Review Reports

Round 1

Reviewer 1 Report

Dear Ilya and co-authors,

I was my great pleasure to read your manuscript. I think it will represent an important contribution to the state of knowledge about the light-screening roles of atranorin and other lichen secondary metabolites. The paper is very detailed and your results are convincing. The science seems to be very sound. I only have a few comments/questions about the science in the attached .pdf file. I only have a few minor comments about the clarity of the methods, and the results. Throughout the manscript, I also noticed that the english grammar was poor in some paragraphs making it very difficult to read and interpret what you were trying to say. I would recommend that you work closely with an editor to improve the english, if possible.

This is very interesting work, thank you for sharing it and good luck with the review process.

Best,

Anonymous

See the attached file.

Comments for author File: Comments.pdf

Author Response

We cordially thank the reviewer for the attention paid to our work, for their constructive sugestions and for the important comments that helped to improve the manuscript. Below we respond to reviewer’s comments point-by-point and specify the edits that were introduced to the manuscript.

 

Comment 1, L20–21: It might be nice to start by saying that while lichens are exposed to fluctuating light and must protect themselves, they must also present their algal symbionts to the light in an efficient manner to facilitate photosynthesis.

RESPONSE 1: 
We thank the reviewer for this elegant suggestion to further highlight the dilemma for lichen protection against excessive radiation. Still we are formally limited by the abstract length of 200 words and hope that the editor will kindly allow us to slightly exceed the limit in favor for scientific precision. The first sentence was rewritten as follows:
“Epiphytic lichens are naturally subjected to highly fluctuating light and must protect their algal symbionts from harmful short wavelengths while facilitating efficient photosynthesis.”

Comment 2, L33: This is awkward.

RESPONSE 2:
This piece was rewritten as well as the first sentence of the Abstract.

Comment 3, L99: Was the spectrometer calibrated regularly using a reflectance standard? If so, describe these methods.

RESPONSE 3:
Yes, the spectrophotometer was calibrated against its internal reflectance standard prior to each measurement. This behavior is standard for both the ordinary software by X-Rite and for ‘spotread’ console utility from ArgyllCMS. There is an option to suppress such calibration while the instrument is still connected to the computer during the repeated measurements, however we never discarded this initial step for sake of the measurements precision and stability. Below we provide a sample terminal output from ‘spotread’ to illustrate that the calibration is performed prior to any measurement:

$ spotread -v -H -s tmp/20260928-sample-reflectance.txt
Connecting to the instrument ..
Instrument Type:   ColorMunki
Serial Number:     2037289
Firmware version:  288
Chip ID:           01-88BEB6130000AD
Version string:    'colormunki FW V1.32  Build Nr. 1303'
Calibration Ver.:  6
Production No.:    58319
Init instrument success !

Spot read needs a calibration before continuing
Set instrument sensor to calibration position,
and then hit any key to continue,
or hit Esc or Q to abort:
Calibration complete

Place instrument on spot to be measured,
and hit [A-Z] to read white and setup FWA compensation (keyed to letter)
[a-z] to read and make FWA compensated reading from keyed reference
'r' to set reference, 's' to save spectrum,
'f' to report cal. refresh rate, 'F' to measure refresh rate
'h' to toggle high res., 'k' to do a calibration
Hit ESC or Q to exit, instrument switch or any other key to take a reading:

Spectrum from 380.000 to 730.000 nm in 106 steps
19.0752, 19.0752, 19.0752, 19.0752, 19.0752, 19.0752, 19.0752, 19.0818, 19.0832, 19.0704,
19.0341, 19.0237, 19.0885, 19.9059, 20.6422, 21.2669, 21.8762, 22.4992, 23.0869, 23.6067,
24.0293, 24.335, 24.5257, 24.6118, 24.6261, 24.5567, 24.4715, 24.4158, 24.3522, 24.2485,
24.1069, 23.9747, 23.8549, 23.7075, 23.5244, 23.2141, 22.8444, 22.4109, 21.9071, 21.304,
20.652, 19.938, 19.1667, 18.3548, 17.5455, 16.7961, 16.1387, 15.5608, 15.0258, 14.4891,
13.9265, 13.3264, 12.6762, 11.9939, 11.3292, 10.7346, 10.2204, 9.78156, 9.42394, 9.18935,
9.12674, 9.30595, 9.75234, 10.4446, 11.2821, 12.1224, 12.8526, 13.4222, 13.8369, 14.1255,
14.3253, 14.4738, 14.5919, 14.7032, 14.8158, 14.9242, 15.0454, 15.1802, 15.3249, 15.5017,
15.7049, 15.9172, 16.1537, 16.3929, 16.6253, 16.804, 16.923, 16.9758, 16.9655, 16.9242,
16.8426, 16.7247, 16.6053, 16.4993, 16.3577, 16.2277, 16.0962, 15.9706, 15.8699, 15.8218,
15.8202, 15.9006, 16.049, 16.2686, 16.54, 16.9309
Peak value 24.626085 at (aprox.) 460.0 nm

Result is XYZ: 13.824663 14.486878 19.233993, D50 Lab: 44.923231 -0.903151 -18.058164

Place instrument on spot to be measured,
and hit [A-Z] to read white and setup FWA compensation (keyed to letter)
[a-z] to read and make FWA compensated reading from keyed reference
'r' to set reference, 's' to save spectrum,
'f' to report cal. refresh rate, 'F' to measure refresh rate
'h' to toggle high res., 'k' to do a calibration
Hit ESC or Q to exit, instrument switch or any other key to take a reading: q

Spot read stopped at user request!
Hit Esc or Q to give up, any other key to retry:

Short sentence with corresponding statement was added to revised manuscript on lines 106–108.

Comment 4, L108–109: These are extracted lichen substances? I would mention something like (see extraction methods below), becuase it is not immediately clear how you obtained these substances.

RESPONSE 4:
These individual lichen substances were obtained by our colleagues from the Laboratory of Briology and Lichenology of the same Komarov Botanical Institute in 197x–202x and verified by either NMR or FTIR spectra. Currently the collection of these pure substances is maintained by Dr. Ilya Prokopiev (one of the two corresponding authors) in Lab. of Analytical Phytochemistry. We have not extracted and purified these substances ourselves from the same Hypogymnia physodes or H. bitteri samples. It would be a large-scale and time demanding work beyond the aims of the present study.
In particular, pure samples from our collection have previously been used for the qualitative and quantitative analysis of secondary metabolites in the following studies: Ravinskaya A.P., Weinstein E.A. Influence of Some Environmental Factors on the Content of Lichen Substances. Ekologia = Russian Journal of Ecology, 1975, no. 3, pp. 82–85. (In Russ.); Prokopiev, I., Chesnokov, S., Serebryakov, E., Konoreva, L., 2022. Chemical variation in the Arctoparmelia separata (Parmeliaceae, Lichenized Ascomycota). Biochemical Systematics and Ecology 102, 104418. https://doi.org/10.1016/j.bse.2022.104418 and Sleptsov, I.V., Rozhina, S.M., Prokopiev, I.A., Mikhailov, V.V., Mestnikova, A.A., Alekseev, K.V., Zholobova, Z.O., Frolova, D.A., 2025. The Allelopathic Effect of the Epiphytic Lichen Physcia alnophila on Biochemical and Growth Processes in the Tissues of Larix gmelinii in the Cryolithozone. Forests 16, 711. https://doi.org/10.3390/f16050711
We have edited lines 117–118 to add the follows: “…lichen substances from the Komarov Botanical Institute pure lichen secondary substances collection…”

Comment 5, L112–114: It is unclear why this was done, if the thalli or extracts were presented so close to the spectrometer and they were completely opaque to the light, why subtract the background? How much does it effect the final spectrum to subtract these blanks? It would be nice to include a brief explanation of why you chose to do this.

RESPONSE 5:
Background substraction was performed mainly to obtain clean fluorescence spectra of pure lichen substances. These substances were placed as even as possible on the surface of a glass microscopy coverslip, and illuminated by UV source light at a sharp angle, however some rays could reflect from the glass surface or subtle dust particles and reach the spectrophotometer sensor. In addition, the stock UV-pass filter supplied by manufacturer allowed to pass some far-red and infrared radiation from UV LED. When obtaining lichen thalli reflectance spectra, we had to take into account that the spectrophotometer working spot was 5 mm in diameter. That is larger than some of thin Hypogymnia physodes peripheral parts, so a small fraction of light from the spectrophotometer internal lampcould reach the background and reflect from fabric or dust. For these reasons described above, we substracted background signal in both modes. Please refer to the attached example spectra showing raw UV source light reflected from glass, raw atranorin fluorescence spectrum and ‘clean’ (background-substracted) atranorin fluorescence spectrum. All three curves supplied with error bars showing standard error, n=10.

Comment 6, L123: It's ambiguous, were there just three steps, no light, 66, then 285?

RESPONSE 6:
We thank the reviewer for pointing out this issue and apologize for unclear statement. “10 s” in the manuscript stands for “ten seconds”, not “ten steps”. ‘No light’ state serves for the establishment of F₀ (initial chlorophyll fluorescence of dark-adapted sample) and F_m (maximal fluorescence after saturating pulse) which are used to calculate quantum yield, photochemical and non-photochemical quenching [18] during illumination of lichen with actinic light of step-wise increasing intensity. The three actinic light levels for which we report Y(II), qP, NPQ and ETR values are the following: 66, 125 and 285 μM photons ⋅ m^–2 ⋅ s^–1. We made the corresponding edit to line 133 in the manuscript. This ambiguity highlighted by the reviewer also explains the reason for the following reviewer’s question to figure 5, which we address below.

Comment 7, L151–153: which standards specifically? what is the RAS collection, spell it out?

RESPONSE 7:
‘RAS’ stands for “Russian Academy of Sciences”, it is the part of the full institution name and not the abbreviation for the pure substances collection name. We have addressed this issue in detail in our reply above, and removed ‘RAS’ from line 167just to clarify.

Comment 8, L189–L191: give precise measurements for what you mean by near-complete and less effective

RESPONSE 8:
near-complete removal was 90–99% based on the comparison of peak areas. The corresponding edit was introduced to line 206.

Comment 9, L199: grammar

RESPONSE 9:
We thank the reviewer for highlighting the mistake and decided to just remove the mistaken plural form ‘lichens’ since the simple mention of ‘thalli’ is clear from the context.

Comment 10, L268–272: This is difficult to read.

RESPONSE 10:
We thank the reviewer for pointing this out. Indeed, these sentences contained various information in mixed order. We have reordered and simplified these sentences as follows:
“Lower cortical layer was slightly thicker and heavily melanized in both H. physodes (35.3±5.6 μm) and H. bitteri (43.0±4.5 μm). Importantly, atranorin and chloroatranorin were detected there as well (Fig. S1e). However, fluorescence signal from lower cortical layer never reached its level from the upper cortical layer, being slightly higher than from the medullary layer.”

Comment 11, L301: I find it very interesting that the medullary compounds fluoresce UV-light as blue light, especially physodic acid. I wonder if this is to facilitate photosynthesis of the algal layer from below, as well as from above. I understand that these compounds are not the focus of the study, but I would like to read something about this in the discussion.

RESPONSE 11:
We thank the reviewer for rising such an interesting question. On the one hand, this topic has been in focus in some studies, i.e. [15, 16]. On the other hand, in our opinion, the context and interrelations and localization of all compounds capable of fluorescence are important. Indeed, physodic acid emit fluorescence in blue range and potentially is able to transfer energy to chlorophyll. However it receives a light beam that is already heavily screened by atranorin, and emits fluorescence omnidirectionally, which further decrease the efficiency of energy transfer to photosystems. In addition, our data on the overall fluorescence of lichen thalli support the hypothesis that short-wavelength light is mainly discarded in the form of fluorescence (Fig. 3b).

Nevertheless we agree with the reviewer in that this topic deserves attention, and we added corresponding fragment to discussion (lines 459–469).

Comment 12, L315–318: This is difficult to read.

RESPONSE 12:
We have simplified this structure and split it into two sentences (lines 337–339).

Comment 13, L340: It's unclear what each of these box plots mean. Why does each treatment x species have three boxes?

RESPONSE 13:
We thank the reviewer for pointing this out. Each three sequential boxes correspond to three increasing actinic light levels, namely, 66, 125 and 285 μM photons ⋅ m^–2 ⋅ s^–1. In fact, we rendered box plot in slightly different color intensities, however we now realize that was far from obvious. To make it clear, we revised the figure 5 to supplement it with actinic light indication, and edited figure 5 caption correspondingly.

Comment 14, L365–369: Very interesting, makes sense to me.

RESPONSE 14:
We thank the reviewer for this remark and feel deeply satisfied if the research outcome may be employed that fast.

Comment 15, L390–391: This is confusing, are you saying that the increase in reflectance of the thallus is because the machine used to measure reflectance emits wavelengths that excite the fluorescence of chloroatranorin and atranorin?

RESPONSE 15:
We thank the reviewer for pointing this out. On the one hand, this may feel confusing, but indeed the light from the internal light source of spectrophotometer emit short wavelengths by design (as well as the longer ones) to fully illuminate various types of samples and record their spectral response (reflectance). These short wavelengths naturally facilitate atranorin fluorescence, either in form of pure crystalline substance or in form of microscopic crystals in lichen thallus. On the other hand, we relied on the same property of atranorin to fluoresce from short wavelength to visualize its localization under laser scanning confocal microscope. Laser wavelength of 405 nm effectively elicited bright atranorin fluorescence in thalli sections (figure 3 and S1). Therefore, we believe that this phrase may be left as is, as it is fully supported by the experimental data.

Comment 16, L393–394, L396–398: grammar

RESPONSE 16:
These sentences were rewritten as follows:
“Earlier studies revealed a considerable reduction in the reflectance of dark-brown lichen surfaces [27–29].”
and
“The thallus of H. bitteri not only contained a smaller amount of atranorin and chloroatranorin, but these substances were also likely replaced by melanin in the older parts of the thallus (Fig. S1d).”

Comment 17, L409–413: This is very interesting, what about their crystalline vs. liquid state changes their reflectance?

RESPONSE 17:
Indeed, the fluorescence spectra of previously studied lichen substances differ substantially between solution and crystalline states. This difference is fundamentally photophysical: fluorescence is emitted not by an isolated molecule but by a molecule coupled to its microenvironment. In solution versus the crystal, the local environment, intermolecular interactions, and excited-state relaxation pathways differ, which consequently changes transition probabilities, emission wavelengths, and spectral profiles. For example, in solvents such as ethanol, parietin absorbs blue and UV-B light and emits fluorescence at 480–540 nm, whereas in intact lichen thalli this fluorescence is red-shifted by approximately 90 nm [16]. However, most lichen secondary metabolites are lipophilic compounds that are very poorly water-soluble and therefore occur in lichens almost exclusively as small crystals on the surface of hyphae. Accordingly, when investigating the physiological role of secondary metabolites in lichens, it is more appropriate to use solid-state fluorescence spectra rather than solution spectra

Comment 18, L479–482: I agree

Response 18:
We are happy to see positive reply and are looking forward to further investigate this topic in the nearest future.

Author Response File: Author Response.pdf

Reviewer 2 Report

This article examines the photoprotective role of two, mainly, secondary metabolites – atranorin and chloroatranorin – against UV-A and Vis (blue-violet radiation) in the thalli of two epiphytic species, Hypogymnia physodes and H. bitteri.

The article presents high-quality results and highlights the hitherto poorly understood photoprotective role of secondary metabolites in lichens, which exhibit weak reflection of radiation through its scattering in the form of fluorescence.

The article is suitable for publication following a few revisions and the rectification of a serious methodological error, namely the failure to include a control sample for the thallus treated with acetone, which should have been tested immediately in order to detect any potential influence of acetone on the research results obtained, and consequently on the conclusions. Although the authors address this issue in the discussion, it could have been taken into account, at least in part, in the research methodology.

Furthermore, the following issues should be addressed in the discussion:

What might be the photoprotective role of atranorin and chloroatranorin against UV-B radiation, and in the context of the results obtained for UV-A and Vis radiation?

The need for further experiments on the photoprotective mechanisms of atranorin and chloroatranorin should be addressed.

Detailed comments are provided in the attached PDF file.

Please, see the attachment.

Comments for author File: Comments.pdf

Author Response

We cordially thank the reviewer for the attention paid to our work, for their constructive sugestions and for the important comments that helped to improve the manuscript. Below we respond to reviewer’s comments point-by-point and specify the edits that were introduced to the manuscript.

Specific comments to the manuscript:

Comment 1, L85–88: This description is too scarce. It is ambigous if the thalli was intact or in another form. Moreover, according to the figures in the text, the thalli was on bark. The given description should be more precised. Moreover, the biololigal replicates of the tested samples and control samples (N), expressed in number or mass, should be provided. Furthermore, based on the description, thalli which were conducted to rinse with acetone, should be also a secondr control sample as thalli conducted to rinse with acetone and immediately applied to the main experiments. Such a control sample should show an impact of acetone on metabolite profile compared to the evaporated ones. This control sample should be included and the main text revised.

RESPONSE 1:
We would like to address the two parts of the reviewer’s comment separately. On the first question regarding sampling of lichen thalli, we already mentioned in section 2.1 that Hypogymnia thalli were collected from bark of Pinus sylvestris, Larix gmelinii and Duschekia fructitosa trees. The number of samples was at least 5 for each group, which we have mentioned in the revised version of the manuscript in the last sentence of section 2.1.

On the second remark, we hope we understood correctly the reviewer’s concern about including second control immediately after acetone rinsing. We wish it was possible to include that group into analysis. But in reality, acetone treatment is fully incompatible with living (wet) state of lichen thallus. On the one hand, lichen secondary substances are insoluble in water and are soluble in various organic solvents including acetone. On the other hand, photosynthesis requires humidity and intracellular membrane intactness. Among different organic solvents, acetone seems to give the least impact to chloroplast membrane structures given that the rinsing is applied to strictly dry thallus. If we try to humidify lichen thalli before acetone have been fully evaporated, photosynthetical apparatus in algae became damaged, which immediately results in zero response to PAM survey. Chlorophyll does still exhibit fluorescence, but samples demonstrate no response to saturating light pulses. During preliminary experiments, we faced with this problem in the field: lichen thallidried in the dessicator were subjected to acetone rinsing and then left to evaporate the acetone in humid air due to rainy weather. Nomeaningful PAM light curves were obtained at all. To continue with experimental procedure with acetone-rinsed thalli, one must fully get rid of acetone remains in dry conditions, and attempt to rehydrate the lichen thalli strictly after that. Therefore, other experimental procedures with thalli immediately after acetone rinsing make no sense. We value the reviewer’s remark about the possible impact of the acetone rinsing on the lichen metabolic profile. Indeed, that could be an interesting direction of research if only lichens could perform normally in wet state in presence of acetone remains, which is apparently not the case.

Comment 2, L131: the RPM should be provided.

RESPONSE 2:
The RM-1L vortex operated in “F5” cycle mode, performing 135° clockwise rotation, high-amplitude shake for 1.5 s, 90° clockwise rotation, high-amplitude shake for 1.5 s, 135° clockwise rotation, then 1.5 s rest. The whole cycle lasted for 6 s, so 10 RPM in total. The corresponding edit was done in the manuscript on lines 144–146.

Comment 3, L149, L152: in italic

RESPONSE 3: we thank the reviewer for the remark, the formatting is corrected in the revised manuscript.

Comment 4, L179–L183: The description of statistics should be more precised and included descriptions for all the conducted experiments (rinsing, extractions, spectra, fluorimetry tests).
Moreover, if the tested biological replicates (N) are below 10 (small sample), the test for normality and homogeneity might be ommitted due to low draws of findings value to meet the normality distribution. The non-parametric test of Mann-Whitney indicates that the parametric values had not been met; however, taking into consideration, that the second control sample should be included (thalli treated with acetone and applied immediately to further experiments) a non-parametric test of Kruskall-Wallis test and post-hoc test of Dunn for multi-group comparison, should be performed.
Moreover, the "at p<0.05" should be revised to significant level (alpha).

RESPONSE 4:
We fully agree with the reviewer's comment regarding the small sample sizes used in the study and have removed references to tests for normality and homogeneity of variance. We agree that Kruskal-Wallis test with post-hoc Dunn’s test for multi-group comparison would serve best if we aimed to perform cross-comparisons between experimental conditions and two lichen species. However, we performed and discussed comparisons between two groups each time: the control (intact) one and the acetone-rinsed one,and have not addressed the dynamics of PAM response under increasing light, for instance. Since all experiments included appropriate controls, the significance of differences was assessed in all cases using the Mann-Whitney test.
We also clarified the choice of significance level (alpha).

Comment 5, L206 (table 1 header row): a literature reference to the metabolites clasess should be provided.

RESPONSE 5:
We agree with the reviewer’s remark and believe that the fundamental work by Huneck and Yoshimura, 1996 serves best as the corresponding reference (Huneck, S.; Yoshimura, I. Identification of Lichen Substances. In Identification of Lichen Substances; Springer Berlin Heidel-berg: Berlin, Heidelberg, 1996; pp. 11–123 ISBN 978-3-642-85245-9). Since this book chapter is already referenced in the manuscript, we supplemented table 1 footer row with reference number.

Comment 6, L209: This issue is ambigous from the methodological viewpoint. It should be described in detail in methodological secion, in which way, the sample for chromatographical analysis before acetone rinsing was obtained.

RESPONSE 6:
We have made clarifications to Section 2.6 (Materials and Methods). As described in Section 2.2, thalli were longitudinally dissected into two equal parts, one of which served as control sample, and the other one was subjected to acetone rinsing. Then dry samples were divided into 5 equal parts and analyzed by HPLC.

Comment 7, L287–289: this fragment should be transferred to methodological section.

RESPONSE 7:
We agree with the reviewer in that this part is better moved to Materials and Methods. The sentence is appended to Section 2.4 of Materials and Methods (lines 124–125).

Comment 8, L348: The discussion should be divided into relevant subsection to better readibility.

RESPONSE 8:
We agree with the reviewer that Discussion needs partitioning, especially with newly added fragments. Please refer to section 4 of the revised manuscript.

Comment 9, L365–369: This issue should be more discussed from perspective of limitations of the study, included an ecological perspective.

RESPONSE 9:
Thank you for the comment. We have expanded the discussion of limitations of the study and included an ecological perspective.

Round 2

Reviewer 2 Report

None.

None.