Functional Traits Associated with Drought Tolerance Exhibit Low Variability in 21 Provenances of a Montane Tree Species—Eucalyptus delegatensis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article entitled "Functional traits associated with drought tolerance exhibit low variability in 21 provenances of a montane tree species - Eucalyptus delegatensis" present results on morphological and physiological traits linked to drought tolerance from alpine ash from different provenance across Australia. The authors showed that there was no variability between the different provenance as well as only weak correlation between mophological and physiological traits and between climatic and traits. They concluded that this species did not exhibit difference in expression of drought tolerance related functional traits and no relation to the climate of origin. However, I think that this study did not present that data to conclude that. 

All saplings were grown under the same climatic conditions. Even though there was no significant difference, drought adapatation mechanisms could have been hidden by those conditions since individuals did not have to adapt themselves to this favourable conditions. With that, it is impossible to give any advice about forest management, especially because this study did not highlight any difference.

To really conclude about origin provenance/climate adaptation to drought, authors should have studied physiological and morphological traits responses to drought through the application of water stress gradient, corresponding on the precipitation range of all provenances (800 - 1850 mm/yr). Without those data, it is very difficult to conclude anything except that there is no genetic adaptation under good conditions of growing which the authors did. But I feel like those results did not bring that much information and this study should be completed before publication. 

Then, to go further into data analyses, multivariate analyses (PCA, RDA) should be done with provenance as factor (each site or at the region level) including origin climatic conditions. Then, I would do the same for correlations between morphological and physiological traits and between origin climate and traits by taking expressing them according to provenance (each site or at the region level). And then to compare the response, perform slope comparisons. By the way, I think that if the authors cannot add water stress response data on this paper, they should perform those analyse to improve this article.

Finally, correlations are not discussed at all and it is not clear why the authors did that.

Minor comments:

• line 26 => change mm in mmol in unity if stomatal conductance
• precise unity of each measurement you performed into material and methods section

Author Response

Reviewer 1

Reviewer comment: The article entitled "Functional traits associated with drought tolerance exhibit low variability in 21 provenances of a montane tree species - Eucalyptus delegatensis" present results on morphological and physiological traits linked to drought tolerance from alpine ash from different provenance across Australia. The authors showed that there was no variability between the different provenance as well as only weak correlation between mophological and physiological traits and between climatic and traits. They concluded that this species did not exhibit difference in expression of drought tolerance related functional traits and no relation to the climate of origin. However, I think that this study did not present that data to conclude that. 

All saplings were grown under the same climatic conditions. Even though there was no significant difference, drought adapatation mechanisms could have been hidden by those conditions since individuals did not have to adapt themselves to this favourable conditions. With that, it is impossible to give any advice about forest management, especially because this study did not highlight any difference.

Author response: This study aimed to investigate the variability of functional trait expressions in 21 provenances of Eucalyptus delegatensis that were sourced along a climate gradient. The study clearly showed that the provenances have not adapted to the climatic conditions because there was no trait variation among the provenances. We highlight in the discission that there is a difference between adaptation (genetically controlled expression of traits) and adjustment or plasticity (the expression of trait variation that is triggered by environmental cues). We investigated the former as we were interested if the long-term exposure to different climatic conditions had led to adaptation, i.e. genetically controlled differences in trait expression, which evidently was not the case. We explain why that is likely the case: lack of sufficient environmental stressors that would lead to a selection of traits. We highlight that plasticity of trait expression is possible or likely in E. delegatensis (L421-424 Discussion, new submission, no tracked changes). And we comment on the relevance of these results for forest management: it is clear that provenances of E. delegatensis do not differ in the genetically controlled expression of drought tolerance traits, likely because drought tolerance is not selective stress. In other words: E. delegatensis is likely not very stressed in its current environment and has not evolved differences in drought tolerance. It is possible that the trees express trait differences in the natural environment, but these are based on differences in trait plasticity.

 

Reviewer comment: To really conclude about origin provenance/climate adaptation to drought, authors should have studied physiological and morphological traits responses to drought through the application of water stress gradient, corresponding on the precipitation range of all provenances (800 - 1850 mm/yr). Without those data, it is very difficult to conclude anything except that there is no genetic adaptation under good conditions of growing which the authors did. But I feel like those results did not bring that much information and this study should be completed before publication. 

Author response: The reviewer is asking for a different experiment that was not the aim of this study. We clearly identify the aim in the introduction and highlight it also in the Discussion L409-414: “It is important to note that we studied the expression of functional traits in a common garden experiment under non-stressed conditions. This means that all provenances were exposed to the same environmental conditions. It is therefore expected that trait expression was mainly controlled by plant genetics as there would be very little (or similar) environmental influence on trait expression. This will be different in studies that investigate plants along environmental gradients in situ.” What the reviewer is asking to perform a different study and such a study can be done as a follow up. But a stress experiment does not invalidate this study that had a clear knowledge gap and clear results. A study should be evaluated on its merits and not invalidated by asking for a different experiment that has different aims.

 

Reviewer comment: Then, to go further into data analyses, multivariate analyses (PCA, RDA) should be done with provenance as factor (each site or at the region level) including origin climatic conditions. Then, I would do the same for correlations between morphological and physiological traits and between origin climate and traits by taking expressing them according to provenance (each site or at the region level). And then to compare the response, perform slope comparisons. By the way, I think that if the authors cannot add water stress response data on this paper, they should perform those analyse to improve this article.

Author reply: Thank you for the suggestion. We have added two new figures to the manuscript, Fig 6 (Quartile coefficient of variation) and Fig 7 (Relationship btw QCV and climatic variables) to investigate the relationship between traits and climate in more depth. We have also completed two PCA. One with traits and one with traits & climate, see graphs below. We removed precipitation from the second PCA analysis, as it was highly correlated with elevation. The trait-only PCA indicated PCA to have a strong positive association with mean OP and a strong negative association with mean SD. PC2 was driven gmin (negative) and SLA (positive). About 30% of variance was explained by PC1 and 2 respectively, indicating a strong model. In the second PCA including climate variables, PC1 was associated with all climate variables (positively) and PC2 was driven by mean SD (negatively) and OP (positively). PCA explained 28% of variance, PC@ 17%, making for slightly less statistical power. These PCA do not indicate novel insights due to an absence of clear grouping among provenances, or state of origin. This likely stems from the fact that the variances between traits among the provenances are too small to elicit any changes in the results. The variation of the traits were very small and we discuss this at length in the Discussion (L381-394). For example, the osmotic potential at full turgor varied by 0.1 MPa among all provenances and the minimum stomatal conductance varied by 2 mmol m2 s-1. We therefore decided not to include the PCA analyses and results into the manuscript.

 

 

We also carried out an RDA analysis using traits as response and climate variables as constraining variables. The constraining variables only explained ~6% of variance, indicating that climate was weak driver of drought trait expression (see graph below). RDA 1 was driven by elevation (negatively) and AHMI (positively), while RD2 was driven by temperature (neg.). The Model was significant, but the adjusted R-squared was low at 0.03. More detailed model statistics indicated that only RD1 was significant in describing trait composition and on the term level, only elevation had a significant effect. These more detailed results shed more light on the low variance explained by the constrained variables in the RDA. Therefore, we elected not to present these results in the manuscript but added them here in response to the suggestion.

 

Reviewer comment: Finally, correlations are not discussed at all and it is not clear why the authors did that.

Author reply: The correlations are discussed in L368-378

 

 

Reviewer comment: line 26 => change mm in mmol in unity if stomatal conductance

Author reply: Change made

 

Reviewer comment: precise unity of each measurement you performed into material and methods section

Author reply: The units of each measurement are displayed in the results as it is custom. There is no need to give the units of measurements as they depend on the way they are calculated and displayed. Thus, units ought to be displayed in the results.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Drought stress is a main abiotic factor that affects physiological, morphological, molecular adaptation, and evolution in plants. This manuscript reported that functional traits associated with drought tolerance exhibit low variability in 21 provenances of a montane tree species-Eucalyptus delegatensis, and the authors did not find that based on functional trait expression, provenances would have different drought tolerant responses than others. It is important to understand the adaptive mechanisms to changing environments especially drought stress. However, I think the following comments should be considered.

• ABSTRACT: First, the research GAP or hypothesis and significance should be clearly stated in the ABSTRACT. Second, what is SLA? Whose full name should be appeared.
• INTRODUCTION: First, line 46, the reference ‘[8] observed a strong correlation between leaf water potential at the turgor loss point and water availability within and across the biomes”, this citation is improper. Second, the relationship between drought tolerance with morphological and physiological traits should be hypothesized.
• METHODS: The differences of drought tolerance in 21 provenances of a montane tree species should be further determined and then estimated other relationship.
• RESULTS: The results of the differences of drought tolerance in 21 provenances of a montane tree species s and the relationship between drought tolerance with morphological and physiological traits should be supplemented
• DISCUSSION: First, the relationship between drought tolerance with morphological and physiological traits should be further discussed. And then, the causes that provenances would not have different drought tolerant responses should be further discussed according to physiological, morphological, molecular, and genetic traits.
• New relative references published in 2024-2025 should be suitably supplemented.

 

 

 

Comments on the Quality of English Language

no

Author Response

Reviewer 2

Reviewer comments: Drought stress is a main abiotic factor that affects physiological, morphological, molecular adaptation, and evolution in plants. This manuscript reported that functional traits associated with drought tolerance exhibit low variability in 21 provenances of a montane tree species-Eucalyptus delegatensis, and the authors did not find that based on functional trait expression, provenances would have different drought tolerant responses than others. It is important to understand the adaptive mechanisms to changing environments especially drought stress. However, I think the following comments should be considered.

 

Reviewer comment: ABSTRACT: First, the research GAP or hypothesis and significance should be clearly stated in the ABSTRACT. Second, what is SLA? Whose full name should be appeared.

Author reply: We agree and have added the following sentence to the Abstract: “While alpine ash forests are vulnerable to climate-change, it is unclear if different provenances have adapted to the climatic conditions in which they grow.” (L17-18, new submission, no tracked changes). We have added the term “specific leaf area” to the abstract (L28) to explain the abbreviation SLA.

 

Reviewer comment: INTRODUCTION: First, line 46, the reference ‘[8] observed a strong correlation between leaf water potential at the turgor loss point and water availability within and across the biomes”, this citation is improper.

Author reply: We agree and have changed the text so the sentence now starts as followed (L53): “For example, Bartlett et al (2012) observed a strong…”

Second, the relationship between drought tolerance with morphological and physiological traits should be hypothesized.

Author reply: We have stated the following hypothesis (L112-115): (i) Alpine ash provenances will exhibit variability in their functional traits across a climatic gradient, (ii) Morpho-physiological traits would be significantly correlated to each other, and (iii), there would be a significant correlation between functional trait expressions and climate of origin. These hypotheses are based upon the literature we cited in the introduction. The term “drought tolerance” is not easily defined and can mean different things, depending on the context. The traits we measured are often used to infer “drought tolerance” but it would be incorrect to hypothesise that there is a correlation between traits and drought tolerance.

 

 

Reviewer comment: METHODS: The differences of drought tolerance in 21 provenances of a montane tree species should be further determined and then estimated other relationship.

Author reply: It is not clear what the reviewer is asking. We evaluated the differences in trait expression among 21 provenances of E. delegatensis in order to evaluate if there is any evidence for genetic adaptation of traits that could lead to differences in drought tolerance. We clearly demonstrate that this is not the case. Functional trait expression did not vary and there is no evidence that drought tolerance would be different between the provenances. What “further determinations” would improve this clear result?

 

Reviewer comment: RESULTS: The results of the differences of drought tolerance in 21 provenances of a montane tree species s and the relationship between drought tolerance with morphological and physiological traits should be supplemented

Author reply: The term “drought tolerance” is difficult to define and we caution to use it in an over-simplified way. We have been very careful throughout the manuscript and very clear that we investigate differences in functional trait expression among the 21 provenances of E. delegatensis. Measuring “drought tolerance” per se is very difficult and usually requires a different experimental design. We can conclude, with confidence and evidence, that these 21 provenances did not differ in their expression of functional traits that are related to drought tolerance and that their “drought tolerance” is therefore likely not genetically different. However, we also make it clear that expression of traits is also influenced by environmental conditions like periods of drought or heat that can be locally different. This can lead to plasticity in trait expression and that can influence “drought tolerance”. We have discussed these relationships in the Discission (L410-427).

 

Reviewer comment: DISCUSSION: First, the relationship between drought tolerance with morphological and physiological traits should be further discussed. And then, the causes that provenances would not have different drought tolerant responses should be further discussed according to physiological, morphological, molecular, and genetic traits.

Author reply: We believe that we have discussed the results in sufficient depth. If there are any specific aspects that we can add then we would be happy to consider. We explain why the variation between traits is so small among the provenances (L381-394) and conclude that the conditions of the natural environment in which E. delegatensis is naturally occurring are not leading to sufficient selective pressure that would have selected for functional traits that are associated with greater drought tolerance. We highlight the difference between genetic and environmental influences of trait expression (L410-427) and conclude that trait expression in our experiment was mainly controlled by plant genetics and thus that we have no evidence for adaptive processes. We make it clear that trait expression could be plastic and that in the field under natural conditions trait expression could be different. Thus, we explain that “drought tolerance” (as concluded from trait expression) does not differ between provenances and we also explain why that is the case.

 

Reviewer comment: New relative references published in 2024-2025 should be suitably supplemented.

Author reply: We have added three additional references published in 2024-25 that are relevant to the topic (see below). We have also added additional references in relation to the additional analyses carried out.

 

Blackman, C. J., Halliwell, B., & Brodribb, T. J. (2024). All together now: A mixed-planting experiment reveals adaptive drought tolerance in seedlings of 10 Eucalyptus species. Plant Physiol, 197(1). doi:10.1093/plphys/kiae632

 

Kibria, M. G., Tjoelker, M. G., Marchin, R. M., Arndt, S. K., & Rymer, P. D. (2024). Can species climate niche predict canopy growth, functional traits and phenotypic plasticity in urban trees? Urban Forestry & Urban Greening, 98. doi:10.1016/j.ufug.2024.128417

 

Anderson, N. S., Fontaine, J. B., Lewandrowski, W., Walden, L., & Ruthrof, K. X. (2025). Drought and Wildfire Legacies Highlight Vulnerability of a Mediterranean Climate‐Type Forest. Austral Ecology, 50(1). doi:10.1111/aec.70011

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I agree that your study was not the response of alpine ash to drought but only to see if there are genetical adpatations of functional traits to climate origin under a good watering. You showed that there almost no differences betwwen the different seedlings origins. But with these data, I think you cannot conclude about drought tolerance for this species since you did not have test it. You are not able to show the plasticity of each seedling provenance, crucial parameter to assess the resistance/resilience to drought.

Because, it is highly possible that when well watered, tested individuals did not have to put in place any resistance mechanisms to drought whereas, you could observe differences when water stress (with a range mimicking the expected climate change) is applied, especially for those coming from wetter area (up to 1800mm/year in precipitation). Thus, you miss a part of the global picture.

I totally understand that you are probably not able to redo this experiment but at least I would be more carefull in the discussion/conclusion. I would add a part on the discussion about traits responses between control/stress trees

Author Response

Reviewer comment: I agree that your study was not the response of alpine ash to drought but only to see if there are genetical adpatations of functional traits to climate origin under a good watering. You showed that there almost no differences betwwen the different seedlings origins. But with these data, I think you cannot conclude about drought tolerance for this species since you did not have test it. You are not able to show the plasticity of each seedling provenance, crucial parameter to assess the resistance/resilience to drought.

Response: Thank you for the comment. We disagree that we cannot comment on the drought tolerance of the species because we have not tested its drought response in an experiment. There is plenty of evidence in the literature that the drought resistance of a species is conveyed by the expression of traits and by the ‘strategy’ a plant is employing. E.g. a plant can be drought resistant by being a drought avoider: e.g. by deep roots systems or by growing near rivers or in ecosystems with shallow ground water. Plants can also be drought resistant by expressing a tolerance to drought. This drought tolerance will be conveyed through trait expressions that will allow the plant to tolerate low water potentials, otherwise it would not be drought tolerant. We measured four functional traits that are related to drought tolerance and none of these traits are, if compared to other species, indicative of a substantial drought tolerance. Thus, we can conclude that the species is not very drought tolerant and that would also not be expected given its environment. However, we have added a new paragraph in the discussion to explain this in more detail (L424-435, see text below).

Reviewer comment: Because, it is highly possible that when well watered, tested individuals did not have to put in place any resistance mechanisms to drought whereas, you could observe differences when water stress (with a range mimicking the expected climate change) is applied, especially for those coming from wetter area (up to 1800mm/year in precipitation). Thus, you miss a part of the global picture.

Response: Yes, we agree with this statement that trait expression could change if plants are stressed. But we disagree that we did not discuss it. In fact, we discuss this in an entire paragraph (L409-424) where we explicitly discuss that trait expression may change if tree would be studied in their natural environment. We also discuss the likely response mechanism of the species to drought in the environment (L396-407) and argue that the drought avoidance mechanisms (tight stomatal control of water loss, stem water storage) and the generally wet environment did not select for more drought tolerant traits. Thus, the reader is getting the full picture, and we are not claiming anything that is not substantiated by data.

Reviewer comment: I totally understand that you are probably not able to redo this experiment but at least I would be more carefull in the discussion/conclusion. I would add a part on the discussion about traits responses between control/stress trees

Response: We only measured trees under controlled conditions in a common garden and that is what we can discuss. However, we have added a new paragraph to the Discussion in order to address the concerns raised by the reviewer, which other readers may share (L424-435):

It is possible that all four functional traits could exhibit trait plasticity and adjust to local conditions. Yet, the environmental conditions in the natural environment with high elevation, low MAT and high MAP are not very drought prone. Thus, it is likely that any adjustments would be transient and drought avoidance responses outlined above are more likely than plasticity mediated drought tolerance. The functional traits of E. delegatensis are typical of a less drought tolerant species: high p₁₀₀., low g_min, large leaves, tall trees and a hydraulic architecture with large xylem vessels enabling an efficient water transport - and hydraulic traits in eucalypts appear to be largely genotypic in origin rather than environmentally plastic [16]. Thus, it is unlikely that a eucalypt could become drought tolerant by means of trait plasticity alone. However, future experiments could explore the drought tolerance of the species in stress experiments to further explore plastic trait responses.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript has been revised according to the comments.

Comments on the Quality of English Language

no

Author Response

Thank you!