This article presents investigations into lichen of the genus Peltigera as producers of ice nucleators (INs). It is well-conceived, methodologically sound, and enjoyable to read. The insights gained through these investigations are new and interesting, so they merit publication in Biogeosciences. There are a few minor issues I recommend the authors to consider in a revision:

1.) Peltigera are mostly ground-dwelling and have a compact morphology. Which process could dislocate particles small enough from them to escape the surface layer and reach higher altitudes? I would appreciate to a sentence or two on that issue in in the Conclusions.

2.) Lines 31-33 and lines 330-333 state: "Our analysis suggests that the INs released from this fungus in culture are 1000 times more efficient than the most potent bacterial INs from Pseudomonas syringae." I find the term "efficient" problematic in this context because "efficient" often refers to the activation temperature of INs, i.e., INs active > -10°C (e.g., Zhang et al., 2020, https://doi.org/10.1016/j.atmosres.2020.105129). You also define T50 as a measure of efficiency (lines 168-169).

3.) Line 113: What is meant with "bioavailability" here?

4.) Lines 168-169: "The temperature at which 50% of the droplets froze, T50, was recorded as a measure of the efficiency of the INs." I guess it means the same as "freezing efficiency", an expression first used in line 201? If so, please add (in brackets) this term to the end of the sentence in lines 168-169.

5.) Line 170: The T50 value of water is relatively high (-11°C). Were values of Peltigera samples corrected for that and, if so, how?

6.) Line 173: Consider replacing "robust" with "precise".

7.) Lines 212-216: The 96 droplets in TINA experiments may be large enough a number to derive differential IN-spectra from (see Vali, 2019, https://doi.org/10.5194/amt-12-1219-2019). Differential spectra afford clearer interpretation than cumulative spectra, especially in the context of your study.

8.) Table 1: Isn't it surprising that the warmest T50 was found in a species collected in the tropics? Could this be taken as an indication for IN production in Peltigera being similarly incidental as it seems to be in pollen (Kinney et al., 2024, doi.org/10.5194/egusphere-2023-2705)?

9.) There appears to be a contradiction in lines 263-265: "Based on the fast growth rate and

presence of mycelial-like growth, we classified L01-tf-B03 as a lichen-associated fungus. It is notoriously difficult to isolate mycobionts (Cornejo et al., 2015), which are very slow growing, ..." Why should the fast growth rate seen in L01-tf-B03 support your classification when mycobionts are very slow growing?

10.) Lines 279-280: The T50 value of -23.5°C indicated here is much lower than the one mentioned in line 170 (-11°C). Please clarify.

11.) Lines 284-286: " The large decrease of over 4°C in bacterial freezing efficiency is in striking contrast to L01-tf-B03, for which the IN-activity is reduced by less than 1°C at the same concentration." I think this finding merits an attempt at interpretation.

The authors extend their study from Eufemio et al. 2023 which also investigated biological entities as sources of ice nucleators. Their first paper reported a range of lichen species across Alaska, while this manuscript focuses on Peltigera lichens from various regions, highlighting their efficiency as IN agents. The new knowledge is therefore incremental, but the authors can improve their story line by addressing the following criticisms and specific comments.

Major criticism to address prior to publication decision:

The conclusion that “Our analysis suggests that the INs released from this fungus in culture are 1000 times more efficient than the most potent bacterial INs from Pseudomonas syringae” (in abstract, but also repeated on lines 290-291) is misleading. Figure S1 suggests that at warmer temperatures P. Syringue is most efficient. The authors can certainly describe the temperature range and the concentration range at which L01-tf-B03 becomes competitive. These conclusions would need to be revised and clarified that these statements are specifically due to a temperature range to be considered for publication.

The conclusion that “the ice nucleation activity of lichens does not seem to strongly correlate with the geographic region or climate zone where the samples were collected” (lines 202-203) is not supported by the presented data set. The season, storage, age of the samples likely is too variable to draw this conclusion and such a sample size.

Handling blanks should be shown. To be clear, handling blanks represent water going through the same procedure and steps, but without the lichen. I find the reported -11 oC (line 170) to be an unacceptably high background freezing temperature. I would expect background freezing of 3 microliter dropets to be more around -25 oC. Can the authors explain and show data to support this high background freezing? Otherwise, it’s difficult to ensure that the freezing temperatures reported are due to the experimental set up vs the lichen.

Why were the sites chosen? Were they part of a decadal study? I have the impression that these samples were rather opportunistically chosen (rather than intentional sites) and I think this point needs to be discussed in the manuscript (it’s fine if they made use of other sampling campaigns or other projects – but it should be stated explicitly). The point on different biomes is well taken and well demonstrated, but the reasoning behind why these specific sites would need to be justified.

The “N/A” values for the TINA measurements are unexplained. If the authors admit that the Vali-type temperatures are for “initial tests” (line 173), then why are there TINA measurements missing? (Same comment for Table S2 – why are TINA measurements not reported if the authors think this instrument is better?

The comparison between this study and their former study (2023) needs to be made clearer. Here are some suggested:

• Locations from Eufemio et al. 2023 should be included in Figure 1.
• Samples and freezing temperatures should be included in Table 1 (and see my point below about turning Table 1 into a figure)
• The authors could consider comparing quantitatively and visually the claim on lines 191-192. This comparison would be important for context.

Heat treatments were already done in Eufemio et al. 2023 and so what is new/different about the heat treatments in this study? Data from 2023 could be included in the heat treatment Table S1 (should rather be a figure). Are heat treatments the best method for identifying proteinaceous material? I think there are multiple additional experiments (even elemental analysis) that could help further support the claim for INPs.

Storage certainly plays a role in this study (the authors mention this issue only on lines 113-114). Could the authors add this information to Table 1? According to my comment below about turning Table 1 into a figure, one could add a dashed/solid outline to showcase the potential impact on freezing temperature. This point and discussion would merit a new and separate subsection and connecting to literature.

Why do the authors think that these lichen have had so much less attention if they’ve been known since the 80s? (Line 55-56 and again line 82) The motivation for this study should be more than because something is unknown. I’d be interested to hear about why the authors think P. Syringae has dominated attention. For example, in its use in Snomax.

The microscope data is missing and should be included in the SI. Including the images to support the claim on line 260.

Specific comments:

Lines 50-53 mention aggregates and the first sentence is missing a reference. Consider also the paper referenced and citing (Bieber and Borduas-Dedekind, 2024; Hartmann et al., 2022; Lukas et al., 2020, 2022; Renzer et al., 2024) (most of these papers have shared authors with this manuscript, so I’m surprised they haven’t been included.)

Line 66: dominate 10% of what? Lichens cover 10% of the entire surface of the earth? This point could be clarified.

Lines 68-69: the future reader would likely benefit from having this list of references explicitly stated. What each found and how the authors built on this previous work.

Line 94: it would be worth deleting the cardinals for the US (the directions are not listed for the other countries)

Line 102: could the authors add to their SI the vegetation identification guides that were used?

Table 1 can be converted into a figure to better visualize the breadth as well as the differences between location, symbiosis type and T50. T50 vs species in ascending/descending order of T50. (for example, using markers to represent symbiosis and colour to represent T50).

Were some of the lands sampled from indigenous/aboriginal lands? Are there land acknowledgements to be made?

Line 148: Could the authors clarify what the “hydration state of the thalli” signify?

Line 167-168: how were the freezing temperatures recorded? Manually? How many replicates?

Line 170: what was the positive control demonstrating? In other words, what was it controlling?

Line 173: show data to support the claim.

Line 180: write equation explicitly.

Line 195: the term “undiluted” confused me, because the solutions needed to be made. Perhaps worth deleting? The concentration is reported further down.

Lines 198-199: why do the authors think that the tri- and bi-membered lichens would show difference T50 values?

Line 210: Key point of the paper in my opinion! This conclusion is indeed well supported in this manuscript.

Line 225: Why would some samples be more sensitive to heat?

Line 318: Why was the threshold of -6.2 oC specifically chosen? Why is this temperature remarkable?

Figure S1 which should be included in the main text. Figure 3b has a peculiar decreasing value for the x-axis. It wasn’t clear to me (and likely to the future reader) what this figure was intended to highlight. Figure S1 or a figure of T50s with increasing temp could also be considered.

More of a curiosity question, but would the authors suggest that lichens replace P. syringae in Snomax? Could be worth discussing in the implications section?

References:

Bieber, P. and Borduas-Dedekind, N.: High-speed cryo-microscopy reveals that ice-nucleating proteins of Pseudomonas syringae trigger freezing at hydrophobic interfaces, Sci. Adv., 10, eadn6606, https://doi.org/10.1126/sciadv.adn6606, 2024.

Hartmann, S., Ling, M., Dreyer, L. S. A., Zipori, A., Finster, K., Grawe, S., Jensen, L. Z., Borck, S., Reicher, N., Drace, T., Niedermeier, D., Jones, N. C., Hoffmann, S. V., Wex, H., Rudich, Y., Boesen, T., and Šantl-Temkiv, T.: Structure and Protein-Protein Interactions of Ice Nucleation Proteins Drive Their Activity, Front. Microbiol., 13, 2022.

Lukas, M., Schwidetzky, R., Kunert, A. T., Pöschl, U., Fröhlich-Nowoisky, J., Bonn, M., and Meister, K.: Electrostatic Interactions Control the Functionality of Bacterial Ice Nucleators, J. Am. Chem. Soc., 142, 6842–6846, https://doi.org/10.1021/jacs.9b13069, 2020.

Lukas, M., Schwidetzky, R., Eufemio, R. J., Bonn, M., and Meister, K.: Toward Understanding Bacterial Ice Nucleation, J. Phys. Chem. B, 126, 1861–1867, https://doi.org/10.1021/acs.jpcb.1c09342, 2022.

Renzer, G., de Almeida Ribeiro, I., Guo, H.-B., Fröhlich-Nowoisky, J., Berry, R. J., Bonn, M., Molinero, V., and Meister, K.: Hierarchical assembly and environmental enhancement of bacterial ice nucleators, Proc. Natl. Acad. Sci., 121, e2409283121, https://doi.org/10.1073/pnas.2409283121, 2024.