Thank you for comments provided.

Please find here the answers on comments:

1. Figure legends need to stand alone from the text, currently they do not provide enough information (particularly Fig. 6, which is complex and needs to include qualitative information for context).

Response:  Changed, the revised version for Figure 6 legend is:

Fig. 6. Distance-based redundancy analysis (tbRDA) showing relationships between invertebrate taxa (response variables, red arrows) and environmental variables (explanatory variables, black arrows; see Table 1). The direction of arrows indicate the direction of relationships: angles between arrows reflect correlations among variables, with smaller angles indicating positive relationships and opposite directions indicating negative relationships. Solid black arrows indicate environmental variables that significantly explain variation in community composition (Monte Carlo permutation test, p < 0.05), dashed arrows represent non-significant variables (p > 0.05). Sites (n = 32) are shown as grey numbers. Response variables: Aca – Acari, Ara – Araneae, Col – Collembola, Ins – Insecta, Nem – Nematoda, Rot – Rotifera, Tar – Tardigrada; Explanatory variables: CC – canopy cover, CHA – chlorophyll a, CHL – Chlorophyceae biomass, CON – conductivity, CORG – organic carbon, CYA – Cyanophyceae biomass, DEN – tree density, DIS – distance to trees, OD – organic debris.

1. Is it possible that monogononts were overlooked? These often co-occur with bdelloids in cold environments but can be misclassified as protists due to their small size.

        Response: No, likelihood that monogonont rotifers were overlooked is extremely low. First author of the manuscript can identify rotifers, and all individuals were identified as bdelloids based on morphological characteristics. Even if they are not-active, their characteristic ‘tun’-shaped external view and ramate mastax were clearly distinguishable. Furthermore, this observation is consistent with our understanding of snow rotifer communities, which are thought to be colonized primarily from mosses and lichens (e.g., those growing on tree bark), habitats typically dominated by bdelloid rotifers.

1. The interaction/contribution of the underlying soil is not considered. It seems that in recurring years there will be large depositions of invertebrates into the soil once the snow has melted. Is there any data available that compares invertebrate composition/density in underlying soil from a snow patch vs. other soil areas? Also, is it possible that the underlying soil is a major contributor to the invertebrates that appear each year in seasonal snow, instead of them being deposited from the peripheral surroundings, as suggested?

         Response: We fully agree that the potential contribution of the underlying soil was not explicitly considered in this study. However, our aim was not to determine the source(s) of microfauna present on snow surfaces, as these are likely to be multiple and interacting. Instead, we focused on identifying the key environmental variables that explain variation in invertebrate community composition.

 In the article, we initially considered the SSPs as habitats influenced by their most visible and closest context, which are trees (since almost all SSPs were located in sheltered places in forest). All sampled SSPs were covered by organic debris indicating the great influence of trees on snow surface. This interpretation is further supported by our statistical analyses, which highlighted the importance of variables associated with this close surrounding.

To accept the idea that snow ecosystem can be influenced by soil, we have to assume that soil particles can be transported from the soil surface through entire snow column (up to 30 cm in some cases) and then be deposited on the surface where microfauna lives. It appears less likely compared to downward deposition processes.

Regarding the source of microfauna itself – we agree that multiple pathways are possible, and we intentionally avoid making conclusions on this aspect. However, active upward migration of rotifers and tardigrades from the soil through the snow to the surface seems unlikely. In contrast, deposition from above represents a more plausible mechanism. Conversely, the deposition of microfauna from melting snow into the underlying soil, as suggested by the reviewer, is indeed possible but falls beyond the scope of the present study and would require targeted studies.

1. What is the evidence that these invertebrates are reproducing in the snow? Could it be that reproduction occurs without snow and they are just tolerating the snow conditions?

Response:  We made this suggestion basing mostly on numbers of microscopic invertebrates – for example, in case of thousands of all three main groups at one spot. Please note that these habitats are short-living. Given this, it appears unlikely that such densities could be explained solely by sporadic inputs of individual organisms.  However, we absolutely agree that ability to reproduce should be tested experimentally, hence our interpretation, potentially explaining the large presence of microfauna on snow surface, remains hypothetical. However, we do not claim that reproduction is restricted to snow environments; rather, we suggest that snow is inhabited by ‘normal’ microfauna which representatives may be capable of both surviving and potentially reproducing. And this assumption explains the observation we made. Importantly, this was not used as a basis for any formal hypothesis in the manuscript. We fully understand that other mechanisms also can work, at least partially.

1. Can anything be said about the diversity of species within each phylum? Are these mostly the same species of rotifers, tardigrades, nematodes, etc or is there notable biodiversity within phyla?  Are these species different from those which inhabit glacial ecosystems? I understand there is no molecular data, but some morphological characters would give a sense of species diversity within phyla.

         Response: Yes, the molecular studies aimed to reveal the species composition in snow patches and in the surroundings (mosses, lichens nearby) are still in process. However, based on preliminary morphological examination, some initial conclusions can be drawn. Both rotifers and tardigrades found in snow appear to belong to common, European taxa, particularly rotifers typical of limno-terrestrial environments. So far, no glacial (polar, Arctic, etc.) species have been identified. However, there is only partial overlap between species found in snow and those in the immediate surroundings. We found some rare rotifer species (previously recorded in Europe but not considered common). These results are still preliminary and have to be supported using molecular approach, which supposed to be another study (focused on diversity and connectivity with other types of cryosphere). Regarding other groups – unfortunately, the specialists in nematodes and arthropods were not involved ; therefore, we refrain from further interpretation for these taxa.

1. Is there any evidence that these snow invertebrates are more similar to glacial or soil counterparts?

        Response: Please, see above. In general – we did not observe similarities of SSP fauna with the glacial one.

Minor comments:

Line 71: is --> was

Line 74: onto --> on

Line 78: for understanding the SSPs

Line 117: Figure 1 legend needs more information (e.g., cross-reference numbers to Table with coordinates)

Line 138: Figure 2 legend needs more details

Line 173: “...contents of tubes were slowly refrozen at +5 –...” do you mean “unfrozen” or “thawed”?

Line 189: canopies --> canopy

Line 379: is --> was

Line 446: characterized “to” be…

471: in Figure 6

Response: All comments were considered, and appropriate changes were made.

New legends for figures 1 and 2:

Fig. 1. Locations of sampling sites ## 1–40 in Lithuania, Latvia, and Estonia in 2023–2024 (a); SSPs at sampling sites # 11 (b), # 20 (c), and # 36 (d). For coordinates of sampling sites, see Table S1

Fig. 2. Sampling sites # 16 (a), # 22 (b), and # 36 (c) representing different levels of canopy cover: 5 %, 24 %, and 58 %, respectively. For coordinates of sampling sites, see Table S1

Thank you so much

Thank you for comments provided.

Please find here the answers on comments:

line 34: Snowfields in general receive much attention, but here authors should rather specify the uniqueness of their research in having a look on "snowfields and snow patches in LOWLANDS" (of course not in capital letters)

Response: Yes, we tried to specify that our study focuses exclusively on lowland snow in a temperate climate. This is stated several times later in the introduction, however, some corrections were made in these lines.

line 123: Great that only the upper layer of snow was harvested. This may be due to the avoidance of the snow-soil interface. This is according to the best practice to collect "true" (autochthonous) algae and avoid soil algae as it is shown in Fig. S2 in Lutz et al. 2018.



Stefanie Lutz, Lenka Procházková, Liane G. Benning, Linda Nedbalová & Daniel Remias (2019): Evaluating amplicon high–throughput sequencing data of microalgae living in melting snow: improvements and limitations. Fottea , 19(2), 115-131. DOI: 10.5507/fot.2019.003



However, the authors of the paper under review collected the uppermost layer of snow, so the airborne algae were most likely part of the sampled snow.

Response. Yes, exactly. We sampled only upper layer of the snow, no soil / ground / litter from underlay was collected.



line 123 - Was the depth of the sampled snowfield measured before sampling? Or the snow cover was always much thicker than 4 cm, making it possible to safely avoid any sampling of the soil to snow interface?



Response. We did not measure the thickness of the snow; however, we do confirm that all sampled snow patches were thicker than 4 cm (typically ranging from 8 to 12 cm). This information has now been added to the ‘Methods’.



line 163-165: Could the authors briefly describe the protocol for microalgae enumeration in the Utermöhl chamber according to HELCOM guidelines? For instance, what was the minimum number of cells counted per species? Furthermore, how many cells of each species were measured to determine the mean cell volume?

Response: The total number of cells counted in the transect ranged from 111 to 1025. According to the recommendations by LST EN 15204, at least 400 units per sample should be counted to ensure statistical reliability, while dominant species (biomass relevant taxa), at least 50 cells of each taxon should be counted. Moreover, the precision can be decided according to the selected level, which correlates with the number of microalgae counted. In samples where the number of cells in the transect was less than 400 (9 samples out of 32), the most of the representatives belonged to the same genus (considered as dominant), therefore, it was aimed to count more than 50 units to ensure quantification uncertainty (according to the same procedure recommended by Utermöhl (1958) and HELCOM (2021)). Since only two microalgal genera dominated the snow samples based on microscopic analysis, the abundance of these taxa in some samples was very low. Therefore, samples were concentrated as much as possible to ensure that a statistically sufficient number of cells could be counted. The same number of cells was used to calculate the cell biovolume (varied from 111 to 1025 cells).

We have added more detailed information about phytoplankton abundance and biomass calculations. In addition, the methodology is now supported by a reference to LST EN 15204. The paragraph has been updated (Lines 65-80) in following way:

The composition of the microalgal communities was analyzed under an inverted microscope (LEICA DMI 3000, Leica Microsystems) at magnifications of 400×, according to the method described by Utermöhl (1958). According to HELCOM (2017) and European Committee for Standardization recommendations (2006), the abundance of microalgae (counts L^-1) was calculated by multiplying the number of units counted with the coefficient C (dm³), calculated using Eq. (1):

C = (A x 1000) / (N x a x V),                                                                                                                                                                           (1)

where A is the cross-section area of the top cylinder of the combined sedimentation chamber (the usual inner diameter is 25.0 mm, giving A= 491 mm²), N is the number of counted fields or transects, a is the area of single field or transect, and V is the volume (cm³) of sedimented aliquot.

The biomass of microalgae (mg L^-1) was calculated by the allocation of algal species (genus) to size classes according to the scheme of Olenina et al. (2006) and updated appendix available at ICES website (https://www.ices.dk/data/vocabularies/Pages/default.aspx). The biomass of microalgae (mg L^-1) was calculated based on Eq. (2), as recommended by HELCOM (2021):

Biomass = abundance x VCU x 10^-6,                                                                              (2)

where VCU is the volume of the counting unit (mkg).

Line 218-220 It would be appreciated if the authors could also include algal cell abundances in the manuscript, rather than only biomass levels. This would facilitate comparison with existing literature on  Chloromonas snow algae, such as e.g. in Procházková et al. (2020). I assume the abundance was lower than 10,000 cells/ml, as the sampled snow patches did not appear to show any visible snow algal discoloration (if I understood correctly).



Lenka Procházková , Daniel Remias, Wolfgang Bilger, Heda Křížková, Tomáš Řezanka, Linda Nedbalová (2020): Cysts of the Snow Alga Chloromonas krienitzii (Chlorophyceae) Show Increased Tolerance to Ultraviolet Radiation and Elevated Visible Light. Frontiers in Plant Science 11: 617250. DOI: 10.3389/fpls.2020.617250



Response: Algal cell abundances have now been added to the revised manuscript, and all abundance data are also provided in the Supplementary Table. No visibly coloured snow patches were observed. Algal abundances were much lower than those reported by Procházková et al. (2020).

 line 305 - In which order the sites are shown? Sample number? The authors shall specify that density of animals is shown by grey bar chart (it is currently not clear on the first sign). What about showing an alternative figure of animal density in relation to  chlorophyll-a?

Response: Sites are shown simply in order from #1 to #40. Clarifications have now beed added in the legend



line 272 - 273 - The results of the analysis is regarded by the authors as "weak positive correlation" if Rs=0.48  while on the lines 349-350 it is regarded by the authors as "moderate positive correlation" if Rs=0.38, Rs=0.46 and Rs=0.39. This is a bit inconsistent, especially if the common interpretation scale of Spearman Correlation coefficient is the following: weak for 0.20 – 0.39, moderate for 0.40 – 0.59.



Response: Corrected (from 'weak' to 'moderate')



line 436: in this study, I wonder, were there sampled any visible colored snow patches (due to snow algal blooms)? If not, it is better to mention this detail here or in the methods clearly.

Response: No, we did not observe any colored areas. In general, algal blooms were not even expected as they are not typical for seasonal snow in lowlands. Actually, we highlighted this in the Introduction (Lines 56-57: Unlike the seasonal mountain snow studied by Ono et al. (2021) in Japan, which persists from November to June, the last snow in lowlands of temperate Europe typically vanishes by late March and usually does not have algal blooms.). However, we agree that it should also be stated in the Results section, and this has now been added.

line 483- 484 - The authors suggest that rotifers primarily originated from the surrounding trees. However, the images in Fig. 1b-d suggest that the sampled snowfields were quite shallow. Although the manuscript does not specify the total depth of the snow patches (noting only that the uppermost 2-4 cm were collected), is it possible to entirely exclude the possibility that the detected rotifers also originated from the soil beneath these shallow snowfields?

Response: Of course, all snow patches were more shallow than alpine or polar snow, however all of them were thicker than 5 cm. During sampling, only the upper snow layer, together with surface debris, was collected, and care was taken to avoid any picking up of underlying soil or litter. As shown in the photo (attached), snow remained beneath the sampled layer after collection, with no exposed ground.