A Role for Secondary Metabolites in Desiccation Tolerance in Lichens

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

 

The study is well designed and straightforward therefore the conclusions are solidly based. The manuscript is well written and correctly describes the results obtained. However in this kind of experiments it has to be taken into account that removal of secondary metabolites by the acetone method can be a source of undesirable artefacts and this is something that has to be specifically checked in each particular study. In this sense the values of most parameters (water content, Fv/Fm and NPQ) previous to the onset of desiccation treatments seem to differ substantially between control and acetone-rinsed samples. For example in the Results this effect is described “Lichen substance removal had little effect on the maximum water holding capacity…” but this is not quantified or statistically tested. However the statistical analyses performed do not allow to verify whether such differences are significant or not. Apart from the ANOVA, I suggest to compare control and acetone-rinsed samples at each time-point with a post-hoc analysis. Even if the initial values differ, the results are valuable itself and merit publication if this aspect is conveniently considered.

Some important details of the experimental design are missing or I can’t easily find them. For example it is said that experimental desiccations were performed at 5 µmol photons m-2 s-1. However If Fv/Fm was monitored during desiccation it requires dark-adaptation of the studied samples. As desiccation treatments were quite fast (60 min) and measurements were done at 10 min interval, it is difficult to understand how they were performed at low light and simultaneously Fv/Fm was measured. For clarity this needs a better description. In fact in the description of the experimental treatments it is indicated that measurements were taken at intervals of 60 min, but what I see in figures is that measurements were done every 10 minutes. It is not clear whether such incubation in darkness was performed over silica or under the lab conditions once the samples were removed from the chamber. Other point that needs clarification is how NPQ was measured, was it performed immediately after measuring Fv/Fm  and in the same spots?. 

I find water content in lichens after 1h in silicagel higher than what I could expect (e.g.  Crocodia), I wonder how was the water content after two weeks in silica gel when the rehydration experiments started (please provide these data).

Finally it is somewhat strange the pattern of response observed (and, consequently the role of secondary metabolites) differs between species. This point has been sufficiently addressed in the discussion.

Author Response

Reviewer 1

The study is well designed and straightforward therefore the conclusions are solidly based. The manuscript is well written and correctly describes the results obtained. However in this kind of experiments it has to be taken into account that removal of secondary metabolites by the acetone method can be a source of undesirable artefacts and this is something that has to be specifically checked in each particular study. In this sense the values of most parameters (water content, Fv/Fm and NPQ) previous to the onset of desiccation treatments seem to differ substantially between control and acetone-rinsed samples. For example in the Results this effect is described “Lichen substance removal had little effect on the maximum water holding capacity…” but this is not quantified or statistically tested. However the statistical analyses performed do not allow to verify whether such differences are significant or not. Apart from the ANOVA, I suggest to compare control and acetone-rinsed samples at each time-point with a post-hoc analysis. Even if the initial values differ, the results are valuable itself and merit publication if this aspect is conveniently considered.

Response: Yes, we accept that acetone rinsing can cause some damage. Careful inspection for example of Figure 2 shows that for several species, acetone rinsing caused a small reduction in Fv/Fm (which we acknowledge in the text). However, such reductions were very small relative to the reductions caused by desiccation. I suppose that in theory we could have carried out t-tests to confirm this, but as the reviewer indicates, even if significant this would not change the substance of the findings.

 

Some important details of the experimental design are missing or I can’t easily find them. For example it is said that experimental desiccations were performed at 5 µmol photons m-2 s-1. However If Fv/Fm was monitored during desiccation it requires dark-adaptation of the studied samples. As desiccation treatments were quite fast (60 min) and measurements were done at 10 min interval, it is difficult to understand how they were performed at low light and simultaneously Fv/Fm was measured. For clarity this needs a better description. In fact in the description of the experimental treatments it is indicated that measurements were taken at intervals of 60 min, but what I see in figures is that measurements were done every 10 minutes. It is not clear whether such incubation in darkness was performed over silica or under the lab conditions once the samples were removed from the chamber. Other point that needs clarification is how NPQ was measured, was it performed immediately after measuring Fv/Fm  and in the same spots?.

 

Thanks, yes, we should have acknowledged that in practice, the requirement for a 10 min dark adaptation period meant that much of the actual desiccation and rehydration were carried out in the dark. We have amended our text accordingly. And yes, NPQ was measured immediately after measuring Fv/Fm, again we have amended our text to reflect this.

 

I find water content in lichens after 1h in silicagel higher than what I could expect (e.g.  Crocodia), I wonder how was the water content after two weeks in silica gel when the rehydration experiments started (please provide these data).

 

Yes, the RWC did decline between 1 h and 2 weeks storage over silica gel, typically to RWCs of around 2-5% (a note to this effect has been added to the text).

 

Finally it is somewhat strange the pattern of response observed (and, consequently the role of secondary metabolites) differs between species. This point has been sufficiently addressed in the discussion.

 

Agree, the changes are not always 100% intuitive.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The topic is very interesting, however, the manuscript could be improved in various ways.

It is not explained why the given species were investigated by the authors. The authors background knowledge on these species is not convincing. The information on secondary metabolites is insufficient and mentioned as originating from literature sources. Nevertheless, these sources are not referred. Analysing the substances that were actually present in the investigated samples would be more informative, since the secondary metabolite content may change by habitats in various geographic areas.

The chemical content is indicated by mistakes, e.g. Parmotrema perlatum contains atranorin, but it is not mentioned. Cladoni conicraea contains fumarprotocetraric acid,  Cldaonia foliace contains usnic acid and fumarprotocetraric acid. It is not clear if any of these species or both of them were investigated.

It is not considered that the structure, growth form and symbiont partner (green alga/cynobacterium) has and influence on water uptake and also the form of water available for lichens - The time is not mentioned if it was standard or differed by species (as in nature) - have you checked it before application?

Lichens with green alga photobiont prefer water vapour, the liquid water might be ideal for lichens with cyanobacteria photobiont.

 

The experimental arrrangement should be explained in more details – e.g. why it was carried out in dark.

The literature applied for the study of ion leakage should be cited.

The description of the statistical analysis is incomplete and I hope, that you used ANOVA on the continuous variables (Fv/Fm, NPQ, % of the toal conductivity loss) which is missing from the text. One of the most important assumptions of the ANOVA: "One continuous (interval or ratio) dependent variable and two categorical (nominal or ordinal) independent variables with two or more levels."

How did you test that other assumptions of the ANOVA are met on your data? (homogeneity of variance, normal distribution)

It would be useful and informative to run an ANOVA on the water content data in case of the different species. Since you only draw conclusions on desiccation rate from Figure 1. However, the species respoded differently to the treatment. It would be interesting to know how significant statistically the differences in water content are between treated and control thalli  during desiccation as you wrote in case of Fv/Fm, NPQ and ion leakage. After that you could mark the significant differences (*) on Fig.1 in every time of measurement.

 

- Fig1: The data would be better comparable if the Y axes would have the same maximum values. The labels of the Y axes are Water content and the unit of the measurement is the g water g-1 dry mass in brackets.

 

- Fig.1, Fig.2: Cladonia coniocraea or Cl. foliacea (materials and methods)?

 

- Fig.4: You mentioned two weeks over silica gel in the text

 

- Table1: I think the effect of treatment on the desiccation and rehydration would also be very interesting or more interesting helping the reader to understand the responses of mycobiont and photobiont. Since your investigation is focusing on the effect of LSMs on desiccation stress.

 

- Discussion: Did you considered that the investigated lichens are characterised by different thallus structure (Usnea, Cladonia) that strongly influences the rate of desiccation and rehydration?

line 348-349 it would be more informative if you mentioned that (if it is documented) the investigated LSMs in your study are antioxidants or not. The same to line 353-354, if your investigated materials have a documented light screening effect.

line 364: if you mean Cl. foliacea, the species has an additional protecting strategy: curling during desiccation (Barták et al. 2006)

line 380-383: Dietz et al. 2000, Gauslaa et al. 2017

 

There are further notes directly in the manuscript.

As an overall evaluation the manuscript needs major revision.

Comments for author File: Comments.pdf

Author Response

Reviewer 2

 

The topic is very interesting, however, the manuscript could be improved in various ways.

 

It is not explained why the given species were investigated by the authors. The authors background knowledge on these species is not convincing. The information on secondary metabolites is insufficient and mentioned as originating from literature sources. Nevertheless, these sources are not referred. Analysing the substances that were actually present in the investigated samples would be more informative, since the secondary metabolite content may change by habitats in various geographic areas.

 

We have added some rationale to the choice of lichens used. Briefly, they represented the main macrolichens in the region we work (Afromontane / Savanna), with the exception on Cetraria which was collected during some field work by one of the authors while overseas. Yes, we fully accept that the study would have been stronger if the specific lichen substances present had been analysed. Unfortunately, we don’t have a working HPLC at the moment.

 

The chemical content is indicated by mistakes, e.g. Parmotrema perlatum contains atranorin, but it is not mentioned. Cladoni conicraea contains fumarprotocetraric acid,  Cldaonia foliace contains usnic acid and fumarprotocetraric acid. It is not clear if any of these species or both of them were investigated.

 

We searched the literature for substances typically present in the lichen species we used. As the Reviewer indicates, even within a single species, lichen substance composition is well-known to vary considerably both quantitatively and qualitatively between locations. Therefore, we simply indicated common substances reported in the literature for the species used, without making too much of potential effects of specific compounds.

 

It is not considered that the structure, growth form and symbiont partner (green alga/cynobacterium) has and influence on water uptake and also the form of water available for lichens - The time is not mentioned if it was standard or differed by species (as in nature) - have you checked it before application?

 

As indicated in the M&M, material was always rehydrated by keeping it on wet filter paper overnight. It is our experience that this will allow full turgor to be reached in all species.

 

Lichens with green alga photobiont prefer water vapour, the liquid water might be ideal for lichens with cyanobacteria photobiont.

 

This is quite true, although all the species used here were Chlorophycean (a note to this effect has been added to the text).

 

The experimental arrrangement should be explained in more details – e.g. why it was carried out in dark.

 

Agree, see response to Reviewer 1, these details have now been included.

 

The literature applied for the study of ion leakage should be cited.

 

Agree, an appropriate reference has now been cited (Munzi et al. 2009).

 

The description of the statistical analysis is incomplete and I hope, that you used ANOVA on the continuous variables (Fv/Fm, NPQ, % of the toal conductivity loss) which is missing from the text. One of the most important assumptions of the ANOVA: "One continuous (interval or ratio) dependent variable and two categorical (nominal or ordinal) independent variables with two or more levels." How did you test that other assumptions of the ANOVA are met on your data? (homogeneity of variance, normal distribution).

 

Classical two-way ANOVAs with the usual checks were carried out, more details added to the methods.

 

It would be useful and informative to run an ANOVA on the water content data in case of the different species. Since you only draw conclusions on desiccation rate from Figure 1. However, the species respoded differently to the treatment. It would be interesting to know how significant statistically the differences in water content are between treated and control thalli  during desiccation as you wrote in case of Fv/Fm, NPQ and ion leakage. After that you could mark the significant differences (*) on Fig.1 in every time of measurement.

 

We have now carried out ANOVAs on the water relations data, and the results appear in Table 1. Text also amended in the appropriate places.

 

- Fig1: The data would be better comparable if the Y axes would have the same maximum values. The labels of the Y axes are Water content and the unit of the measurement is the g water g-1 dry mass in brackets.

 

Unfortunately, water holding capacity varies considerably between species, so we prefer to keep the maximum water contents on the y-axes different.

 

- Fig.1, Fig.2: Cladonia coniocraea or Cl. foliacea (materials and methods)?

 

Apologies, should be C. coniocraea throughout.

 

- Fig.4: You mentioned two weeks over silica gel in the text

 

Thanks, there was an error in the legend to Figure 4, text should have read two weeks.

 

- Table1: I think the effect of treatment on the desiccation and rehydration would also be very interesting or more interesting helping the reader to understand the responses of mycobiont and photobiont. Since your investigation is focusing on the effect of LSMs on desiccation stress.

 

In Table 1, the effects of desiccation and rehydration on Fv/Fm and NPQ are considered separately, but for water contents only desiccation was considered, and for ion leakage only rehydration was considered.

 

- Discussion: Did you considered that the investigated lichens are characterised by different thallus structure (Usnea, Cladonia) that strongly influences the rate of desiccation and rehydration?

 

Yes, this is certainly true. However, the main point of the present study was to compare the effects of lichen substance removal on desiccation tolerance, rather than to compare rates of drying in different species.

 

line 348-349 it would be more informative if you mentioned that (if it is documented) the investigated LSMs in your study are antioxidants or not. The same to line 353-354, if your investigated materials have a documented light screening effect.

 

We understand what the reviewer is saying, and in theory it would be nice to relate specific abilities of substances to protect from desiccation with antioxidant capacity.

However, when we started to check we found that for many of the compounds that the literature indicated should be present in our species e.g., pulvinic, salizanic and stictic acids there was no or almost no specific data on antioxidant activities for these compounds. Furthermore, as we discuss in the Introduction, some compounds such as usnic acid appear to act as pro-oxidants or antioxidants depending on the species or probably presence of other compounds. Thus while there is a wealth of data suggesting that in general lichen substances can act as antioxidants, in many of these studies specific compounds are not identified, and in particular those present in some of the species tested here have not received enough attention.

 

line 364: if you mean Cl. foliacea, the species has an additional protecting strategy: curling during desiccation (Barták et al. 2006)

 

Apologies, as indicated above there was an error in the species (was coniocraea rather than foliacea)

 

line 380-383: Dietz et al. 2000, Gauslaa et al. 2017

 

Yes, there are several studies showing increased NPQ during drying (we reviewed them recently in Beckett et al. 2021), but here cited just one classical reference (Calatayud et al. 1997).

 

There are further notes directly in the manuscript.

 

Comments on the m/s

 

Lines 76 – 78. Yes, the comments about the need in future studies to actually determine which substances are present is true, and has been discussed above.

 

Line 131. We did not measure rehydration rates, they all appeared to be very rapid. However, we take the point about measuring more slow rehydration in future e.g. above air at 100% RH. This may allow more sensitive separation between material with and without lichen substances.

 

Line 169. “Have you thought of that this species contains a large amount of lichenin - not removed by acetone?”

This is an interesting point, but we searched the literature and could find no references to lichenin having an antioxidative effect.

 

Line 328. “According to Swinscow and Krog (1988) this species (Ramalina celastri) does not contain any medullary substances, while usnic acid is in the cortex only.”

Also a good point, the location within the thallus of the secondary metabolites probably does influence their effectiveness at acting as antioxidants, but to investigate this further would be beyond the scope of the present study.

 

Line 362. “it must be a mistake, it contains fumarprotocetraric acid - soluble in water, thus if it was collected in a humid period, not just usnic acid was missing, but very little amount of fumarprotocetratic could be present”

Thanks, yes, it was a mistake, the major lichen substance in C. coniocreaea is indeed  fumarprotocetraric acid. Text has been amended accordingly.

 

Line 430. This variation is very important - there could be more emphasis on this fact. There are several thousands of lichen secondary metablolites of various chemical structures that may cause alterations in effects. Current authors mention also that future work is needed in this direction. It is also mentioned above that the amount (concentration) of substances should also be known.

We agree, future studies need to consider the actual levels of lichen substances as well as the specific substances present, we have amended the text.

 

Reviewer 3 Report

Comments and Suggestions for Authors

Please see the attached file.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Please see the attached file.

Author Response

Please see the attached file.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Lines 62-64

In the Intro remove the comments about the Methods

"The tolerance of the mycobiont to desiccation was assessed by measuring membrane leakage of electrolytes."

Line 65 -66-

In the Intro remove the comments about Results

"Results showed that while generally secondary metabolites appear to reduce the effects of desiccation stress, in some species the substances apparently act as prooxidants and exacerbate stress."

Line 76 --- this section of the methods would be better as a table format. "From the literature, the main lichen substances in these species have been reported to be for Crocodia aurata: calycin and pulvinic acid; for Parmotrema perlata: salizanic and stictic acid; for Ramalina celastri, Usnea undulata and Cladonia coniocraea: usnic acid; and for Cetraria islandica: fumaprotetraric acid."

The figures are well done.

In the Diss.

Ramalina celastri and Parmotrema perlata are often associated with marine environments and the Ramalina has be reported to be somewhat salt tolerant. So it is interesting that they have reacted different that the other lichen taxa to this treatment. This could be associated with salt tolerance? 

Author Response

Reviewer 4

 

Lines 62-64

In the Intro remove the comments about the Methods. "The tolerance of the mycobiont to desiccation was assessed by measuring membrane leakage of electrolytes."

Our aim of mentioning “methods” in the Introduction is to emphasize that we measured the effect of desiccation on both the mycobiont and photobiont. We therefore prefer to leave the comments in the Introduction.

 

Line 65 -66-

In the Intro remove the comments about Results: "Results showed that while generally secondary metabolites appear to reduce the effects of desiccation stress, in some species the substances apparently act as prooxidants and exacerbate stress."

It is standard practice to very briefly summarise the main conclusions of the m/s at the end of the Introduction. While some may view this as repetition, it does help the reader to understand what we consider to be the main findings of our study.

 

Line 76 --- this section of the methods would be better as a table format. "From the literature, the main lichen substances in these species have been reported to be for Crocodia aurata: calycin and pulvinic acid; for Parmotrema perlata: salizanic and stictic acid; for Ramalina celastri, Usnea undulata and Cladonia coniocraea: usnic acid; and for Cetraria islandica: fumaprotetraric acid."

We could present this information as a Table but feel that it is probably unnecessary in this instance.

 

The figures are well done.

 

In the Diss. Ramalina celastri and Parmotrema perlata are often associated with marine environments and the Ramalina has be reported to be somewhat salt tolerant. So it is interesting that they have reacted different that the other lichen taxa to this treatment. This could be associated with salt tolerance?

There are certain similarities between salt tolerance and desiccation, with salt tolerance often described as inducing “physiological drought”. While it is possible that R. celastri and P. perlata may have additional stress tolerance mechanisms that confound the results presented here, we feel that to comment on this would be rather speculative.

 

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

 

The paper has been improved in several places, however some remarks were neglected.

Also the new texts need corrections.

 

line 78 – literature on species and their main metabolites is not mentioned – please refer Swinscow and Krog 1988; Smith et al. 2009 or any identification key, monographs you used for this information

line 85 – change „few d” to „few days”

line 108 – change „occasional” to „occasionally”

 

Considering that the investigated lichens are characterised by different thallus structure (Usnea, Cladonia) that strongly influences the rate of desiccation and rehydration

authors answer that

the main point of the present study was to compare the effects of lichen substance removal on desiccation tolerance, rather than to compare rates of drying in different species.

 

It is a conceptionally important assumption if the effect of LSMs and thallus structure on the desiccation rate of the lichen thallus is separated or not. For a further study the effect of one variable shoud be investigated on the organism only while the other variables are kept the same. For example, you could investigate lichens characterised by different thallus structures and containing the same LSMs or lichens containing different LSMs, but their thallus structure is the same. In your study, both thallus structures and LSMs are different, and the LSM content is unknown. The suggested arrangemens would add to your current results.

Author Response

line 78 – literature on species and their main metabolites is not mentioned – please refer Swinscow and Krog 1988; Smith et al. 2009 or any identification key, monographs you used for this information

 

Agree, have added.

 

line 85 – change „few d” to „few days”

 

Changed as requested

 

line 108 – change „occasional” to „occasionally”

 

Changed as requested

 

“Considering that the investigated lichens are characterised by different thallus structure (Usnea, Cladonia) that strongly influences the rate of desiccation and rehydration”, authors answer that: “the main point of the present study was to compare the effects of lichen substance removal on desiccation tolerance, rather than to compare rates of drying in different species.”

It is a conceptionally important assumption if the effect of LSMs and thallus structure on the desiccation rate of the lichen thallus is separated or not. For a further study the effect of one variable shoud be investigated on the organism only while the other variables are kept the same. For example, you could investigate lichens characterised by different thallus structures and containing the same LSMs or lichens containing different LSMs, but their thallus structure is the same. In your study, both thallus structures and LSMs are different, and the LSM content is unknown. The suggested arrangemens would add to your current results.

 

We certainly accept that thallus structure may affect the rates of drying. It is possible that the effect of lichen substance removal may vary according to drying rate. However, if, as we suggest, and major part of the reason why substances are generally protective is due to their antioxidative properties, then it is hard to imagine why there should be an interaction between drying rate and substance removal. The authors have discussed this question, and on reflection we think it would be useful in future to take a single species that responds well to substance removal, and then to dry this species at different rates with and without lichen substances. This would provide useful information as to whether the effect of substance removal differs according to thallus structure. We have added some sentences to the Conclusions to this effect.

Reviewer 3 Report

Comments and Suggestions for Authors

Please see the attachment.

Comments for author File: Comments.pdf

Author Response

We have carefully viewed the attached file, but can find no comments to respond to.