Responses to Drought in Seedlings of European Larch (Larix decidua Mill.) from Several Carpathian Provenances
, Adriana F. Sestras 1, Oscar Vicente 2, Jaime Prohens 4, Monica Boscaiu 3,* and Radu E. Sestras 1Round 1
Reviewer 1 Report
Dear authors,
in this study six European larch population were tested for drought tolerance. The drought reaction was measured based on several growth and biochemical traits. In general, the article brings interesting results, which increase our knowledge about drought tolerance and biochemical reaction of European larch populations. Nevertheless, I think that the paper needs major revision, before it could be accepted for publication. Overall, the very minor drought reaction of two month old larch seedlings after period of one month without water is surprising. It seems that the drought stress was very very mild as could be indicated by very high needle WC% (around 77%) at the end of experiment. Although you measured that the soil humidity decreased by 50%, it doesn´t mean that your seedlings were subjected to drought stress. It will be very helpful for next experiments, if some physiological traits as shoot water potential and stomatal conductance will be measured to identify drought stress level for each population. I think you have to be more critical at this point and you have to write in your discussion and results that seedlings were subjected to very mild drought stress. Maybe, you can calculate soil water potential based on your soil data. It is also not clear if your seedlings were subjected to the same environmental condition during drought period -i.e. 16 h light with 23 °C and 8 h dark with 17 °C and RH around 50-80%. If you know this environmental condition you can also calculate vapour pressure deficit and potential evapotranspiration. It will give us more information about eventual drought stress, but anyhow we will be not sure about the stress level in seedlings. The other point is, that you wrote that your analyzed European larch populations, except BVVP, are relatively tolerant to drought stress. I´m not sure about this statement. Although the growth reduction of BVVP population was higher than in the other populations, the growth traits of BVVP were still higher after one month of "drought" than in the other populations. It seems that BVVP population has higher plasticity than the other populations as it grows better under control as well as drought conditions than the other population. See my other comments below:
line 2 - TITLE: Although water stress is usually connected to drought stress it could be also connected with hypoxia. In context of your paper I will prefer to use the term "drought stress" here and elsewhere in the paper.
line 33 Drought stress lead to stem length decrease in all population, whereas ....
line 33 - "fresh weight" of what??
line34 - water content of what??
line 38 delete "practically" and "significantly"
line 41 - Can you give some example of trait correlations?
line 42 - see my comment above
line 43 - the last sentence could be deleted. It is similar to former one.
line 50-52 - It is also true for Central Europe?
line 59 provenances x populations - be consistent and use only one term
line 62 Delete this sentence. Similar to bellow sentence.
line 102-104 - The large variability for drought resistance of tree could also exist within population
line 122 - give space between number and unit (23 °C)
line 120-123 - This condition was also keep during drought period?
line 134 - fresh weight of the shoots. Does it mean FW of five shoots? Does it mean fresh weight of needles+stem? Figure 2 shows only fresh weight of needles .
line 137-141 -be consistent and use only one term - soil moisture x soil water content x soil humidity % x substrate humidity
line 143-185 - How much needle dry weight did you use for each analysis?
line 201 - humidity of what?
line 213 give reference to Fig.
line 241-241 - or due to very very mild drought stress
line 246 - give reference to Fig.
line 247-248 the last sentence could be deleted
line 284-285 - delete
line 289-292 - Do you have some idea how from when the soluble sugar came from? Starch, sugar translocation from roots?
line 288 delete "(even though slight)"
line 305 - ... an increase in studied populations, except of Anin and Sace, ...
line 307-308 - delete
line 312-313 - delete the last sentence
line 315-316 - delete the last sentence
line 363-364 - Did HCA confirm PCA results? It seems that both clearly separated control and drought treated seedlings, but the grouping within treatment is different.
line 370 - This is new abbreviation -"WC%"?
line 381-397 - This is partly repetition of Introduction. Make this part shorten and focus on your results.
line 398-399 - This pattern is also valid for control trees?
line 400 - write "salt stress" instead of "150 mM NaCl"
line 406-409 - connect these sentences
line 409-413 - Make these sentences more readable
line 417-418 - These biochemical markers were tested in the field on adult trees? Do adult trees have similar biochemical reaction as seedlings?
line 419-429 - You should also mentioned leaf phenology as leaf development is important factor for photosynthetic pigments content.
line 433 - Why it was expected?
line 434-436 - Can you write some physiological meaning of these correlations?
line 442 - see paper (45). There is no increment of Pro with drought stress.
line 442-447 - Make this part shorter as it is mostly connected to salt stress.
line 450-451 - or your drought stress was very mild
line 452-456 - Describe possible ways of soluble sugar increase (starch - root reserves??)
line 476 - only minor differences between populations --- but according to HCA and PCA they were differ
line 476-478 - your drought stress was very mild!!!!
line 482-484 - Again they were subjected to very mild drought stress.
Author Response
Thank you very much for your comments and suggestions that allowed us to improve the quality of the manuscript. We agree with all your comments, and we corrected point by point the manuscript accordingly. Please find our comments in italics and red color.
In this study six European larch population were tested for drought tolerance. The drought reaction was measured based on several growth and biochemical traits. In general, the article brings interesting results, which increase our knowledge about drought tolerance and biochemical reaction of European larch populations. Nevertheless, I think that the paper needs major revision, before it could be accepted for publication. Overall, the very minor drought reaction of two month old larch seedlings after period of one month without water is surprising. It seems that the drought stress was very very mild as could be indicated by very high needle WC% (around 77%) at the end of experiment. Although you measured that the soil humidity decreased by 50%, it doesn´t mean that your seedlings were subjected to drought stress. It will be very helpful for next experiments, if some physiological traits as shoot water potential and stomatal conductance will be measured to identify drought stress level for each population. I think you have to be more critical at this point and you have to write in your discussion and results that seedlings were subjected to very mild drought stress. Maybe, you can calculate soil water potential based on your soil data. It is also not clear if your seedlings were subjected to the same environmental condition during drought period -i.e. 16 h light with 23 °C and 8 h dark with 17 °C and RH around 50-80%. If you know this environmental condition you can also calculate vapour pressure deficit and potential evapotranspiration. It will give us more information about eventual drought stress, but anyhow we will be not sure about the stress level in seedlings. The other point is, that you wrote that your analyzed European larch populations, except BVVP, are relatively tolerant to drought stress. I´m not sure about this statement. Although the growth reduction of BVVP population was higher than in the other populations, the growth traits of BVVP were still higher after one month of "drought" than in the other populations. It seems that BVVP population has higher plasticity than the other populations as it grows better under control as well as drought conditions than the other population. See my other comments below:
We agree with the reviewer’s point of view, that although the irrigation was withhold for one month, the loss of water in the substrate was not so pronounced. We calculated as suggested the vapour pressure deficit and approximate the potential evapotranspiration and this new information was introduced in in the Sections Material and Methods and Results. We introduced as suggested the term mild drought and we explained in the Discussion of the results that the stress was not so accentuated.
We also agree that population BVVC (by mistake we have written in the first version BVVP) shows a higher plasticity as its Fresh weight is significantly higher than of the others in both control and drought treatments.,
line 2 - TITLE: Although water stress is usually connected to drought stress it could be also connected with hypoxia. In context of your paper I will prefer to use the term "drought stress" here and elsewhere in the paper.
The term water stress has been replaced by drought stress in the title and throughout the text.
line 33 Drought stress lead to stem length decrease in all population, whereas
Corrected
line 33 - "fresh weight" of what??
Needles, corrected
line34 - water content of what??
Needles, corrected
line 38 delete "practically" and "significantly"
Deleted
line 41 - Can you give some example of trait correlations?
A new sentence has been added. Substrate moisture is positively correlated with growth parameters of seedlings and K levels in needles and negatively with MDA, total soluble sugars, total phenolic compounds and total flavonoids.
line 42 - see my comment above
According to the reviewer’s point of view, we deleted the part of the sentence stating that population BVVC was the most affected by stress.
line 43 - the last sentence could be deleted. It is similar to former one.
The sentence has been deleted
line 50-52 - It is also true for Central Europe?
Considering also the comments of the second reviewer we deleted this sentence and we reformulated the paragraph.
. line 59 provenances x populations - be consistent and use only one term
The term provenance was used throughout the text, replacing that of population
line 62 Delete this sentence. Similar to bellow sentence.
The sentence has been deleted
line 102-104 - The large variability for drought resistance of tree could also exist within population
Individual variability was mentioned in text as suggested (line 117)
line 122 - give space between number and unit (23 °C)
Done
line 120-123 - This condition was also keep during drought period?
The same conditions were maintained throughout the whole experiment. We added this information to the text
line 134 - fresh weight of the shoots. Does it mean FW of five shoots? Does it mean fresh weight of needles+stem? Figure 2 shows only fresh weight of needles .
It was corrected. Fresh weight of the needles
line 137-141 -be consistent and use only one term - soil moisture x soil water content x soil humidity % x substrate humidity
The term Substrate humidity was chosen and used throughout the text.
line 143-185 - How much needle dry weight did you use for each analysis?
0.05 g. The amount of material was specified in Material and Methods
line 201 - humidity of what?
Substrate, corrected
line 213 give reference to Fig.
Done
line 241-241 - or due to very very mild drought stress
Modified
line 246 - give reference to Fig.
Done
line 247-248 the last sentence could be deleted
Deleted
line 284-285 – delete
Done
line 289-292 - Do you have some idea how from when the soluble sugar came from? Starch, sugar translocation from roots?
Soluble sugars are primary products of photosynthesis, but their levels can also increase by remobilization from storage reserves in stem and roots and translocation to the leaves. In the early stages or under mild drought stress the concentration of TSS usually increases because growth declines earlier than photosynthesis. It has been answered in the Discussion (lines 519-527)
line 288 delete "(even though slight)"
Done
line 305 - ... an increase in studied populations, except of Anin and Sace, .
Done..
line 307-308 – delete
Done
line 312-313 - delete the last sentence
Done
line 315-316 - delete the last sentence
Done
line 363-364 - Did HCA confirm PCA results? It seems that both clearly separated control and drought treated seedlings, but the grouping within treatment is different.
HCA confirmed the PCA as they indeed separated the two treatments but they are not completely overlapping, as the PCA is an analysis of principal components in two dimensions, whereas the HCA is a cluster in only one dimension, that uses a different computational method based on correlations. However, as the second reviewer observed, the grouping of provenances is similar. We modified accordingly the text, to avoid confusions (lines 403-405)
line 370 - This is new abbreviation -"WC%"?
We replaced WC% by WC (the initially used abbreviation) throughout the text
line 381-397 - This is partly repetition of Introduction. Make this part shorten and focus on your results.
This part has been shortened to avoid repetitions
line 398-399 - This pattern is also valid for control trees?
The pattern is the same in both treatments. This observation has been added (line 439).
line 400 - write "salt stress" instead of "150 mM NaCl"
Done
line 406-409 - connect these sentences
Done
line 409-413 - Make these sentences more readable
The sentences have been changed
line 417-418 - These biochemical markers were tested in the field on adult trees? Do adult trees have similar biochemical reaction as seedlings?
Yes, we introduced two new citations referring to the effects of stress in adult trees from natural environments
line 419-429 - You should also mentioned leaf phenology as leaf development is important factor for photosynthetic pigments content.
Leaf phenology
line 433 - Why it was expected?
As expected has been deleted
line 434-436 - Can you write some physiological meaning of these correlations?
We would like to apologise for a mistake in the previous version, K levels are positively correlated (not negatively) with growth parameters and the humidity of the substrate. We have reformulated the sentence, eliminating comments on TSS, MDA and antioxidants that are commented further in the Discussion.
line 442 - see paper (45). There is no increment of Pro with drought stress.
It has been added to the text, line 495
line 442-447 - Make this part shorter as it is mostly connected to salt stress.
It has been reduced
line 450-451 - or your drought stress was very mild.
It has been added, lines 496-497
line 452-456 - Describe possible ways of soluble sugar increase (starch - root reserves??)
It has been answered in the Discussion (lines 519-527)
line 476 - only minor differences between populations --- but according to HCA and PCA they were differ
As explained above
line 476-478 - your drought stress was very mild!!!!
We use the term mild stress, line 526
line 482-484 - Again they were subjected to very mild drought stress.
We used the term Mild drought stress, line 32
Reviewer 2 Report
The paper is clear and interesting. It compares seedlings’ responses of six larch’s provenances to a one-month long drought in order to assess if differences in responses could be detected by biochemical traits.
Since the provenances are all located in a quite narrow geographic space, the authors have to be clearer when presenting the background and proposing their study’s hypotheses. In fact, the work is based on a previous paper about population genetic of larch in Romania. Since the paper reported lack of a strong genetic structure, with low differentiation among populations, I think it is important to justify the usefulness of the experiment the authors are presenting here. Otherwise, I’d retain it is a no-sense to compare populations not differing in the genetic pool. Thus, I suggest strengthening the introduction, by adding more relevant elements about population genetics and the possible interaction of the genetics with the environment.
ll. 47-48
“Trees, with long life cycles, cannot easily adjust to the rapid effects of climate change”
It really sounds limiting, mechanisms to adapt and cope with rapid variations exist, thus I do not totally agree on this. I’d be less strict
ll. 49-50
“European coniferous forests are among those ecosystems forecasted to be most susceptible to the effects of global warming”
Can you briefly cite some of these effects and what this susceptibility consists of?
ll. 82-83
Please, be exhaustive when describing the distribution range, since I cannot understand why you report about species’ chorology in Romania. So please, introduce here its presence in Eastern Europe
ll. 99-100
I would like to see in this introduction more details about this genetic diversity, at least to read about the existence of a structure or sub-populations. Actually, the paper you recall does not state the existence of a strong genetic structure. Thus, I invite to be more precise
ll. 111-115
I suggest including the map here since in my opinion it is really important to see the locations of populations right during reading the paper, instead of referring to a different one.
ll. 137-139
Did you measure soil humidity only at the end of the experiment? How have you assessed the conditions throughout the whole experiment? How much water did you add to the control?
l. 152
You have not reported how you measured leaf DW. Moreover, this is the first time you use this label; therefore, you have to explicit its meaning.
ll. 365-366
Why did you assert “However the cluster topology was different under the two conditions and a different grouping of accessions was observed”? I can see somehow figure 7 and 8 collide, and I’d say as expected! In fact, for example, concerning control, the scatter in fig.7 implies that actually you cannot cluster the sites. However, BVVP is isolated and the farthest provenance. In both charts. As regards the drought, I can actually see the same clusters with Anin isolated, Lato and GuHo close to each other, as well as Sace and BVVP really close.
Additionally, I’d be cautious in utilizing the word “accession”, that it is out of this context. I’d rather use the simple “population”.
Fig. 7
The a) figure needs to be better arranged. For example: add the percentage of explained variability on the x- and y-axes, use the dot as decimal separator in all your figures (actually this has to be made in all figures).
I guess the caption presents a mistake at line 358-359 “observed in six Romanian populations of L. decidua from control plants (blue dots) OR submitted to water stress (red dots)”
Author Response
Thank you very much for your comments and suggestions that allowed us to improve the quality of the manuscript. We agree with all your comments, and we corrected point by point the manuscript accordingly. Please find our comments in italics and red color.
The paper is clear and interesting. It compares seedlings’ responses of six larch’s provenances to a one-month long drought in order to assess if differences in responses could be detected by biochemical traits.
Since the provenances are all located in a quite narrow geographic space, the authors have to be clearer when presenting the background and proposing their study’s hypotheses. In fact, the work is based on a previous paper about population genetic of larch in Romania. Since the paper reported lack of a strong genetic structure, with low differentiation among populations, I think it is important to justify the usefulness of the experiment the authors are presenting here. Otherwise, I’d retain it is a no-sense to compare populations not differing in the genetic pool. Thus, I suggest strengthening the introduction, by adding more relevant elements about population genetics and the possible interaction of the genetics with the environment.
You have raised an interesting issue. It is true that in a former study by our group [21] we found that only one-sixth of the total variation detected with SSR markers was due to variation among population. However, one-sixth is not a negligible proportion and the differentiated genomic regions may harbor genes with important adaptive functions that may be of relevance for adaptation to local conditions. In this way, we have added the following sentences to the Introduction section: “A previous study [21] in which these populations were genetically characterized revealed that these populations, despite displaying high levels of heterozygosis at the individual level and variation among individuals within the same population, had a moderate degree of genetic differentiation, indicating that some alleles are more frequent in certain populations than in others. This genetic differentiation, albeit limited, may have important implications for adaptation to specific environments, as natural selection and other microevolutive forces may have favored the fixation or increase of frequency of certain adaptive alleles or genomic regions for adaptation to local conditions in the different populations evaluated.”
ll. 47-48
“Trees, with long life cycles, cannot easily adjust to the rapid effects of climate change”
It really sounds limiting, mechanisms to adapt and cope with rapid variations exist, thus I do not totally agree on this. I’d be less strict
We deleted the complete sentence
ll. 49-50
“European coniferous forests are among those ecosystems forecasted to be most susceptible to the effects of global warming”
Can you briefly cite some of these effects and what this susceptibility consists of?
Although global warming results in increasing atmospheric CO2 and warmer temperatures, the negative effects, such as longer and more frequent drought episodes or potentiated risk of disturbances will overweigh the positive trends and may cause increased tree mortality in forests stands in southern and eastern Europe. Lines 54-56.
ll. 82-83
Please, be exhaustive when describing the distribution range, since I cannot understand why you report about species’ chorology in Romania. So please, introduce here its presence in Eastern Europe.
We reduced information about the chorology of L. decidua in Romania and referred to its European distribution. Accordingly the literature cited has been changed
ll. 99-100
I would like to see in this introduction more details about this genetic diversity, at least to read about the existence of a structure or sub-populations. Actually, the paper you recall does not state the existence of a strong genetic structure. Thus, I invite to be more precise.
Please read the answer to your general commentary. The text has been changed according to your suggestion. A new paragraph has been introduced (lines 106-116).
ll. 111-115
I suggest including the map here since in my opinion it is really important to see the locations of populations right during reading the paper, instead of referring to a different one.
A map has been included (Figure 1) replacing Table 1.
ll. 137-139
Did you measure soil humidity only at the end of the experiment? How have you assessed the conditions throughout the whole experiment? How much water did you add to the control?
Soil humidity was measured at the end of the treatments. Controls were watered twice per week with 1L of nutrient solution added to the plastic trays that contained 10 pots of 1L. This explanation was added in the Material and Methods section (lines 148-151).
l. 152
You have not reported how you measured leaf DW. Moreover, this is the first time you use this label; therefore, you have to explicit its meaning.
At the end of the treatments, seedlings were sampled, and the length of stems and the total weight of needles were measured. Part of the needles were oven-dried at 65 ºC for 72 h and weighted again for the calculation of the percentage of dry weight (DW). This information has been added (lines 152-154)
ll. 365-366
Why did you assert “However the cluster topology was different under the two conditions and a different grouping of accessions was observed”? I can see somehow figure 7 and 8 collide, and I’d say as expected! In fact, for example, concerning control, the scatter in fig.7 implies that actually you cannot cluster the sites. However, BVVP is isolated and the farthest provenance. In both charts. As regards the drought, I can actually see the same clusters with Anin isolated, Lato and GuHo close to each other, as well as Sace and BVVP really close.
We completely agree with you. Our initial comment was misleading. The text has been changed (lines 418-422).
Additionally, I’d be cautious in utilizing the word “accession”, that it is out of this context. I’d rather use the simple “population”.
The word accession has been deleted.
Fig. 7
The a) figure needs to be better arranged. For example: add the percentage of explained variability on the x- and y-axes, use the dot as decimal separator in all your figures (actually this has to be made in all figures).
All figures have been changed
I guess the caption presents a mistake at line 358-359 “observed in six Romanian populations of L. decidua from control plants (blue dots) OR submitted to water stress (red dots)
You are right, it has been corrected”
I
Round 2
Reviewer 1 Report
The authors answered all my questions and the article is now greatly improved. I have only few minor points:
Check once more your terms and abbreviations (WC% x WC; substrate humidity x substrate water content).
Is your equation for substrate humidity correct? Should it be (SW - DWS)??
Author Response
Once again many thanks for your comments. You were right, there was a mistake in the formula, and the correct one is the one indicated by you..
We have checked and used throughout the text the abbreviation WC for water content instead of WC% and substrate humidity instead of soil humidity or soil (substrate) moisture.
