Polyploid Advantage? Comparing Salt Stress Responses of Di- and Tetraploid Acacia senegal (L.) Willd. Seedlings

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors

I was pleased to read the manuscript of Adja Madjiguene Diallo et al “Response of di- and tetraploid Acacia senegal (L.) Willd. seed-2 lings to salt stress”. The objective formulated by the authors in this study is concrete. It boils down to testing the hypothesis that tetraploid Acacia senegal plants should have greater salt tolerance than diploid plants. The idea that polyploid forms of plants have higher stress tolerance is not new.  It is enough to recall, for example, tetraploid forms of aspen, which have high resistance to heart rot and a number of abiotic factors. To test the hypothesis, the authors used two approaches (1) establishing a correlation between the polyploidy of plants and the level of salinity of their growing sites. (2) Studying the response of di- and tetraploid plants to salt stress under the conditions of a vegetation experiment. To evaluate the response of plants to salt stress, the authors used some integral morphometric indices such as plant height and diameter, dry and wet weight, biomass ratio of underground and aboveground organs, sodium and chlorine ion content in stems, and plant mortality.

Without going into a detailed analysis of the obtained data, it should be said that the authors experimentally confirmed the hypothesis that A. senegal tetraploids have increased tolerance to salinity compared to diploids.  It should be noted that the authors are very careful in interpreting their data, which is respectable.

I have no fundamental comments on this manuscript. For a physiological journal, it would be necessary to characterize water status and ion homeostasis in more detail, while in this case, I think it is not required.

It would be useful to indicate on what signs the authors evaluated dead plants, since the methodological part does not say anything about it.

Photographs of tetraploid and diploid plants subjected to salt stress would facilitate the perception of the material.

It is not very clear to me why the authors determined in soil samples the content of KCl, but used NaCl for the vegetation experiment.

It seems strange to me to start a paragraph (line 276) as follows:  “[24] studied....”.

There are minor technical typos in some places of the text. This requires careful reading of the manuscript.

Kind regards

Author Response

Comment 1. It would be useful to indicate on what signs the authors evaluated dead plants, since the methodological part does not say anything about it.

Response 1. Dead plants were evaluated by leaf loss as well as total absence of green materiel (dryness). This information has been added to the methodology description in lines 102-103, page number 3.

Comment 2. Photographs of tetraploid and diploid plants subjected to salt stress would facilitate the perception of the material.

Response 2. Yes, photos added in lines 83-86, page number 3.

Photo 1. A) whole experiment: pots with blue labels represent controls; pots with red labels plants exposed to salinity. B) Tetraploid (left) and diploid (right) Acacia senegal subjected to salt stress.

Comment 3. It is not very clear to me why the authors determined in soil samples the content of KCl, but used NaCl for the vegetation experiment.

Response. This is a misunderstanding. We assessed soil pH using suspensions of water and KCl. This has been clarified in the methods in line 66, page number 2. Measurements of pH in KCl solutions will release bound protons in the soil and thus complement assessments of acidity using at water suspension.

Comment 4. It seems strange to me to start a paragraph (line 276) as follows:  “[24] studied....”.

Response 4. These sentences have been reformulated thoroughly in the text  i.e in lines 43-45, page number 2.

Comment 5. There are minor technical typos in some places of the text. This requires careful reading of the manuscript.

Response 5. Careful reading has been performed to edit minor technical typos. Thank you.

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Please improve manuscript as suggested

Comments for author File: Comments.pdf

Author Response

We thank the reviewer for the opportunity for revising the manuscript, and for the constructive comments to improve it. Our responses to the comments are detailed below. Please do not hesitate if you have questions in this regard.

Reviewer 2. Reviewer Suggestions Section Wise

Comments 1. Suggestions for Title Revision (Pick any one from following)

Response 1. Thank you for the suggestions. We have opted for a revised version of the last suggestion in lines 2-3, page number 1. “Polyploid Advantage? Comparing Salt Stress Responses of Di- and Tetraploid Acacia senegal ( L.) Willd. seedlings”.

Suggestions for Abstract

Comments 2. The phrasing of the results, particularly regarding the correlation between polyploid presence and soil salinity, could be more precisely added. Consider specifying how strong the correlation was.

Response 2: Sentence has been rephrased in line 17, page number 1: “We found polyploids in six stands, and the proportion of polyploids in these stands were significantly and positively correlated with their level of soil salinity”.

Comments 3. In last line of abstract explicit statement on the practical implications of the findings for reforestation efforts.

Response 3. The sentence has been reformulated in lines 21-23, page number 1: “The findings of this study may have practical implications for reforestation of saline soils with polyploid A. senegal, and we recommend further studies to illuminate their potential”.

Suggestions for Introduction Section

Comments 4. Discuss whether polyploidy inherently confers stress tolerance or if observed benefits are context dependent could be more explicitly linked to the specific case of Acacia senegal.

Response 4. This has been discussed in the manuscript in lines 36-46, page number 2. However, we added the following statement “and studies based on trees are scarce” in line 46 to justify our study in polyploid A. senegal.  

Comments 5. Additionally, some references are introduced without much discussion (e.g., [10] and [12]), and it would be helpful to briefly explain their relevance.

Response 5. Brief explanation of cited references has been added in the manuscript in lines 41-44, page number 2.

Comments 6. The transition to the study hypothesis is logical, but strengthening the justification for why A. senegal makes a suitable model for testing polyploid advantages under salinity stress would improve the argument.

Response 6. The following paragraph has been added in lines 50-54, page number 2. Thank you for pointed this out: “A. senegal is reported to naturally grow in salt affected lands in Senegal, estimated at about 1.7 million hectares [14]. Several studies highlighted the ability of A. senegal to tolerate high level of salinity [15-18] without considering potential effect of ploidy level within the species. Thus, understanding and clarifying the basis of observed tolerance in A. senegal with respect to genome size is of prime importance especially in the context of selecting appropriate genotypes for reforestation of these saline zones.”

Suggestions for Materials and Methods Section

The Materials and Methods section provides a detailed and structured description of the study design, including field sampling, greenhouse experiments, and statistical analysis. However, a few aspects could be clarified or improved:

1. Site Selection and Soil Sampling:

Comments 7. It would be helpful to explain why a single composite soil sample was used per site, as this could influence variability in salinity and pH measurements.

Response 7. While we agree that microsite variations may influence the occurrence of di- and polyploids, we were mainly interested in population responses. Thus, the composite sample was made as we used site as the experimental unit, comparing polyploid frequency with site salinity. We have explained this briefly in the text in lines 63-64, page number 2. Replications of 10 soil samples collected at fixed-depth intervals and mixed to constitute a bulk sample is a general procedure mainly used for soil chemical analyses (electric conductivity and pH).  Thank you for this relevant comment.

Comments 8. Clarifying whether intra-site variability was considered (e.g., multiple independent samples) would strengthen the methodology.

Response 8. Please see above. The objective of this study was to globally characterize soil in each site without taking into account intra site variability.  

2. Experimental Design and Controls:

Comments 9. The choice of planting diploid and tetraploid seedlings in shared pots is a strength, but it would be beneficial to explicitly address potential competition effects.

Response 9. Agree. Sentences information have been added in lines 80-81, page number 3 and 234-235, page number 9. Many thanks for pointed this out.

Comments 10. The gradual increase in NaCl concentration is well-justified, but specifying whether the final 0.20 mM concentration is within the range of natural salinity levels in A. senegal habitats would provide context.

Response 10. We have stated that 0.20 mM NaCl corresponds to a highly saline soil in lines 93-94, page number 3.   

3. Statistical Analysis:

Comments 11. The regression and ANOVA approaches are sound, but it would be useful to mention if any adjustments were made for multiple testing.

Response 11. It is correct that we did not apply Bonferroni corrections for multiple testing. We find this justified since we only performed tests of biological meaningful hypotheses closely linked to research questions behind the study. However, it does mean that significant results based on tests statistics close to the 5% significance level should be interpreted with some care. We have added this to the M&M, in paragraph 144-147, page number 4.

Comments 12. The Chi-square test for mortality is appropriate, but discussing whether survival differences remained significant when controlling for initial seedling size or other covariates would strengthen the analysis.

Response 12. As raised by the reviewer, mortality could be influenced by seedling size. However, diploid seedlings in the control treatment were small that polyploids, but has higher mortality (potential negative size effect). But under salt treatment, the polyploids were also larger than diploid but here with lower mortality (potential positive size effect). Although this may cover some interesting biological differences, we did not pursue this aspect based on logistic regression or similar, but only applied the simple and robust Chi-square test. This is because we were careful to limit the number of performed tests given the sample size.

Suggestions for Discussion Section

Comments 13. The results indicate that tetraploid A. senegal exhibited lower mortality and greater root allocation under salt stress, supporting the hypothesis of better adaptation. However, additional discussion on whether these traits translate to long-term survival and reproductive success under field conditions would strengthen the ecological implications.

Response 13. Agree. We have accordingly discussed this aspect in paragraphs 245-250, page number 9 and 270-282, page number 10. 

Comments 14. The contrasting findings with [21] should be discussed in greater depth. Were the differences due to genetic variation among provenances, differences in experimental conditions, or interactions with other environmental factors?

Comments 15. Clarifying whether the observed polyploid advantage is specific to early life stages or extends to mature trees would enhance the discussion.

Response 15. Agree. We have addressed this aspect in the revised version, in paragraph 239-250, page number 9.

Comments 16. The mention of increased root allocation as a potential adaptive mechanism is valuable, but the discussion could benefit from linking this to physiological processes (e.g., ion homeostasis, osmotic adjustment).

Response 16. Agree. This has been considered in the revised version, in paragraph 260-265, page number 10.

Comments 17. Considering whether polyploidy affects hormone regulation or gene expression related to stress tolerance would add depth to the explanation.

Response 17. Agreed. We have added the paragraph 260-265, page number 10 to address this comment. Thank you.

Comments 18. While polyploidy has been associated with increased tolerance to various stresses, the discussion acknowledges that this is not a universal rule. Expanding on how polyploidy interacts with local adaptation and genetic diversity would provide a broader perspective.

Response 18. Agreed. We have included this information in paragraph 270-281, page number 10.

Comments 19. It would be useful to mention any potential trade-offs associated with polyploidy, such as reduced reproductive success or slower establishment under certain conditions.

Response 19. Added in lines 273-2756: “However, polyploidization often comes with a cost in terms of reduced reproductive success and high levels of apomixis [36] which seems also to be the case for A. senegal [20,22], potentially reducing the long-term adaptive potential of this cytotype”.

 

Suggested Improvements on conclusion

Comments 20. Reinforce the Key Findings with a Stronger Statement - Explicitly state that tetraploid A. senegal exhibited higher survival rates and root biomass allocation under salinity stress, supporting the hypothesis of better salt adaptation.

Response 20. Agreed. The statement has been included in the conclusion in lines 286-287, page number 10: “Our results revealed that. tetraploid A. senegal exhibited higher survival rates and root biomass allocation under salinity stress, supporting the hypothesis of better salt tolerance and adaptation” Many thanks for this relevant comment.

 Comments 21. Address Context-Specific Advantages - Highlight that the observed differences in shoot/root ratios suggest that diploids and tetraploids may have different ecological niches or adaptive strategies.

Response 21. Ok; this point has been addressed in lines 287-290, page number 10: “the observed differences in shoot/root ratios suggest that diploids and tetraploids may have different adaptive strategies to cope with salt expositure. Thus, stands presenting higher frequency of polyploid trees could be considered as suitable seed sources for future reforestation actions of salt affected lands”

Comments 22.  Emphasize Early-Stage vs. Long-Term Implications - Acknowledge that while early-stage survival is crucial for population dynamics, further studies on mature trees are necessary to confirm long-term adaptive benefits.

Response 22. Agreed, this has been highlighted in lines 290-293, page number 10:” Our results also document early stage increased survival of tetraploid A. senegal under controlled conditions, which is crucial for population establishment and dynamics. However, it remains crucial to verify if superiority of tetraploids under salt stress is expressed only at early stage or applies more generally in mature trees”.

Comments 23. Call for Further Research with Specific Directions Instead of a general statement about follow-up studies, suggest specific research approaches, such as field trials, genetic studies on salt tolerance mechanisms, or comparisons between A. senegal populations in different habitats.

Response 23. Agreed and information included in the conclusion in lines 293-295, page number 10. “Thus, further studies focusing on comparisons of mature A.senegal cytotypes in different habitats or field trials are needed. More research on understanding and clarifying genetic basis underlying salt tolerance mechanisms is necessary to confirm long-term adaptive benefits”.

 

Author Response File: Author Response.pdf