The Possibility of Regenerating a Pine Stand through Natural Regeneration

Round 1

Reviewer 1 Report

Przybyski and collaborators report the results of a study on the potential of natural regeneration of Scots pine in old forest stands in Poland. This the second time that I review this paper. I still have some concerns that I want to share with the authors.

• 82-87: Molecular markers are gene alleles for which associations with quantitative traits or environment variables are sought. The objective is essentially to identify gene variants that are linked with adaptive traits or are under natural selection. It is not clear the way it is presently written.
• 93-94: What do you mean by differences detected in the gene pool using SSRs can lead to severe adaptive consequences? Sorry, I do not understand.
• 115: What do you mean by natural regeneration incidents? Do you mean occurrence or incidence?
• 116-117: SSRs are neutral markers. They tell nothing about natural selection and adaptation. They just give us an estimate of the level of genetic diversity and of the population structure.
• 222-223: In the equation, I guess that location is represented by s_i, but you do not define it in the following sentence. Moreover, you talk about the number of cones per tree (pi), but it is not considered in the ANOVA model (equation). Your model does not make sense. The s_i should represent the location i and e_ij the number of cones per tree, knowing that 50 trees were sampled in each location.
• 230: You should first present the results of your ANOVA. Are there significant differences between the locations? If ii is so, what is the p-value? Moreover, my understanding is that you counted the number of cones only in 2018. Was the 2018 cone production representative? It would have been much better to collect data over a couple of years to obtain a more accurate information on cone production of each tree
• 250-252: Please provide information on how were the germination capacity and germination energy estimated (supplementary material). Most of the readers do not know the ISTA standards and do not have the time to read them.
• On line 160, you write that seeds were collected from 11 trees in each location. However, you report in Table S1, data for 13 and 12 trees, for Wi and W locations, respectively.
• 364-365: You say that the obtained value of null alleles did not significantly affect the obtained results of genetic variability. What statistical test have you carried out to come to this conclusion, and what is the p-value?
• 378: Please clearly define how natural regeneration was estimated. Is this the number of stems shorter than 200 cm that was counted per 400 m² for instance? It is not clearly defined in the document unless I missed something.

What do you mean by an additional correlation? Is this the only one that is statistically significant?

• 383: This high correlation (r=0.94) is not surprising, knowing that the number of seedlings was estimated using the number of cones (L. 262-264).
• 386: It is a clear indication that the individuals in most locations are highly related.
• 386-389: I am lost here. The correlation existing between natural regeneration and seedlings (Fig. 4) is equal to 0.24. This correlation is still positive, although nothing is said about its significance.
• 482: the excess of homozygotes in a young population cannot be attributed to natural selection. Natural selection plays its role when it eliminates the excess of homozygous individuals during the stand’s development. It has been reported in many studies in the past, an excess of homozygotes being observed young stands and an excess of heterozygotes in the same stand among the trees that have survived over the years.
• 493-494: I am not sure about what you call reproductive potential. I think that this negative relationship is true for the number of full seeds per cone but not for the number of seedlings per 50 trees, when I compare the results of all the locations.
• 495-496: I do not see that correlation with Ne when I look at Fig. 4.
• 508-511: You must present the results of a statistical test to support this statement. Based on the numbers presented in Table 4, I am afraid that there is no significant differences among the locations.
• 560-566: Are these correlations statistically significant? You have not indicated in the manuscript that they were significant, unless I missed something. If they are not significant, I am afraid that you cannot make this conclusion. Moreover, when I look at Table 4, I am quite sure that the locations are not significantly different for any of the parameters, except for Fis.

Author Response

Thank you for your review of the publication. I can assure you that all the reviews received have significantly improved the quality of the results presented, the present work is also after the linguistic correction carried out by MDPI. The suggestions made in this review have been incorporated into the text as described below.

82-87: Molecular markers are gene alleles for which associations with quantitative traits or environment variables are sought. The objective is essentially to identify gene variants that are linked with adaptive traits or are under natural selection. It is not clear the way it is presently written.

The sentence has been corrected according to the reviewer's suggestions.

93-94: What do you mean by differences detected in the gene pool using SSRs can lead to severe adaptive consequences? Sorry, I do not understand.

The sentence has been corrected according to the reviewer's suggestions.

115: What do you mean by natural regeneration incidents? Do you mean occurrence or incidence

Thank you for your suggestion on an important part of the text. The use of the word incidence was due to a lack of feeling for the intent of the word, which is a shortcoming of people for whom English is not the first language. The current sentence probably reads as the authors intended.

116-117: SSRs are neutral markers. They tell nothing about natural selection and adaptation. They just give us an estimate of the level of genetic diversity and of the population structure.

The results presented, as well as the works [Bastien, Alía 2000, Grivet et al. 2009], confirm the usefulness of the microsatellite markers used to study genetic variability and structure at the neutral DNA level. They allow the evaluation of the correlation of genetic variation with the variation of phenotypic traits, whose positive results may indicate the close position of specific microsatellite loci with genomic regions under selection. Neutral DNA variation analysed in populations allows the study of genetic background to distinguish the influence of demographic processes and natural selection acting on population variation.

Bastien C., Alía R. 2000. What might be useful measures of genetic variability for adaptive traits within populations of Scots pine? Invest. Agr.: Sist. Recur. For. 9: 97−110

Grivet D., Sebastiani F., González−Martínez S. C., Vendramin G. G. 2009. Patterns of polymorphism resulting from long−range colonization in the Mediterranean conifer Aleppo pine. New Phytologist 184 (4): 1016−1028

222-223: In the equation, I guess that location is represented by si, but you do not define it in the following sentence. Moreover, you talk about the number of cones per tree (pi), but it is not considered in the ANOVA model (equation). Your model does not make sense. The si should represent the location i and eij the number of cones per tree, knowing that 50 trees were sampled in each location.

Unfortunately, there was a mistake in the model description, "si" should have been instead of "pi". However, the model and all other parts of the description are correct. The model was used to compare the average number of cones per one dominant tree between studied locations.  The significant differences between the average number of cones per dominant tree indicate that cones yield for 50 trees will also be statistically different because cone yield is a mean number of cones multiplied by the number of trees. So, in our model yij it is the number of cones observed at i-th location on j-th sample tree, si it is the average number of cones per one dominant tree in i-th location and eij is the normally distributed independent random errors, or in another word, the difference between an observed value yij and the average number of cones per one dominant tree in i-th location. In the last version of the model, the si was replaced by μi that is more commonly used in models as a symbol of an average of a group level (in our case groups it's studied locations).

Some references about one way ANOVA models application:

Montgomery, D. C., & Runger, G. C. (2010). Applied statistics and probability for engineers. John Wiley & Sons.

Ostertagová, E. , & Ostertag, O. (2013). Methodology and Application of Oneway ANOVA. American Journal of Mechanical Engineering, 1(7), 256-261.

 

230: You should first present the results of your ANOVA. Are there significant differences between the locations? If ii is so, what is the p-value? Moreover, my understanding is that you counted the number of cones only in 2018. Was the 2018 cone production representative? It would have been much better to collect data over a couple of years to obtain a more accurate information on cone production of each tree

The introduction to the results was supplemented by information on the ANOVA analysis. The differences between the locations are significant, as shown in Figure 3. As suggested by the reviewer, the caption of Figure 3 has been supplemented with information on the post-hoc test performed. The authors of the publication suggest not to include another table with statistical differences between locations, if it is visible in Figure 3. Regarding the comment on one year of observation (2018), we note that all factors described in the publication were evaluated in one year, which allows us to perform the data correlation under uniform ecological conditions.

250-252: Please provide information on how were the germination capacity and germination energy estimated (supplementary material). Most of the readers do not know the ISTA standards and do not have the time to read them.

In accordance with Reviewer remark we added information on how germination energy and capacity were calculated.

On line 160, you write that seeds were collected from 11 trees in each location. However, you report in Table S1, data for 13 and 12 trees, for Wi and W locations, respectively.

Thank you very much and I am very impressed with your perceptiveness. I made an (uncorrected) error by including data with information from trees that serve as reserves for selected stands. This data only increases the accuracy of the estimates for the two populations. Scientific accuracy requires that this information be included in the material section, which it was.

364-365: You say that the obtained value of null alleles did not significantly affect the obtained results of genetic variability. What statistical test have you carried out to come to this conclusion, and what is the p-value?

We agree with the reviewer that a p-value should be provided when reporting statistical significance, so the sentence has been removed from the publication text. Analysis of the presence of null alleles using the software Micro-Checker [35] is a commonly accepted method of analysis for this phase of the study. The results indicate the presence of null alleles at only one locus, which we consider a success, as previous international studies of pine SSR markers [Dabrowski et al. 2013] indicate a high frequency of null alleles.

Dąbrowski N. J., M. Pilot M., M. Kruczyk M., M. Żmihorski M., Umer H. M., Gliwicz J. (2013). Reliability assessment of null allele detection: inconsistencies between and within 2 different methods. Mol. Ecol. Resour. Mar,14(2):361-73. doi: 10.1111/1755-0998.12177.

378: Please clearly define how natural regeneration was estimated. Is this the number of stems shorter than 200 cm that was counted per 400 m2 for instance? It is not clearly defined in the document unless I missed something.

It was explained in the chapter 2.3. Species diversity sampling: : “Shorter trees and shrubs were counted in circular experimental plots (Figure 2) with a radius of 5.64 m (area: 100 m²)”

What do you mean by an additional correlation? Is this the only one that is statistically significant?

I agree with the reviewer the word additional is unnecessary in the sentence and it has been removed. The correlation value in the sentence is given and is 0.57

383: This high correlation (r=0.94) is not surprising, knowing that the number of seedlings was estimated using the number of cones (L. 262-264).

I agree with the reviewer.

386: It is a clear indication that the individuals in most locations are highly related.

Genetic analyses were performed on seedlings in which external pollen transfer played an important role. According to the authors, natural selection mechanisms occurring in the stands are also of great importance, although to varying degrees. Data on the relationship of the studied stands are available in the publication by Przybylski et al. (2021), which I recommend you to read.

Przybylski P., Mohytych V., Rutkowski P., Tereba A., Tyburski Ł., Fyalkowska K. 2021. Relationships between Some Biodiversity Indicators and Crown Damage of Pinus sylvestris L. in Natural Old Growth Pine Forests. Sustainability 13,1239, https://doi.org/10.3390/su13031239.

   

386-389: I am lost here. The correlation existing between natural regeneration and seedlings (Fig. 4) is equal to 0.24. This correlation is still positive, although nothing is said about its significance.

The sentence has been corrected to guide the reader logically through the results obtained according to the authors' concept.

482: the excess of homozygotes in a young population cannot be attributed to natural selection. Natural selection plays its role when it eliminates the excess of homozygous individuals during the stand’s development. It has been reported in many studies in the past, an excess of homozygotes being observed young stands and an excess of heterozygotes in the same stand among the trees that have survived over the years.

I agree with the reviewer about general knowledge. The sentence on selection processes has been changed to avoid direct statements. However, the results presented should be considered unique because the analyses were conducted in pine stands that are nearly 200 years old and probably natural in character. Such work is rare, and in the context of previous articles (Przybylski 2021), the question of the occurrence of natural selection in them seems logical.

493-494: I am not sure about what you call reproductive potential. I think that this negative relationship is true for the number of full seeds per cone but not for the number of seedlings per 50 trees, when I compare the results of all the locations.

We agree with Reviewer that reproductive potential should be considered as the number of viable seeds per cone (cf. Andrew J. Trant, Ryan G. Jameson & Luise Hermanutz (2018) Variation in reproductive potential across a multi-species treeline, Arctic, Antarctic, and Alpine Research, 50:1, DOI: 10.1080/15230430.2018.1524191). In our study, we found that the highest inbreeding coefficient was in the population with the lowest regeneration potential (Wiersze population: 14 viable seeds per cone). For this reason, we used the term "reproductive potential" in this sentence.

495-496: I do not see that correlation with Ne when I look at Fig. 4.

The sentence was deleted from the text.

508-511: You must present the results of a statistical test to support this statement. Based on the numbers presented in Table 4, I am afraid that there is no significant differences among the locations.

The sentence has been corrected to suggest no statistical significance between Ne (as this test is not available). However, statistical differences between populations and significant inbreeding between populations were demonstrated in Tables 4 and 6, which of course has implications for the genetic parameters of the studied stands.

560-566: Are these correlations statistically significant? You have not indicated in the manuscript that they were significant, unless I missed something. If they are not significant, I am afraid that you cannot make this conclusion. Moreover, when I look at Table 4, I am quite sure that the locations are not significantly different for any of the parameters, except for Fis.

The value of the correlation between the data is shown in Fig. 4. All the parameters compiled have strong correlations. The conclusion presented does not refer to Table 4, where the general genetic characteristics of the site are presented and the statistical significance analysis is performed for the parameter Fis. I thank the reviewer for pointing out the caution in formulating research conclusions, but I believe it is warranted in this case.

Author Response File: Author Response.docx

Reviewer 2 Report

Drodzy Autorzy,
Proszę uważnie przeczytać moje komentarze i poprawić swój rękopis:

Line 43-44: Please add a link to the publication or electronic resource with "91D0 Natura 2000" and "91T0 Natura 2000" codes.
Line 54; 57-58: The "State Forests (lasy.gov.pl)" link must be in the references and be formatted according to the requirements of the journal.
Line 74-77: Please add a link to published data confirming this information or remove this sentence from the text.
Line 82-84: The sentence is not correct. Molecular markers cannot influence traits. They are just tools for detecting genetic differences. If you mean the relationship between the studied traits and molecular markers, then rewrite this sentence correctly.
Line 86-87: Please change "Genetic markers of pine have been studied for survival [12], growth [13], resistance to drought [14], and fungi and insects [15]" to "Genetic markers were used for studyind survival [12], growth [13], resistance to drought [14], fungi and insects [15] in pine."
Line 90: Add a description of the nature of the SSR loci and how the markers work. Explain what null alleles are.
Line 105: Please remove the underline.
Line 116-117: Please delete the sentence "Additional SSR analyses of the occurring natural regeneration allow us  to determine the direction of selection and its adaptive potential."
Line 151: Please remove the extra point.
Line 151-154; 229-270: I have great doubts about the correctness of using this method of counting pine cones. Explain why you used the method of the 40s of the 20th century? I believe that your results are very inaccurate. Prove otherwise, or delete the pine cone counts altogether.
Lint 200: Why were these markers chosen for analysis? Why didn't you choose other markers? Why did you choose five markers and not more? Write me a detailed answer to these questions. Better yet, if you edit the text in such a way that these questions do not arise from the reader.
Table 2: You write about polymorphic markers (line 187), but only give one value of product size. Present, please, size ranges among samples or product lengths for each allelic variant.

Z najserdeczniejszymi życzeniami,
Dr. Oleg Aleksandrow

 

Author Response

Dziękujemy za przeprowadzoną recenzję, wniosła ona cenne uwagi i poprawiła publikację. Poniżej zamieszczam szczegółowe odpowiedzi na uwagi do tekstu. 

Line 43-44: Please add a link to the publication or electronic resource with "91D0 Natura 2000" and "91T0 Natura 2000" codes.

Link has been added

Line 54; 57-58: The "State Forests (lasy.gov.pl)" link must be in the references and be formatted according to the requirements of the journal.

The link has been corrected.

Line 74-77: Please add a link to published data confirming this information or remove this sentence from the text.

The sentence has been deleted, the data has not been published.

Line 82-84: The sentence is not correct. Molecular markers cannot influence traits. They are just tools for detecting genetic differences. If you mean the relationship between the studied traits and molecular markers, then rewrite this sentence correctly.

The sentence has been corrected according to the reviewer's suggestions.

Line 86-87: Please change "Genetic markers of pine have been studied for survival [12], growth [13], resistance to drought [14], and fungi and insects [15]" to "Genetic markers were used for studyind survival [12], growth [13], resistance to drought [14], fungi and insects [15] in pine."

The sentence has been corrected according to the reviewer's suggestions.

Line 90: Add a description of the nature of the SSR loci and how the markers work. Explain what null alleles are.

The sentence has been corrected according to the reviewer's suggestions.

Line 105: Please remove the underline.

The sentence has been corrected according to the reviewer's suggestions.

Line 116-117: Please delete the sentence "Additional SSR analyses of the occurring natural regeneration allow us  to determine the direction of selection and its adaptive potential."

The sentence has been corrected according to the reviewer's suggestions.

Line 151: Please remove the extra point.

The sentence has been corrected according to the reviewer's suggestions.

Line 151-154; 229-270: I have great doubts about the correctness of using this method of counting pine cones. Explain why you used the method of the 40s of the 20th century? I believe that your results are very inaccurate. Prove otherwise, or delete the pine cone counts altogether.

Sometimes we have to use solutions that could be considered obsolete. In the case of our research, the cone counting method proved to be the best method due to the location where our study was conducted (Kampinoski National Park). We thought of using UAVs (drones) but we did not get permission. National Park Service denied us permission because of the noise that UAVs produce. The park authorities felt that the use of drones would have a negative impact on the fauna of the park. In our opinion, these arguments were ridiculous, but we had to abide by these conditions.

Regarding the inaccuracy of our counting method, we have to disagree. After counting from the forest floor, we had an arborist collect all the cones from the selected trees. Our estimate proved to be much more accurate than we thought (see table):

Population	Number of cones (estimated)	Number of cones (collected)
Czerwińskie Góry	999	820
Granica	1752	1056
Sieraków	2103	899
Wiersze	2560	1603
Wilków	1506	1103

 

Considering that the estimation was done on 50 trees and the collection was done on 11 trees with the largest amount of cones, you can see that our method (yes, from the 40's of the 20th century) was the best method we had (in the case of no permission for UAV flight), except for cutting the trees, but as you know, that is prohibited in the strict nature reserves.

Lint 200: Why were these markers chosen for analysis? Why didn't you choose other markers? Why did you choose five markers and not more? Write me a detailed answer to these questions. Better yet, if you edit the text in such a way that these questions do not arise from the reader.

The SSR markers selected were identical to previous studies of the maternal generation of the populations studied. The information has been included in the text, thank you. The homogeneity of the markers allows the correlation of results obtained at different stages of the projects. The kit used contains polymorphic loci that can be linked by multiplex PCR. This measure ensures maximum variability of results while minimising costs.

Table 2: You write about polymorphic markers (line 187), but only give one value of product size. Present, please, size ranges among samples or product lengths for each allelic variant.

The reviewer's comment has been taken into account in Table 2.

z serdecznymi pozdrowieniami

Paweł Przybylski z współautorami

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Dear Paweł and coauthors,

I have read your responses to my comments and I am quite satisfied with them. I believe that the manuscript has become better after the additions and corrections.

Let me just allow myself to make another small remark related to the design of links:

Lines 46-47. Thank you for adding the link. However, this link should be made according to the instructions for authors (see clause 9-10 for references at https://www.mdpi.com/journal/forests/instructions). In the text, this link should be presented as [1].

Lines 56-57. In the text, this link should be presented as [3]. In the References part, it should be as "3. Title of Site. Available online: URL (accessed on Day Month Year)."

Hope it won't be too difficult for you to make these corrections.

Best regards

Author Response

Dear Reviewer

The comments on improving the linkage of information available online have been fully addressed and the text has been revised as suggested.
Many thanks for the improvement of the text.

Best regards

Paweł Przybylski

Author Response File: Author Response.docx

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Dear Authors,

I received the manuscript entitled ''Genetic variability of pine seedlings with respect to its occurrence in old-growth pine forests''. Overall, the paper has weaknesses and shortcomings that strongly limit its suitability for publishing in the journal Forests. The proposed paper is marginal in scope, addressed only to those readers interested in the studied species, on the regional level. In my opinion, the paper is more suitable for Q3 or Q4 journals.

In addition, the number of microsatellites for the minimum necessary to correctly detect the genetic diversity is ten. In this paper, authors used only five microsatellite markers. Therefore, the results of this research are not likely enough to confirm the tested hypothesis. For this reason, a major revision cannot resolve the issue.

Reviewer 2 Report

This is an interesting study on potential natural regeneration of Scots pine stands and its genetic diversity. The study has been carried out in protected areas of a National Park in Poland. I have several concerns that I have listed below:

Material & Methods

1. 116-121: Some clarifications are needed. 1) It is not clear whether seed traits were estimated using cones collected on a subset of 11 trees among the 50 trees sampled in each of the five stands. This should be clarified. 2) Even if it were indicated that germination energy and capacity were determined in accordance with the ISTA standards, a short description of how the tests were carried out would be welcome. 3) I do not understand how a sample of 1000 seeds can help determine average cone weight, total yield of seeds from cones and the average number of seeds per cone. Please rewrite.
2. 132: You should define that you call a seedling. Is it a Scots pine shorter than 20 cm, for instance?
3. 135: More details about the procedure of DNA extraction should be provided. Were the needles put in liquid nitrogen and then ground, for instance?
4. 157: No results of PCoA are presented in this paper.

Results

Figure 3 caption: The statistical used should be indicated.

3. 177: I seems to me that Gr does not differ from Wi and S, based on the letter used in Fig. 3.
4. 186-196: If an ANOVA was carried out for the number of cones , why was it not done for seed traits? The average number of seed per cone appears to differ among locations.

Table 4: Add what * means.

Table 5: Add in a footnote what are those references in the Table and add the references in the reference list.

1. 295: What do you call natural regeneration? You have never indicated how it was estimated. Field observations are not enough. It should be determined using a scientific protocol.
2. 297-298: It is just a correlation, and not necessarily cause and effect. Moreover, based on Fig. 4, there are no significant relationships between the traits except for Na and Ne with the Shannon Index and between Ho and Fis.

Discussion

1. 331-332: Data or references should support that statement.
2. 372-373: The excess of homozygotes is not the result of natural selection. Natural selection acts in eliminating homozygous individuals over time to leave an excess of heterozygotes at mature stage. To demonstrate that natural selection is playing a role in your stands, you would have to compare Fis values at both the seedling and the mature stages in your locations as others did.
3. 392-395: I am afraid that you do not have the data to support your statement. Potential number of seedlings is not real regeneration data and based on Table 4, there is no significant differences among locations for Ne.