before 33 and put a bracked „)”.

Response

            Corrected.

Row 159: put a space among „pop” and „7”.

Response

            Corrected.

Row 160: „in populations 7 and 9” not „in population...”

Response

            Corrected.

Row 356: delete one of the two full stops.

Response

            Corrected.

The results presented in rows 157-160 and most of those presented below as comparations, have to be supported by a statistical test of differences significance (T test, ANOVA etc.).

Response

            In addition of such analysis we added the variance analysis by ANOVA.

Fig. 2: I do not know how relevant is to perform a cluster including phenotypical traits but it seems a good ideea when comparing to that based on genetic markers.

Response

            We thankfully acknowledge your appreciation and positive comments regarding our manuscript.

Row 191: „which illustrates” instead of „illustrate”.

Response

            Corrected.

Rows 193, 194: „contains” instead of „contain”.

Response

            Corrected.

Table 3: from the statistical point of view, is not right to note min and max values as means and standard deviations. Therefore, min and max are represented by individual values.

Response

            Corrected.

Row 285: „utilization” instead of „utilized”.

Response

            Corrected.

Row 332: „reflects”.

Response

            Corrected.

Round 2

Reviewer 1 Report

I am satisfied with the changes :)

Author Response

REVIEWER 1

I am satisfied with the changes

Response

We thankfully acknowledge your appreciation and positive comments regarding our manuscript.

Reviewer 3 Report

I have read through the revised version of the manuscript and it appears that authors have addressed most of my concerns. However, the manuscript was uploaded with a track-change marking and it was a bit confusing to read. I think the authors should give the manuscript one more round of careful reading for some mistakes. I have highlighted some mistakes and my comments are highlighted in yellow. I have a major comment on the "data analysis section" please address and correct accordingly. 

Comments for author File: Comments.pdf

Author Response

February 28, 2020 Dear Editor 

Thank you very much for handling the review process of our manuscript “Association of morphological, ecological and genetic diversity of Aerva javanica populations growing in the Eastern Desert of Egypt”. We thankfully acknowledge your comments on our manuscript.

We greatly appreciate the comments and suggestions raised by the Reviewers. All the changes made in response to the Reviewers’ comments were highlighted in yellow in the revised manuscript.

We greatly appreciate your time and consideration, and we hope that you would find our revised manuscript suitable now for publication. We look forward to receiving the positive decision from you soon.

Sincerely,

Hoida Zaki

Botany and Microbiology Department, Faculty of Science, South Valley University, 83523 Qena, Egypt

Email: [email protected]; [email protected]

Responses to the Reviewer’s Comments

Comments to the Author

REVIEWER 3

I have read through the revised version of the manuscript and it appears that authors have addressed most of my concerns. However, the manuscript was uploaded with a track-change marking and it was a bit confusing to read. I think the authors should give the manuscript one more round of careful reading for some mistakes. I have highlighted some mistakes and my comments are highlighted in yellow. I have a major comment on the "data analysis section" please address and correct accordingly. 

Response

We would like to give you special thanks for your careful reading of the whole MS and also for your meaningful corrections that helped us a lot to improve our manuscript significantly.

*. I have highlighted some mistakes and my comments are highlighted in yellow.

Response

We have revised the manuscript according to your suggestions (highlighted these in yellow in the new revised MS).

Comment 1- add "and" in peer-review-6507269.v1.pdf (row 97)

Response

It has been added in new revised MS row 93.

Comment 2- Fig. 1: A map of Egypt on which the sites of populations

Response

It has been changed to "Map of Egypt showing the sites of populations" in new revised MS row 109.

Comment 3- delete. Obviously soil samples will be collected from soil (ROW 125)

Response

The dear reviewer is correct. It has been deleted in new revised MS row 119.

Comment 4- use a comma before "a" (row 139)

Response

It has been added in new revised MS row 131.

Comment 5- Poorly constructed sentence. Make it concise and meaningful. (row 314)

Response

It has been rephrased in new revised MS row 308-310.

Comment 6- Just say, this was in confirmation with the UPGMA result. "corresponeded" is not a good choice. (row 348)

Response

It has been changed in new revised MS row 338.

Comment 7- I have a major comment on the "data analysis section" please address and correct accordingly.

 Response

It has been re titled to "Statistical Analysis" in new revised MS row 150.  In this section, we were presented all the statistical analysis that were used in our MS in a detailed methods.