This study uses microscopy and DNA sequencing to examine the spatial heterogeneity of snow algae across an elevation gradient in Alaska. The study presents an important comparison of supraglacial snow compared to snow over soils and presents evidence that factors related to snowmelt including snow depth and elevation are important factors in snow algae phenotype as well as potential underlying substrate (e.g., ice vs. soil). The manuscript and data are exceptionally well written and presented and the data and findings will be of interest to a broad audience. I have a handful of comments for improvement:

Line 67: Deposition could still be local. Perhaps a more specific description is needed here.

Lines 105-106: Why the different depths? Based on Figure 2, snow algae are in the snowpack below 2 cm. 

Line 130: Is the 100 cells true for each sample, for both geometric size and diameter?

Line 149: Remove reaction (redundant with PCR)

Line 210: I like this comparison but I’m confused about the surface vs. subsurface samples. I think the methods need to be updated to better describe the sample collection. 

For figure 3, nitrate and ammonium cannot be 0. They would be below the limit of detection. These data points should not be shown on the graph because these numbers do not exist to plot. 

Line 232: Abundant is probably more accurate than dominated

The present manuscript explores the morphological and taxonomic variability of red snow algae in different elevational gradients and substrate types (soil-based or ice-based) across Alaskan glacier and its forefield, using molecular as well as microscopic approaches. The study provides valuable insights into the composition of snow algae in various habitats, as well as information on variations in algal communities responsible for red snow across elevation and substrate gradients. I also value the comparison and discussion on different approaches and their outputs. The manuscript is well written, with only minor language inconsistencies that are easy to correct.

My main concern lies in the analysis of soluble nutrients, which are evaluated throughout the study. The approach used to collect soluble nutrients, especially phosphorus, is not commonly used, so validation of this method for these types of samples is needed or needs to be referred in the study. This concern stopped me from further review of the data and results connected to soluble nutrients. Also, when evaluating surface and subsurface data, I miss further information on radiation conditions from the sampling campaigns, as cells of snow algae can migrate within the snow layer. I believe that such data could contribute substantially to a better evaluation of the results. I describe all my comments in point-by-point below.

I must also stress that I am not an algologist by specialization. I recommend this paper to be seen by a specialist in snow and glacier algae, as I am not fully available to review the algal information and data in detail.

Abstract

L16: the sentence, as currently written, is not fully consistent with results which state that (e.g., L195) mean concentration of spherical cells between environments were similar. Please revise it so that its meaning is clear and consistent with the results.

Methods

L110 + Sect. 2.3: For the chemical analysis of PO₄³⁻, acid-prewashed bottles are commonly used to prevent adsorption of P to the vessel surface, for example, and further bias in analyses. Were your vessels acid-washed before sampling?

My main concern is freezing samples for chemical analysis. Even though the samples were filtered, samples for P analysis are commonly stored in the refrigerator, not frozen, as freezing might break down fine particles, for example, and affect the final P concentration in the analysed water. Has your approach been validated for this type of sample? If so, providing a reference to the particular validation or detailed justification for the particular approach would help to increase the reliability of your results.

L136: I might have overlooked it, but it is not clear to me at which section the quantitative comparison of morphotype composition with molecular taxonomic composition by calculating volumetric biomass for each morphotype is present.

L177: As discussed further, all morphotypes can vary in their species composition, also some snow algae species revealed cryptic diversity (i.e. Remias et al. 2023). How was the pooling of all non-spherical cells for further analyses and evaluation justified? They might differ in their metabolic/environmental requirements and pooling such data into a single category may limit the explanatory power of further analyses and evaluation of results.

Results

3.1. I suggest calling the section “Red snow algae morphotypes in snow samples” as the section refers solely to morphotypes.

L190: I would recommend including the scale bar size directly in the picture to help with the orienting in the figure.

Sect. 3.3. While reading this section, I am wondering why subsurface samples weren’t taken and analysed also for soil-based samples if the reported lowest depth was 4 cm in all sites and subsurface samples were taken approx. 3–5 cm deep? I cannot find any justification for this decision as such results might be very valuable for the study.

Fig. 6 + 7. The orientation in the figures while reading the results is rather difficult. For the better orientation, I recommend adding a heading along the y-axis describing the data origin (e.g., 18S rRNA, potentially red pigmented, etc.).

Section: 3.5, as highlighted above. I would like to see evidence that the methodological approach used in this study has been validated for this type of samples as higher P than N in snow samples is at least unexpected.

Figures in overall: The study uses similar colours for different variables in various figures. For example, Fig. 5 and Fig. 6 use the same red colour which make the orientation harder for reader. I would suggest choosing colour-schemes to be unified for the same variables to make the orientation in figures easier.

Discussion

L274–275: The sentence needs to be reworded slightly to make the meaning clearer. I would also recommend dividing it into shorter sentences to make it easier to read. I am not sure what information the authors wanted to convey to the reader, as both the soil-based site and S14 had a higher proportion of non-spherical cells and Chloromonas.

L279: “Previous studies support this explanation.”  I think this sentence is unnecessary, particularly given the paragraph's logic. The authors discuss the fact that the spherical morphotype is taxonomically heterogeneous, yet they continue to refer to studies supporting the spherical cell type for Sanguina.

L284: “Therefore, morphology alone can merge taxonomically distinct algae into the same category, especially for spherical cells.” In my opinion this sentence is redundant with L278.

L322: I miss the reference for the sentence on modulation of local growth.

Sect. 4.4. As radiation can affect the migration of algal cells, I would like to see more information and data on light conditions during sampling, as this information could substantially contribute to evaluating the results. This is particularly important when samples are taken within four days at which the light conditions could potentially differ.