Genetic Diversity of Five Broadleaved Tree Species and Its Spatial Distribution in Self-Regenerating Stands

Round 1

Reviewer 1 Report

The submitted manuscript compares genetic variation between mature trees and their natural regenerations for five economically and ecologically important broadleaved species. The study is scientifically sound and provides useful results for forest management. 

In particular, some methods are not adequately introduced and / or the results are not mentioned at all in the Results part. Comments related to this point:

- Which distance measure is Principal Coordinate Analysis based upon? This information has to be provided (easy to find in the GenAlEx documentation). By the way, I recommend use of the abbreviation PCoA as PCA is an established term for Principal Component (not Coordinate) Analysis.

- MLG is not introduced. Was it defined just by visually inspecting genotypes? If yes, this should be stated. It should also be introduced or at least written in full words (multilocus genotype) the first time of occurrence in the text. Concerning Populus tremula, the size of MLGs would also be of interest (maybe more interesting to see on a map than the structure results). 

- It is crucial to mention which diversity parameter was used. As shown in many papers, the allelic richness after rarefaction is the parameter least affected by population size. Given the asymmetric sampling sizes, this parameter is important. By the way, when looking at AR, it seems to me that there are no significant differences between mature trees and regeneration.

- Also, some parameters (e.g. LGP, first mentioned in l. 431) were used but not even mentioned in the Results and then commented in the Discussion. To me, the rationale behind the use of some measures like LGP is not clear.

Further comments:

l. 24: did P. tremula regenerate exclusively via root suckers. As stated here, it sounds like only vegetative reproduction occurred.

l. 25: "or even higher" sounds vague. Statistical significance is a prerequisite in order to make such statements.

l. 33: "are ensured" -> I would rather say "greatly depend on"

l. 50: put "and" before fellings

l. 102-104: What is meant here? When is the genetic diversity sufficient to secure sustainability and why?

l. 163: "an ongoing epidemic" -> I would say "the ongoing epidemic".

l. 176: "have been chosen" -> "were chosen"

l. 197: "SSR data analysis" -> "fragment length analysis"

l. 208: Put article "the" before linkage disequilibrium

l. 250: I would specify what "distribution" means here (i.e. ditribution/dispersal of points on the scatterplot)

Fig. 3: PCoA should be preferred (rather than PCA)

l. 267: replace "showing" by "shows" and I think that "genetic differentiation among individuals" fits better than "distibution" (which could mean many things...)

l. 280-281: which parameter was compared and differed significantly. It is important to mention this here! 

l. 282-283: well, a careful inspection of Table 3 shows that also for B. pendula natural regeneration has higher values than mature individuals. Please consider revising this statement.

l. 380: I don't agree that deviation from HWE indicates an ongoing change in gene frequencing. Actually, changes in the reproductive system (more or less inbreeding) do not affect allele frequencies, but only the genotype frequencies. Another cause for increased inbreeding coefficients and deviations from HWE are null alleles. Could this be the case here?

l. 447-449: I see a controversy here. Table 3 rather suggests an increase of heterozygosity, not homozygosity, as stated in this sentence. In addition, I would refrain from such a strong statement as no formal test for bottleneck was carried out (this would be possible using the software BOTTLENECK, but I think the number of markers used here is not adequate).

Author Response

Dear Reviewer,

Thank you for your valuable remarks, that helped us to improve the paper. We have added the missing info about the multi-locus genotype (MLG) identification into the materials and methods section. We have improved the results section and corrected the manuscript according to your remarks. The corrections and answers to each remark are given by bullet points after each of your comment below:

Comments and Suggestions for Authors

The submitted manuscript compares genetic variation between mature trees and their natural regenerations for five economically and ecologically important broadleaved species. The study is scientifically sound and provides useful results for forest management. 

In particular, some methods are not adequately introduced and / or the results are not mentioned at all in the Results part. Comments related to this point:

- Which distance measure is Principal Coordinate Analysis based upon? This information has to be provided (easy to find in the GenAlEx documentation). By the way, I recommend use of the abbreviation PCoA as PCA is an established term for Principal Component (not Coordinate) Analysis.

• Principal Coordinate analysis (PCoA) was based on Nei’s genetic distance matrix, this was indicated in the Materials and Methods section (lines 191-192) and in the description of Figure 3.
• The titles of each PCoA in Figure 3 have been corrected and figures with the revised titles were resubmitted as separate files.

- MLG is not introduced. Was it defined just by visually inspecting genotypes? If yes, this should be stated. It should also be introduced or at least written in full words (multilocus genotype) the first time of occurrence in the text. Concerning Populus tremula, the size of MLGs would also be of interest (maybe more interesting to see on a map than the structure results). 

• The abbreviation of MLG is now explained in the Materials and Methods section. The number of MLGs has been determined using GenAlEx software package. We have added this info to the Statistical Analysis section in the Materials and Methods. The distribution of clones (or sampled individuals with the same MLGs) of Populus tremula are presented in Figure 2.

- It is crucial to mention which diversity parameter was used. As shown in many papers, the allelic richness after rarefaction is the parameter least affected by population size. Given the asymmetric sampling sizes, this parameter is important. By the way, when looking at AR, it seems to me that there are no significant differences between mature trees and regeneration.

• The Results section has been revised following Reviewer’s recommendations. The genetic diversity parameters that were used to compare mature and juvenile cohorts have been listed in line 271. The paragraph in the Results section describing Table 3 has been corrected, and statistical significance of differences has been shown.

- Also, some parameters (e.g. LGP, first mentioned in l. 431) were used but not even mentioned in the Results and then commented in the Discussion. To me, the rationale behind the use of some measures like LGP is not clear.

• In the Materials and Methods section (lines 199-200), the Latent genetic potential (LGP) is now presented. The results of LGP calculation are now mentioned in the Results section (lines 290-291.
• The rationale of calculating this parameter is as follows: this measurement allows evaluation of the ability of a certain tree population to sustain its adaptability under changing climatic conditions. The genetic analysis of a certain population reveals its “operating genetic potential” (i.e., part of its genetic composition which guarantees the survival of the population under present realized conditions, which is analogous to the effective number of alleles), while the remaining part in this context is currently “latent”. This portion of genetic diversity is related to low frequency alleles in the population, which can nevertheless play a significant role for future adaptation under drastically changing environmental conditions, which can be of great importance for conservation practices (Aravanopoulos 2011, 2016). Therefore, a change, and especially a reduction, of the LGP may indicate a reduction of the overall adaptive capacity of the population. The rationale for calculating this parameter was shortly introduced in the paper (lines 291-293) “LPG is an important genetic parameter that reflects the aptitude of a population to preserve adaptability under changing environmental conditions [56,57].”.

Further comments:

1. 24: did P. tremula regenerate exclusively via root suckers. As stated here, it sounds like only vegetative reproduction occurred.

• The results of this study show the presence of both vegetative and sexual reproduction in tremula. The respective statement in the Abstract is now rephrased (line 24).

1. 25: "or even higher" sounds vague. Statistical significance is a prerequisite in order to make such statements.

• The statement is now corrected (the wording “or even higher” has been removed).

1. 33: "are ensured" -> I would rather say "greatly depend on"

• corrected

1. 50: put "and" before fellings

• corrected

1. 102-104: What is meant here? When is the genetic diversity sufficient to secure sustainability and why?

• The aim of the presented study (lines 101-105) was rephrased to “The study aimed at answering the question if current genetic diversity of the natural regeneration of five broadleaved tree species in disturbed areas is adequate to that of mature tree populations of the respective species, i.e., if sustainability of Lithuanian broadleaved tree populations (secured by a certain level of their genetic diversity) is not compromised”.

1. 163: "an ongoing epidemic" -> I would say "the ongoing epidemic".

• corrected

1. 176: "have been chosen" -> "were chosen"

• corrected

1. 197: "SSR data analysis" -> "fragment length analysis"

• corrected

1. 208: Put article "the" before linkage disequilibrium

• corrected

1. 250: I would specify what "distribution" means here (i.e. distribution/dispersal of points on the scatterplot)

• The explanation is now added (lines 241-242): “Distribution of points on the scatterplots (Figure 3) shows Nei’s genetic distances among the investigated individuals ”

Fig. 3: PCoA should be preferred (rather than PCA)

• corrected

1. 267: replace "showing" by "shows" and I think that "genetic differentiation among individuals" fits better than "distribution" (which could mean many things...)

• corrected

1. 280-281: which parameter was compared and differed significantly. It is important to mention this here! 

• Compared parameters are now listed in the Results section (line 271). Statistically significant diferences were found only in Na for excelsior and Q. robur. This is now explained in text (lines 275-276).

1. 282-283: well, a careful inspection of Table 3 shows that also for B. pendula natural regeneration has higher values than mature individuals. Please consider revising this statement.

• The sentence has been rephrased: “Higher effective number of alleles and allelic richness was found in mature cohorts, except for B. pendula and F. excelsior for which these parameters were higher in juveniles; however, for all investigated species the differences were statistically non-significant.” Lines 276-279.

1. 380: I don't agree that deviation from HWE indicates an ongoing change in gene frequencing. Actually, changes in the reproductive system (more or less inbreeding) do not affect allele frequencies, but only the genotype frequencies. Another cause for increased inbreeding coefficients and deviations from HWE are null alleles. Could this be the case here?

• We have checked the data for null allele presence. pendula, P. tremula and A. glutinosa showed no signs of null allele presence, while Q. robur showed signs of null allele presence in one of the eight investigated loci; the probability of null allele presence in that locus was 0.12. B. pendula, P. tremula and A. glutinosa didn’t show signs of inbreeding (Fis for these species showed negative values). The inbreeding coefficient for Q. robur was only slightly above zero (0.075).
• For excelsior the increased inbreeding might be due to the presence of null alleles as in this species there were four loci with increased probability of null alleles. However, F. excelsior is undergoing high selection pressure due to the ongoing ash dieback caused by Hymenoscypus fraxineus.
• Fourteen alleles detected in mature excelsior trees were missing in juveniles.There were 128 rare alleles (less than 5% frequency) in juveniles and only 61 rare alleles in mature cohort. We can’t compare allele frequencies in mature and juvenile cohorts due to different sampling effort, but it seems that there are some changes in allele frequencies.

1. 447-449: I see a controversy here. Table 3 rather suggests an increase of heterozygosity, not homozygosity, as stated in this sentence. In addition, I would refrain from such a strong statement as no formal test for bottleneck was carried out (this would be possible using the software BOTTLENECK, but I think the number of markers used here is not adequate).

• The statement is now corrected: “However, during the last decade of the XX^th century some young, naturally regenerated F. excelsior stands emerged, which sanitary condition over the last five years were more or less satisfactory [76]. This brings some hope for survival of Lithuanian F. excelsior populations despite the ongoing epidemic.” Lines 446-449.

Submission Date

28 December 2022

Date of this review

19 Jan 2023 21:43:06

Reviewer 2 Report

The Authors presented an interesting article about the genetic dynamics in Lithuanian populations of five natural forest tree species. Facing global climate changes, in some European countries, the broadleaved tree species are more threatened than conifers. Based on their potential of plasticity and their genetic differentiation at the intra- and interpopulation levels, the broadleaved species may better encounter some environmental discrepancies (pathogen diseases and pest outbreaks, or windfall) than coniferous tree populations. The novelty of the paper consists of a simultaneous study of the five broadleaved species, i.e. European oak, silver birch, black alder, European ash, and European aspen distribution based on genetic polymorphism in chosen managed stands of Eastern Europe (Lithuania).

The manuscript was prepared correctly. The title, abstract, and keywords clearly reflect the paper's content. The introduction explicitly presents the problem. The methodology and analysis of results rather don't raise any objections except for the small number of microsatellite loci applied to the study (only eight markers per species). Nevertheless, the results obtained in the carried study in general suggested the proper transmission of the genes from the mother stands to the progeny. The reader does not have information about the scattered or continuous distribution of each species in the investigated area, which can influence the pollen flow from the neighbor stands. Even if based only on one stand per species, the study suggests that all investigated populations will remain and survive the changing climate and anthropogenic activity in Lithuania.

Discussion and results are justified.

References are complete and adequate.

About Presentation:

Length is commensurate with the paper's content. Moderate English changes are required.

Description of Results could be improved:

The quality of Table 1 needs to be amended.

Line 213 - Check the spelling of “Stjudent’s T test” and change it to “Student’s …”

Line 220 – remove an extra “]” at the end

Line 224 – explain the abbreviation “MGL” when the first time cited, and not later (line 267)

Line 254 – Add a letter “A” to (Figure 3), corresponding to ash species. The same remark for other species' descriptions concerning the PCoA plot (Figure 3B, etc.). By the way, why the title of each plot is “PCA” and not “PCoA”?

Line 256 – please explain the term of “circular plots” for European aspen grouping. Do you rather mean “quasi equal distribution of points (genotypes) in each quadrant”? 

Author Response

Dear Reviewer,

Thank you for the valuable comments that helped us to improve the paper. All your suggestions were included in the paper. The results section was improved, also some small changes were made in the Materials and methods part. Your comments are answered by bullet points after each remark below:

Description of Results could be improved:

The quality of Table 1 needs to be amended.

• corrected

Line 213 - Check the spelling of “Stjudent’s T test” and change it to “Student’s …”

• corrected

Line 220 – remove an extra “]” at the end

• corrected

Line 224 – explain the abbreviation “MGL” when the first time cited, and not later (line 267)

• Description on how multi-locus genotypes were determined and MLG abbreviation explained in lines 188-189.

Line 254 – Add a letter “A” to (Figure 3), corresponding to ash species. The same remark for other species' descriptions concerning the PCoA plot (Figure 3B, etc.). By the way, why the title of each plot is “PCA” and not “PCoA”?

• Letters designating particular species PCoA next to Figure 3 are now added.
• The titles of each PCoA in Figure 3 have been corrected and figures with the revised titles were resubmitted as separate files.

Line 256 – please explain the term of “circular plots” for European aspen grouping. Do you rather mean “quasi equal distribution of points (genotypes) in each quadrant”? 

• The term circular plot refers to circular sampling plots, as presented in principal sampling scheme in Figure 1. Figure 2 was drawn using sampling coordinates and principal sampling scheme of tremula individuals.

Submission Date

28 December 2022

Date of this review

14 Jan 2023 17:37:42