Phyllosphere Fungal Diversity and Community in Pinus sylvestris Progeny Trials and Its Heritability Among Plus Tree Families

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

The manuscript entitled “Phyllosphere Fungal Mycobiome can Affect Pinus sylvestris Growth and Its Heritable Between Similar Host Genotypes.” addresses an important and urgent topic in forest ecology and tree breeding. The study is based on a robust dataset and explores the interactions between fungal communities, genotypes, and environmental factors. However, I identified several issues that need to be addressed.

Comments

• The title contains grammatical error: please change 'Its Heritable Between Similar Host Genotypes' to 'and Its Heritability among Similar Host Genotypes'.
• Regarding the Materials and Methods, subheading 2.2. please Add missing PCR details: cycles, annealing temperature, replicates.
• Regarding the results, separate mere description from interpretation. Some parts read like a discussion.
• In lines 656-658 of the Results section, the interpretation of the heritability test is statistically incorrect. The statement "p = 0.979 shows heritability to the mycobiome population" is misleading. The p-value of 0.979 is too high and does not indicate any statistically significant difference, meaning there is no evidence of heritability. Furthermore, although H² = 0.23 indicates a moderate proportion of variance, this estimate is not statistically supported due to the non-significant p. This section should be carefully rewritten to avoid drawing unsupported genetic inferences and to clarify that environmental factors are likely the dominant factors influencing fungal community structure.
• Conclusion is too long; shortened to key findings and limitations.

Author Response

Comment 1: The title contains grammatical error: please change 'Its Heritable Between Similar Host Genotypes' to 'and Its Heritability among Similar Host Genotypes'.

Response 1: Thank you for this observation, corrected in the manuscript, lines 2-4.

 Comment 2: Regarding the Materials and Methods, subheading 2.2. please Add missing PCR details: cycles, annealing temperature, replicates.

Response 2: We appreciate this suggestion, and we included in the materials and methods, section 2.2 the details requested about the PCR process, in lines186-192

Comment 3: Regarding the results, separate mere description from interpretation. Some parts read like a discussion.

Response 3: Thank you for your comments. The results and discussion section were corrected and improved.

 Comment 4: In lines 656-658 of the Results section, the interpretation of the heritability test is statistically incorrect. The statement "p = 0.979 shows heritability to the mycobiome population" is misleading. The p-value of 0.979 is too high and does not indicate any statistically significant difference, meaning there is no evidence of heritability. Furthermore, although H² = 0.23 indicates a moderate proportion of variance, this estimate is not statistically supported due to the non-significant p. This section should be carefully rewritten to avoid drawing unsupported genetic inferences and to clarify that environmental factors are likely the dominant factors influencing fungal community structure.

Response 4: Thank you for your observation. We apologize for the confusion and misunderstanding. In order to clarify where that p-value comes from and how it was interpreted as an indicator in the evaluation of potential heritability, we corrected accordingly the materials and methods, which explains how to interpret results of heritability, please see lines 314-319, in addition adjustments were made in section 4.3 Heritability (discussion), in lines 759-771.

Regarding heritability in the broad sense, the word confirms in line 658 is altering the meaning of the result, so a modification was also made in line 770-771.

 Comment 5: Conclusion is too long; shortened to key findings and limitations.

Response 5: Thank you for your comments, we followed your observations and improved it in the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

This study aims to investigate the influence of the fungal mycobiome on the growth of Scots pine, as well as its heritability among similar host genotypes.  Experimental trials were conducted in four locations across Estonia. Twelve different genotypes were included in the study, but only one of these was present in all locations. The fungal mycobiome was analysed using PacBio third-generation sequencing technology. Alpha diversity was estimated using the Shannon index. PCoA and NMDS were employed to analyse and visualise beta diversity. ANOSIM and PERMANOVA were employed to identify factors explaining significant differences in mycobiome composition and to test for variations in fungal communities between locations and pine genotypes. The results show that the structure and composition of needle and shoot tissues differ. Location influences the composition and structure of the mycobiome. Evidence of heritability was seen in the composition of saprotrophic fungi in the seed orchard and progeny trial trees.

 

General comment:

This study falls within the scope of the Forests journal. As not all genotypes are present in all sampling locations, only partial conclusions can be drawn. Several sections require improvement. The introduction and discussion sections, in particular, should be carefully revised to improve clarity and avoid repetition. Some parts of the discussion section provide detailed descriptions of the results, referencing figures that should not be reported in this section. Rewriting these sections would improve the quality of the manuscript, which would benefit from being shorter. The grammar and English style also need to be revised for clarity.

Specific comments:

L3: Please change 'heritable' to 'heritability'.

L42–43: Please check the grammar. It seems that some words are missing.

L105–112: Could this part be eliminated from the introduction, or could it be made more concise? It would be more appropriate for the 'Discussion' or 'Conclusions' section.

L118: Please change 'with' to 'on'.

L124: Please change 'are' to 'carried out'.

L126: Please check the temperature values. These values should also be checked in Table 1.

L133–136: Please check the grammar. The description is unclear.

L151–152: Remove the bold style.

L165–169: Please check the grammar to improve clarity.

Lines 172–173. Please check the grammar.

L178: This sentence is unclear. Are some words missing?

L197–198: Please check the grammar.

L243–245: Improve the clarity of the sentence.

L260–262: Please check the grammar.

L269–275. Please improve the grammar.

L290–293. Please improve the grammar.

L297–300. The first result is that most OTUs cannot be functionally assigned. In this section, you are referring to taxonomy. Please explain. Also, please check the percentages; the sum is not 100%.

Figure 2. Add the letter 'A' at the beginning of the sentence in the legend. Improve the resolution of the figure.

Figure 3: Please provide a detailed legend. P-value, description of boxes, etc.

L518–520. This sentence repeats the previous one.

L541–544. This part describes the results whereas L630–645 contains the actual discussion of the results.

L647–662. This section describes the results whereas L630-645 contains the actual discussion of the results.

 L713. Please delete ‘in alpha and beta diversity analyses.

Comments on the Quality of English Language

The grammar and English style also need to be revised for clarity.

Author Response

General comment:

This study falls within the scope of the Forests journal. As not all genotypes are present in all sampling locations, only partial conclusions can be drawn. Several sections require improvement. The introduction and discussion sections, in particular, should be carefully revised to improve clarity and avoid repetition. Some parts of the discussion section provide detailed descriptions of the results, referencing figures that should not be reported in this section. Rewriting these sections would improve the quality of the manuscript, which would benefit from being shorter. The grammar and English style also need to be revised for clarity.

Response-Introduction: Thank you for the insightful observation regarding the length and structure of the introduction. In response, we have revised the section to improve clarity and focus, condensing the background into four concise paragraphs. The updated version appears in the manuscript at lines 38-76; 102-107.

Response-experimental design-methods:

Thank you for your feedback. For alpha diversity, nonparametric tests were performed for comparison and correlation tests because the data did not meet the assumptions of homogeneity and normality. In tour research generalized linear model was used, its allow the inclusion of categorical factors, genotype, and continuous covariates (sum of effective temperature, average temperature, location) under a flexible framework. The dependent variable was the Shannon index. For beta diversity, calculations were performed as a PERMANOVA in which the fixed factor was location and the random factor was genotype. It has clarified in material and methods in lines 263-265 and lines 277-278.

Regarding the analysis of unbalanced data after sampling and sequencing (which can occur), we used robust statistical approaches for unequal sample sizes, such as the Kruskal-Wallis test and Bonferroni-adjusted tests for alpha diversity, Spearman correlation models, and Generalized Linear Models. For beta diversity, in addition to determining the assumptions of normality and homogeneity for alpha diversity, nonparametric tests (Kruskal-Wallis with Bonferroni correction, Spearman correlations) and Generalized Linear Models (GLM) were applied. For beta diversity, PERMANOVA and ANOSIM were used to assess differences between factors, while PERMDISP was used to confirm that the statistically significant effects found in the study were not influenced by dispersion heterogeneity. PERMDISP had already been calculated and is included in the supplementary information for each of the analyses performed. These methods allowed for the inclusion of all available observations without data imputation. See the clarification in the manuscript lines 172-179, 232-248.

Response – Discussion: We have revised the discussion to remove detailed descriptions of results and references to figures. The "Implications for Breeding" section now focuses on interpretation and practical implications, while specific data and functional group analyses remain in Results and Supplementary Information. See revised text on lines 283-28, 475-476, 698-734.

Specific comments:

 Comment 1: L3: Please change 'heritable' to 'heritability'.

Response 1: Thank you for this observation, corrected in the manuscript, line 3.

 Comment 2: L42–43: Please check the grammar. It seems that some words are missing.

Response 2: Thank you for the suggestion, it was corrected on the manuscript we made modifications on the introduction, lines 38-46.

Comment 3: L105–112: Could this part be eliminated from the introduction, or could it be made more concise? It would be more appropriate for the 'Discussion' or 'Conclusions' section.

Response 3: Thanks for the comment. Because this information is part of the justification for our research, we decided to keep it in the introduction, but made it clearer and more concise. The modifications are in the manuscript on the lines 102-107.

Comment 4: L118: Please change 'with' to 'on'.

Response 4: Thank you for this observation, corrected in the manuscript, line 114.

 Comment 5: L124: Please change 'are' to 'carried out'.

Response 5: Thank you for this observation, corrected in the manuscript, line 121-122 .

 Comment 6: L126: Please check the temperature values. These values should also be checked in Table 1.

Response 6: Thank you for this observation. We have checked and corrected the manuscript, line 123-124 and table 1.

 Comment 7: L133–136: Please check the grammar. The description is unclear.

Response 7: Thank you for your valuable comment. We have revised the sentence accordingly, and the correction has been implemented in the manuscript at 131-179.

Comment 8: L151–152: Remove the bold style.

Response 8: Thank you for your observation, the correction has been implemented in the manuscript.

 Comment 9: L165–169: Please check the grammar to improve clarity.

Response 9: Thank you for your comment. We have revised the paragraph to improve clarity and grammar. The updated version now clearly states that mother tree samples were included specifically for heritability analyses and appears in the manuscript at lines 166-179.

 Comment 10: Lines 172–173. Please check the grammar.

Response 10: Thank you for your comment. We have revised the sentence to improve grammar and clarify that each tissue type was processed in separate Eppendorf tubes. The updated version appears in the manuscript at lines 181-184.

 Comment 11: L178: This sentence is unclear. Are some words missing?

Response 11: Thank you for your comment. We have revised the sentence to correct the grammar and clarify the use of primers and gel verification. See in lines 192-195.

 Comment 12: L197–198: Please check the grammar.

Response 12: Thank you for your comment. We have revised the sentence to improve grammar and clarity. The updated version now clearly describes the assignment of species hypotheses and the classification criteria used and appears in the manuscript at lines 217-218.

Comment 13: L243–245: Improve the clarity of the sentence.

Response 13: Thank you for your comment. We have revised the sentence to improve clarity and ensure the correlation between fungal diversity and tree traits is clearly described. The updated version appears in the manuscript at lines 288-290.

Comment 14: L260–262: Please check the grammar.

Response 14: Thank you for your comment. We have revised the paragraph to improve grammar and clarity. The updated version now clearly describes the tree height data and the statistical approach used to identify genotype performance across locations. The revision appears in the manuscript at lines 302-307.

 Comment 15: L269–275. Please improve the grammar.

Response 15: Thank you for your comment. We have clarified the sentence by specifying the groups compared and the structure of the OTU matrices. The revised version appears in the manuscript at lines 309-319.

Comment 16: L290–293. Please improve the grammar.

Response 16: Thank you for your comment. We have revised the sentence to improve grammar and clarity. The updated version now appears in the manuscript at lines 338-340.

 Comment 17: L297–300. The first result is that most OTUs cannot be functionally assigned. In this section, you are referring to taxonomy. Please explain. Also, please check the percentages; the sum is not 100%.

Response 17: Thank you for your comment. We have clarified the distinction between functional and taxonomic classification and added a statement confirming that the percentages shown in Figure 1 sum to 100%, including all minor groups. The updated version appears in the manuscript at lines 344-353.

Comment 18: Figure 2. Add the letter 'A' at the beginning of the sentence in the legend. Improve the resolution of the figure.

Response 18: Thank you for your comment. We have revised the legend to include the letters, lines 367-368. The resolution of the figure has also been improved as requested.

 Comment 19: Figure 3: Please provide a detailed legend. P-value, description of boxes, etc.

Response 19: Thank you for your comment. We have revised the legend for Figure 3 to include a detailed description of the boxplot elements, statistical comparisons, and p-values. The updated legend now clearly explains the Shannon diversity index across tissue types and the significance of observed differences. Lines 380-383.

 Comment 20: L518–520. This sentence repeats the previous one.

Response 20: Thank you for your observation regarding the redundancy between lines 518–520 and the preceding sentence. We improved clarity and avoided repetition; we have revised the paragraph by integrating both ideas into a single. The updated sentence is in the manuscript lines 564-568.

Comment 21: L541–544. This part describes the results whereas L630–645 contains the actual discussion of the results.

Response 21: Thank you for your comments, we followed your observations and improved the manuscript.

 Comment 22: L647–662. This section describes the results whereas L630-645 contains the actual discussion of the results.

Response 22: Thank you for your comments, we followed your observations and improved the manuscript.

 Comment 23:  L713. Please delete ‘in alpha and beta diversity analyses.

Response 23: Thank you for your observation. The indicated content has been removed from the manuscript as suggested.

 Comment on the Quality of English Language: The grammar and English style also need to be revised for clarity.

Response: English was edited by native speaker.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled “Phyllosphere fungal mycobiome can affect Pinus sylvestris growth and its heritable between similar host genotypes” (Manuscript ID: forests-3911241) investigates the phyllosphere fungal mycobiome of Pinus sylvestris and its heritability among genotypes. It employs field sampling, high-throughput sequencing, and statistical analyses to explore the relationship between fungal diversity, host genotype, environmental factors, and tree performance. While the study is original and presents a valuable dataset for understanding tree-microbiome interactions, it suffers from methodological ambiguities, excessive verbosity, and interpretative weaknesses. Some conclusions lack sufficient data support, and the statistical methods require clearer description and justification.

1. Abstract

• The abstract is overly dense and descriptive. It summarizes many details but lacks a clear statement of the main findings and their implications. Please restructure it to highlight (1) aim, (2) methods, (3) key quantitative findings, and (4) their significance in tree breeding or ecology.
• Some sentences are grammatically unclear, for example, lines 22& 23: “Fungal diversity and community by needles and shoots were significantly different.” → should be rephrased for clarity.

2. Introduction

• The introduction provides valuable information but is overly lengthy and contains several redundant or general statements about forests and climate change that detract from the study’s main focus. Please condense the background into 3-4 concise paragraphs that logically lead to the research questions.
• Additionally, the transition from general microbiome concepts to the heritability of phyllosphere fungi needs to be clearer and better supported by relevant literature. Cite more recent studies (for example, research on microbiome heritability in trees or other perennials published after 2020).
• Finally, while the objectives mentioned at the end are clear, they should be presented in bullet or numbered format for improved clarity.

3. Materials and Methods

• This section is comprehensive but has several structural and methodological issues:

Experimental design:

• The sampling scheme is complex but not clearly justified statistically. For example, it is unclear whether genotypes were treated as fixed or random effects, or how replication within sites was handled.
• Clarify how the unbalanced sampling (missing genotypes/sites) was managed in analyses.
• Explicitly describe how biological vs. technical replicates were defined.

Sequencing and bioinformatics:

• The pipeline description (Pipecraft v2.0, VSEARCH, UNITE v.9.0) is adequate, but filtering thresholds, read counts per sample, and quality control steps should be summarized in a short table (currently only in Supplementary).
• Consider adding a concise schematic or flowchart summarizing the bioinformatics workflow.

Statistical analysis:

• The statistical framework needs clarification. Multiple univariate and multivariate tests (Kruskal-Wallis, GLM, PERMANOVA, ANOSIM, BEST) are used, but their assumptions and rationale are not well explained.
• Define dependent and independent variables clearly.
• Justify the use of nonparametric tests for each dataset.
• State how multiple comparisons were controlled to avoid Type I error inflation.

Heritability calculation:

• The formula is given but lacks a proper reference or reasoning for the chosen approach.
• Indicate whether broad-sense heritability was computed per functional group separately and explain why H² was low.
• Mention if the normality of residuals was checked before using ANOVA for H² estimation.

4. Results 

·      The results are comprehensive but contain excessive detail and some repetition between the text, figures, and supplementary materials. It would be beneficial to condense redundant descriptions and highlight key statistical outcomes, including effect sizes, instead of relying solely on p-values.

·      Figures 4 to 8 offer valuable information; however, they are crowded and inadequately labeled. To enhance readability, consider improving the fonts, legends and ensuring consistent color coding for genotypes and sites.

·      The relationship between fungal diversity and tree height/needle retention is intriguing but lacks strong statistical support. It is important to report correlation coefficients (r or P) and sample sizes consistently throughout.

5. Discussion

• The discussion lacks structure and includes extensive narrative repetition of results.
  Reorganize it into thematic subsections:
  (1) Tissue-specific diversity,
  (2) Environmental vs. genetic effects,
  (3) Relationship with tree growth,
  (4) Mycobiome heritability, and
  (5) Implications for breeding.
• Some interpretations are speculative. For instance, attributing differences in growth to “defense trade-offs” or “microbial stability” is not directly supported by data. Please temper these claims or back them with specific references.
• The heritability section overstates its findings: H² values of 0.04–0.07 indicate minimal inheritance; this should be discussed as weak or negligible heritability rather than “evident.”
• The link between fungal diversity and tree performance should acknowledge confounding environmental effects.

6. Conclusions

• The conclusions are too long and partially redundant with the discussion.
• Limit this section to 2-3 concise paragraphs summarizing the core findings and practical implications for forestry or tree breeding.
• Avoid introducing new information not covered in the results.

Minor Comments

• Terminology: Use consistent terms: mycobiome, fungal community, phyllosphere, etc., are alternated inconsistently.
• Units and symbols: Ensure correct spacing and formatting of symbols (e.g., °C, H², p-values, etc.).
• Tables and Figures: Add clear captions indicating what the statistical tests represent (e.g., “Kruskal–Wallis test showing differences in Shannon index among sites”).
• References: Some key works on microbiome heritability and phyllosphere diversity are missing (e.g., recent studies post-2022). Update references accordingly.

 

 

 

Author Response

Thank you for your comments. After receiving your suggestions for improving the manuscript, methodological ambiguities were addressed, and the description was improved to facilitate interpretation of the results. The statistical framework has been rewritten to provide a clear description and justification of each method used, and the conclusions are now fully supported by the data presented. These updates have significantly improved the quality. See the revised text in the Materials and Methods, Results, and Discussion sections.

 

1. Abstract

 

Comment 1: The abstract is overly dense and descriptive. It summarizes many details but lacks a clear statement of the main findings and their implications. Please restructure it to highlight (1) aim, (2) methods, (3) key quantitative findings, and (4) their significance in tree breeding or ecology.

Response 1: Thank you for your comment. We have restructured the abstract to clearly present the aim, methods, key quantitative findings, and their significance for tree breeding and forest ecology. The revised version appears in the manuscript, lines 13-33.

 

Comment 2: Some sentences are grammatically unclear, for example, lines 22& 23: “Fungal diversity and community by needles and shoots were significantly different.” → should be rephrased for clarity.

Response 2: Thank you for your observation and suggestion. The correction has already been made, along these lines, with the reorganization of the abstract, lines 20-22.

 

2. Introduction

 

Comment 3:  The introduction provides valuable information but is overly lengthy and contains several redundant or general statements about forests and climate change that detract from the study’s main focus. Please condense the background into 3-4 concise paragraphs that logically lead to the research questions.

Response 3: Thank you for the suggestions. We have revised the section to improve clarity and focus, condensing the background into four concise paragraphs, see lines 38-117.

 

Comment 4: Additionally, the transition from general microbiome concepts to the heritability of phyllosphere fungi needs to be clearer and better supported by relevant literature. Cite more recent studies (for example, research on microbiome heritability in trees or other perennials published after 2020).

Response 3: Thank you for the suggestion, the transition from general microbiome concepts to the heritability of phyllosphere fungi has been clarified, and we have incorporated recent literature on microbiome heritability in trees and perennial plants to better support this part of the text. See lines 47-76.

 

Comment 5: Finally, while the objectives mentioned at the end are clear, they should be presented in bullet or numbered format for improved clarity.

Response 3: Thank you for the suggestion. Please see the revised version in lines 107-117 of the manuscript.

 

3. Materials and Methods

 

This section is comprehensive but has several structural and methodological issues:

 

Experimental design:

 

Comment 6: The sampling scheme is complex but not clearly justified statistically. For example, it is unclear whether genotypes were treated as fixed or random effects, or how replication within sites was handled. Clarify how the unbalanced sampling (missing genotypes/sites) was managed in analyses.

Response 6: Thank you for your feedback. For alpha diversity, nonparametric tests were performed for comparison and correlation tests because the data did not meet the assumptions of homogeneity and normality. In tour research generalized linear model was used, its allow the inclusion of categorical factors, genotype, and continuous covariates (sum of effective temperature, average temperature, location) under a flexible framework. The dependent variable was the Shannon index. For beta diversity, calculations were performed as a PERMANOVA in which the fixed factor was location and the random factor was genotype. It has clarified in material and methods in lines 263-265 and 277-278.

 

Regarding the analysis of unbalanced data after sampling and sequencing (which can occur), we used robust statistical approaches for unequal sample sizes, such as the Kruskal-Wallis test and Bonferroni-adjusted tests for alpha diversity, Spearman correlation models, and Generalized Linear Models. For beta diversity, in addition to determining the assumptions of normality and homogeneity for alpha diversity, nonparametric tests (Kruskal-Wallis with Bonferroni correction, Spearman correlations) and Generalized Linear Models (GLM) were applied. For beta diversity, PERMANOVA and ANOSIM were used to assess differences between factors, while PERMDISP was used to confirm that the statistically significant effects found in the study were not influenced by dispersion heterogeneity. PERMDISP had already been calculated and is included in the supplementary information for each of the analyses performed. These methods allowed for the inclusion of all available observations without data imputation. See the clarification in the manuscript lines 172-179, 232-248.

 

Comment 7: Explicitly describe how biological vs. technical replicates were defined.

Response 7: thank you for your observation, we clarified on a detail in lines 172-179, and line 188.

 

Sequencing and bioinformatics:

 

Comment 8: The pipeline description (Pipecraft v2.0, VSEARCH, UNITE v.9.0) is adequate, but filtering thresholds, read counts per sample, and quality control steps should be summarized in a short table (currently only in Supplementary).

Response 8: We appreciate your comment. We have already attached the scheme that you requested in the supplement information, as we do not consider it relevant in the main body of the paper, as there have been guide articles where this is not frequently included, Supplementary information 10, Table S10.

 

Table S10. summarizes the main steps of the Pipecraft v2.0 pipeline applied to PacBio ITS reads.

Step	Parameter / Threshold	Result / Notes
Length filtering	>100 bp	Reads retained: ~95%
Demultiplexing	≤3 mismatches in index	All samples retained (n = 1,678)
Quality filtering	mothur default	Reads per sample: 1,200–8,500 (mean ≈3,450)
Chimera removal	UCHIME	Removed: ~3–5% of reads
ITS extraction	ITSx	Full/partial ITS preserved
Clustering	98% similarity (VSEARCH)	OTUs: 26,756; sequences: 5,781,188
Taxonomy assignment	UNITE v9.0 (BLAST)	100% OTUs = fungi; 31.1% species-level (≥98%)

 

Comment 9: Consider adding a concise schematic or flowchart summarizing the bioinformatics workflow.

Response 9: We appreciate your comment. We have already attached the scheme that you requested in the supplement information, as we do not consider it relevant in the main body of the paper, as there have been guide articles where this is not frequently included. Supplementary information 11, figure S19.

Figure S19. General outline of the workflow, from sampling to statistical analysis.

 

Statistical analysis:

 

Comment 10: The statistical framework needs clarification. Multiple univariate and multivariate tests (Kruskal-Wallis, GLM, PERMANOVA, ANOSIM, BEST) are used, but their assumptions and rationale are not well explained.

Response 10: I appreciate your observation and comments, but these were already initially described in the submitted manuscript, in sections 2.4 Statistical Analysis of fungal diversity, 2.4.1 Alpha diversity and 2.4.2 Beta diversity, Lines 231-286. But for greater clarity we added a paragraph to section 2.4 as an introduction to the analyses performed, lines 232-245.

 

Comment 11: Define dependent and independent variables clearly.

Response 11: Thank you for your observation. These variables are already defined in lines 245-248.

 

In addition, a table was added in the statistical analysis section on line 245-246, where the variables used in each statistical test are specified. And we added Table 2, line 247-248.

 

Comment 12: Justify the use of nonparametric tests for each dataset.

Response12: Thank you for your comment. Below, we clarified our choice of nonparametric statistical analysis. Normality and homogeneity of variance were tested using the Shapiro-Wilk and Levene tests, and the alpha diversity data did not meet these assumptions (p < 0.05). Furthermore, the sampling design was unbalanced due to missing genotypes and unequal sample sizes across sites. For these reasons, nonparametric tests (Kruskal-Wallis and Spearman) were used, as they are robust to violations of normality and heteroskedasticity and do not require equal sample sizes. See revised text on lines 233-240.

 

Comment 13: State how multiple comparisons were controlled to avoid Type I error inflation.

Response 13: Thank you for your feedback. Control for Type I errors was already explicitly stated in the revised manuscript. For α diversity, Bonferroni-adjusted post hoc tests were applied after Kruskal-Wallis analyses. For β diversity (PERMANOVA and ANOSIM), significance was determined using 999 permutations, which inherently justifies controlling for multiple comparisons by randomization.

 

Heritability calculation:

 

Comment 14: The formula is given but lacks a proper reference or reasoning for the chosen approach.

Response 14: Thank you for your comment. The formula already had a reference, but we added another one to support the approach, see reference number 116, line 334.

 

Comment 15: Indicate whether broad-sense heritability was computed per functional group separately and explain why H² was low.

Response 15: Thank you for your comment. Broad-sense heritability was calculated for the entire fungal community and for each functional group (saprotrophs and pathogens) separately, as described in lines 334-335.

 

On the other hand, in the discussion section, possibilities were suggested as to why broad-sense heritability could be low; this is described in lines 766-711, 775-783.

 

Comment 16: Mention if the normality of residuals was checked before using ANOVA for H² estimation.

Response 16: Thank you for your comment. The assumptions of normality and homogeneity were met by the Shapiro-Wilk and Levene tests, as well as for the Shanks index and axis 1 of pCoA, for the three groups analyzed (the complete fungal community, the saprotrophic community and the pathogenic community), and were not met. However, according to Falconer and Mackay (1996), ANOVA was applied as a standard method to estimate variance components and broad-sense heritability in quantitative genetics, where H² is considered robust for descriptive purposes even under moderate deviations from these assumptions.

 

Falconer, D. S., & Mackay, T. F. C. (1996). Introduction to Quantitative Genetics (4th ed.). Longman Group Ltd.

4. Results

 

Comment 17: The results are comprehensive but contain excessive detail and some repetition between the text, figures, and supplementary materials. It would be beneficial to condense redundant descriptions and highlight key statistical outcomes, including effect sizes, instead of relying solely on p-values.

Response 17: Thank you for your observation, an adjustment was made following your suggestions in the results. The results section was improved.

 

Comment 18: Figures 4 to 8 offer valuable information; however, they are crowded and inadequately labeled. To enhance readability, consider improving the fonts, legends and ensuring consistent color coding for genotypes and sites.

Response 18: Thank you for your observation, an adjustment was made following your suggestions in the manuscript.

 

Comment 19: The relationship between fungal diversity and tree height/needle retention is intriguing but lacks strong statistical support. It is important to report correlation coefficients (r or P) and sample sizes consistently throughout.

Response 19: Thank you for your observation, an adjustment was made following your suggestions in the results description of this section, 3.6. Analyses of Fungal Diversity Effect on Tree Height (2022) and Needle Retention of Scots Pine Progenies, in the lines 482-501.

5. Discussion

 

Comment 20: The discussion lacks structure and includes extensive narrative repetition of results.
Reorganize it into thematic subsections:
(1) Tissue-specific diversity,
(2) Environmental vs. genetic effects,
(3) Relationship with tree growth,
(4) Mycobiome heritability, and
(5) Implications for breeding.

Response 20: Thank you for your comment, we followed your suggestion and made the modification to the manuscript in the Discussion section.

 

Comment 21: Some interpretations are speculative. For instance, attributing differences in growth to “defense trade-offs” or “microbial stability” is not directly supported by data. Please temper these claims or back them with specific references.

Response 21: Thank you for your comments. We've adjusted the discussion section on lines 706-734. Ad also we added new references, references 117-120.

 

Comment 22: The heritability section overstates its findings: H² values of 0.04–0.07 indicate minimal inheritance; this should be discussed as weak or negligible heritability rather than “evident.”

Response 22: Thanks for your suggestion, we made some modifications to section 4.4; Fungal Communities Heritability, see the lines 758-783.

 

Comment 23: The link between fungal diversity and tree performance should acknowledge confounding environmental effects.

Response 23: Thank you for your comments. We've adjusted the discussion section on lines 755-754 and also, we added new references, references 121, 122.

 

6. Conclusions

 

Comment 24: The conclusions are too long and partially redundant with the discussion.

Response 24: Thank you for your comments. The conclusion section is corrected and improved in the manuscript.

 

Comment 25: Limit this section to 2-3 concise paragraphs summarizing the core findings and practical implications for forestry or tree breeding.

Response 25: Thank you for your comments. The conclusion section is corrected and improved in the manuscript.

 

Comment 26: Avoid introducing new information not covered in the results.

Response 24: Thank you for your comments. The conclusion section is corrected and improved in the manuscript.

 

Minor Comment

 

Comment 27: Terminology: Use consistent terms: mycobiome, fungal community, phyllosphere, etc., are alternated inconsistently.

Response 27: Thank you for your comments. The terminology is corrected and improved.

 

Comment 28: Units and symbols: Ensure correct spacing and formatting of symbols (e.g., °C, H², p-values, etc.).

Response 28: Thank you for your comments. Symbols are corrected and improved.

 

Comment 29: Tables and Figures: Add clear captions indicating what the statistical tests represent (e.g., “Kruskal–Wallis test showing differences in Shannon index among sites”).

Response 29: Thank you for your comments. Are corrected and improved in manuscript.

 

Comment 30: References: Some key works on microbiome heritability and phyllosphere diversity are missing (e.g., recent studies post-2022). Update references accordingly.

Response 30: Thank you for your comments. Are corrected and improved in the section.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript was substantially improved after the authors made the revisions. I have only few suggestions for the authors.

L681-688. This part could be written more concisely, avoiding the list of fungi and adding the relative references.

L750.  I suggest eliminating any reference to supplementary files. 

 

 

Comments on the Quality of English Language

The grammar and English style also need to be revised for clarity.

Author Response

Dear Reviewer,  

We would like to thank you for your time, and valuable  feedback, it was helpful in improving the quality and clarity of our manuscript. Please find attached a revised version of our research article entitled: “Phyllosphere Fungal Mycobiome can Affect Pinus sylvestris Growth and Its Heritable Between Similar Host Genotypes”, by Carel Elizabeth Carvajal-Arias, Ahto Agan, Kalev Adamson, Tiit Maaten, Rein Drenkhan. Below we describe the responses to each of your comments. The responses are in blue, and the correction made to the manuscript are in green highlight.

Sincerely yours, 

Authors

REVIEWER 2

 

Comment 1: L681-688. This part could be written more concisely, avoiding the list of fungi and adding the relative references.

Response 1: Thank you for your comments, we followed your observation and deleted it in the manuscript (lines 682-685).

 

Comment 2: L750.  I suggest eliminating any reference to supplementary files. 

Response 2: Thank you for your comments, we followed your observation and deleted it in the manuscript (line 746).

 

Comments on the Quality of English Language: The grammar and English style also need to be revised for clarity.

Response: The English grammar is already edited by native speaker, please see current paper acknowledgments.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors conducted all the reviews.

Author Response

Dear Reviewer,

Thank you for your positive feedback and for confirming that the manuscript meets the required standards. We appreciate your time and effort in reviewing our work.

Sincerely,

Authors