Melting snow and glacier surfaces host microalgal blooms in polar and mountainous regions. The aim of this study was to determine the dominant taxa at the species level in the European Arctic and the Alps. A standardized protocol for amplicon metabarcoding using the 18S rRNA gene and ITS2 markers was developed. This is important because previous biodiversity studies have been hampered by the dominance of closely related algal taxa in snow and ice. Due to the limited resolution of partial 18S rRNA Illumina sequences, the hypervariable ITS2 region was used to further discriminate between the genotypes. Our results show that red snow was caused by the cosmopolitan Sanguina nivaloides (Chlamydomonadales, Chlorophyta) and two as of yet undescribed Sanguina species. Arctic orange snow was dominated by S. aurantia, which was not found in the Alps. On glaciers, at least three Ancylonema species (Zygnematales, Streptophyta) dominated. Golden-brown blooms consisted of Hydrurus spp. (Hydrurales, Stramenophiles) and these were mainly an Arctic phenomenon. For chrysophytes, only the 18S rRNA gene but not ITS2 sequences were amplified, showcasing how delicate the selection of eukaryotic 'universal' primers for community studies is and that primer specificity will affect diversity results dramatically. We propose our approach as a 'best practice'.

• Keywords
• ITS2 secondary structure, cryosphere, eDNA, glacier ice algae, next generation sequencing, snow algae, species delimitation,
• MeSH
• Chlorophyceae * genetics   MeSH
• Chlorophyta * genetics   MeSH
• Genes, rRNA MeSH
• Ice Cover MeSH
• RNA, Ribosomal, 18S genetics   MeSH
• Snow MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Ribosomal, 18S MeSH

Snow algae blooms often form green or red coloured patches in melting alpine and polar snowfields worldwide, yet little is known about their biology, biogeography, and species diversity. We investigated eight isolates collected from red snow in northern Norway, using a combination of morphology, 18S rRNA gene and internal transcribed spacer 2 (ITS2) genetic markers. Phylogenetic and ITS2 rRNA secondary structure analyses assigned six isolates to the species Raphidonema nivale, Deuterostichococcus epilithicus, Chloromonas reticulata, and Xanthonema bristolianum. Two novel isolates belonging to the family Stichococcaceae (ARK-S05-19) and the genus Chloromonas (ARK-S08-19) were identified as potentially new species. In laboratory cultivation, differences in the growth rate and fatty acid profiles were observed between the strains. Chlorophyta were characterized by abundant C18:3n-3 fatty-acids with increases in C18:1n-9 in the stationary phase, whilst Xanthonema (Ochrophyta) was characterized by a large proportion of C20:5n-3, with increases in C16:1n-7 in the stationary phase. In a further experiment, lipid droplet formation was studied in C. reticulata at the single-cell level using imaging flow cytometry. Our study establishes new cultures of snow algae, reveals novel data on their biodiversity and biogeography, and provides an initial characterization of physiological traits that shape natural communities and their ecophysiological properties.

• Keywords
• 18S rRNA, ITS2 rRNA, fatty acids, imaging flow cytometry, microalgae, phylogeny,
• MeSH
• Chlorophyceae * MeSH
• Chlorophyta * genetics   MeSH
• Phylogeny MeSH
• Lipids MeSH
• Microbiota * genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Norway MeSH
• Names of Substances
• Lipids MeSH

Seasonally slowly melting mountain snowfields are populated by extremophilic microalgae. In alpine habitats, high-light sensitive, green phytoflagellates are usually observed in subsurface layers deeper in the snowpack under dim conditions, while robust orange to reddish cyst stages can be seen exposed on the surface. In this study, uncommon surface green snow was investigated in the High Tatra Mountains (Slovakia). The monospecific community found in the green surface bloom consisted of vegetative Chloromonas cells (Volvocales, Chlorophyta). Molecular data demonstrated that the field sample and the strain isolated and established from the bloom were conspecific, and they represent a new species, Chloromonas kaweckae sp. nov., which is described based on the morphology of the vegetative cells and asexual reproduction and on molecular analyses of the strain. Cells of C. kaweckae accumulated approximately 50% polyunsaturated fatty acids, which is advantageous at low temperatures. In addition, this new species performed active photosynthesis at temperatures close to the freezing point showed a light compensation point of 126 ± 22 μmol photons · m-2 · s-1 and some signs of photoinhibition at irradiances greater than 600 μmol photons · m-2 · s-1 . These data indicate that the photosynthetic apparatus of C. kaweckae could be regarded as adapted to relatively high light intensities, otherwise unusual for most flagellate stages of snow algae.

• Keywords
• biodiversity, cryoflora, environmental sample, fatty acids, fluorometry, vegetative stages,
• MeSH
• Chlorophyceae * MeSH
• Chlorophyta * physiology   MeSH
• Photosynthesis physiology   MeSH
• Cold Temperature MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Slovakia MeSH

UNLABELLED: Red snow caused by spherical cysts can be found worldwide, while an orange snow phenomenon caused by spherical cells is restricted to (Sub-)Arctic climates. Both bloom types, occurring in the same localities at Svalbard, were compared ecophysiologically. Using a combination of molecular markers and light- and transmission electron microscopy, cells were identified as Sanguina nivaloides and Sanguina aurantia (Chlorophyceae). In search for reasons for a cosmopolitan vs. a more restricted distribution of these microbes, significant differences in fatty acid and pigment profiles of field samples were found. S. aurantia accumulated much lower levels of polyunsaturated fatty acids (21% vs. 48% of total fatty acids) and exhibited lower astaxanthin-to-chlorophyll-a ratio (2-8 vs. 12-18). These compounds play an important role in adaptation to extreme conditions at the snow surface and within snow drifts. Accordingly, the performance of photosystem II showed that one third to nearly half of the photosynthetic active irradiation was sufficient in S. aurantia, compared to S. nivaloides, to become light saturated. Furthermore, formation of plastoglobules observed in S. nivaloides but missing in S. aurantia may contribute to photoprotection. The rapid light curves of the two species show to a certain extent the shade-adapted photosynthesis under the light conditions at Svalbard (high α-value 0.16 vs. 0.11, low saturation point I k 59 vs. 86). These results indicate significant physiological and ultrastructural differences of the two genetically closely related cryoflora species, but the reasons why S. aurantia has not been found at conditions outside (Sub-)Arctic climate types remain unknown. SUPPLEMENTARY INFORMATION: The online version of this article (10.1007/s00300-020-02778-0) contains supplementary material, which is available to authorised users.

• Keywords
• Arctic, Astaxanthin, Chlamydomonas nivalis, Cryoflora, Green algae, Polyunsaturated fatty acid,
• Publication type
• Journal Article MeSH

Snow algae are photosynthetic microbes that inhabit the melting snow surface in alpine and polar regions. We analyzed the pigment and species composition of colored snow collected on Mt. Tateyama in Japan during the melting seasons of 2015 and 2016. High-performance liquid chromatographic analyses of the pigments extracted from the colored snow showed that their composition varied within the study area and were classified into four types: Type A (astaxanthin-monoester dominant), Type B (medium astaxanthin-monoester content), Type C (abundant primary carotenoids and free-astaxanthin), and Type D (abundant primary carotenoids and astaxanthin diesters). Types A and B were most commonly observed in the study area, whereas Types C and D appeared only at specific sites. Analysis of the 18S ribosomal RNA (18S rRNA) gene revealed six major amplicon sequence variants (ASVs) of snow algae, belonging to the Sanguina, Chloromonas, and Chlainomonas groups. The relative abundance of the algal ASVs showed that Sanguina was dominant (>48%) in both Types A and B, suggesting that the difference in astaxanthin abundance between the two types was caused by the production of pigments in the algal cells. The algal community structures of Types C and D differed from those of Types A and B, indicating that the primary carotenoids and astaxanthin diesters were derived from certain algal species in these types. Therefore, astaxanthin-rich Sanguina algae mostly induced the red snow that appeared widely in this alpine area; however, they were partially dominated by Chloromonas or Chlainomonas algae, causing different pigment compositions.

• Keywords
• 18S rRNA, Chloromonas, Sanguina, astaxanthin, phylogenetic analysis, pigment composition, red snow, snow algae,
• Publication type
• Journal Article MeSH

Melting mountainous snowfields are populated by extremophilic microorganisms. An alga causing orange snow above timberline in the High Tatra Mountains (Poland) was characterised using multiple methods examining its ultrastructure, genetics, life cycle, photosynthesis and ecophysiology. Based on light and electron microscopy and ITS2 rDNA, the species was identified as Chloromonas krienitzii (Chlorophyceae). Recently, the taxon was described from Japan. However, cellular adaptations to its harsh environment and details about the life cycle were so far unknown. In this study, the snow surface population consisted of egg-shaped cysts containing large numbers of lipid bodies filled presumably with the secondary carotenoid astaxanthin. The outer, spiked cell wall was shed during cell maturation. Before this developmental step, the cysts resembled a different snow alga, Chloromonas brevispina. The remaining, long-lasting smooth cell wall showed a striking UV-induced blue autofluorescence, indicating the presence of short wavelengths absorbing, protective compounds, potentially sporopollenin containing polyphenolic components. Applying a chlorophyll fluorescence assay on intact cells, a significant UV-A and UV-B screening capability of about 30 and 50%, respectively, was measured. Moreover, intracellular secondary carotenoids were responsible for a reduction of blue-green light absorbed by chloroplasts by about 50%. These results revealed the high capacity of cysts to reduce the impact of harmful UV and high visible irradiation to the chloroplast and nucleus when exposed at alpine snow surfaces during melting. Consistently, the observed photosynthetic performance of photosystem II (evaluated by fluorometry) showed no decline up to 2100 μmol photons m-2 s-1. Cysts accumulated high contents of polyunsaturated fatty acids (about 60% of fatty acids), which are advantageous at low temperatures. In the course of this study, C. krienitzii was found also in Slovakia, Italy, Greece and the United States, indicating a widespread distribution in the Northern Hemisphere.

• Keywords
• UV-A radiation, UV-B radiation, astaxanthin, chlorophyll fluorescence, cysts, photosynthesis, polyunsaturated fatty acids, snow algae,
• Publication type
• Journal Article MeSH

Melting snow fields are an extremophilic habitat dominated by closely related Chlamydomonadaceae (Chlorophyta). Microscopy-based classification of these cryophilic microalgae is challenging and may not reveal the true diversity. High-throughput sequencing (HTS) allows for a more comprehensive evaluation of the community. However, HTS approaches have been rarely used in such ecosystems and the output of their application has not been evaluated. Furthermore, there is no consensus on the choice for a suitable DNA marker or data processing workflow. We found that the correct placement of taxonomic strings onto OTUs strongly depends on the quality of the reference databases. We improved the assignments of the HST data by generating additional reference sequences of the locally abundant taxa, guided by light microscopy. Furthermore, a manual inspection of all automated OTU assignments, oligotyping of the most abundant 18S OTUs, as well as ITS2 secondary structure analyses were necessary for accurate species assignments. Moreover, the sole use of one marker can cause misleading results, either because of insufficient variability within the locus (18S) or the scarcity of reference sequences (ITS2). Our evaluation reveals that HTS output needs to be thoroughly checked when the studied habitats or organisms are poorly represented in publicly available databases. We recommend an optimized workflow for an improved biodiversity evaluation of not only snow algal communities, but generally 'exotic' ecosystems where similar problems arise. A consistent sampling strategy, two- molecular marker approach, light microscopy-based guidance, generation of appropriate reference sequences and final manual verification of all taxonomic assignments are highly recommended.

• Keywords
• 18S rDNA, ITS2 rDNA, Illumina, OTU clustering, Sanger, high-throughput sequencing, oligotyping, red snow, secondary structure, snow algae,
• Publication type
• Journal Article MeSH

Slowly melting snowfields in mountain and polar regions are habitats of snow algae. Orange blooms were sampled in three European mountain ranges. The cysts within the blooms morphologically resembled those of Chloromonas nivalis (Chlorophyceae). Molecular and morphological traits of field and cultured material showed that they represent a new species, Chloromonas hindakii sp. nov. The performance of photosystem II was evaluated by fluorometry. For the first time for a snow alga, cyst stages collected in a wide altitudinal gradient and the laboratory strain were compared. The results showed that cysts were well adapted to medium and high irradiance. Cysts from high light conditions became photoinhibited at three times higher irradiances (600 µmol photons m-2 s-1) than those from low light conditions, or likewise compared to cultured flagellates. Therefore, the physiologic light preferences reflected the conditions in the original habitat. A high content of polyunsaturated fatty acids (about 60% of total lipids) and the accumulation of the carotenoid astaxanthin was observed. They are regarded as adaptations to cope with extreme environmental conditions of snow that include low temperatures, freeze-thaw cycles, and variable light intensity. The intraspecific ability of adaptation of the photosynthetic apparatus to different irradiance regimes seems to be advantageous for thriving in different snow habitats.

• Keywords
• astaxanthin, cryoflora, cysts, environmental sample, fatty acids, photosynthesis,
• Publication type
• Journal Article MeSH

Melting snowfields in polar and alpine regions often exhibit a red and orange colouration caused by microalgae. The diversity of these organisms is still poorly understood. We applied a polyphasic approach using three molecular markers and light and electron microscopy to investigate spherical cysts sampled from alpine mountains in Europe, North America and South America as well as from both polar regions. Molecular analyses revealed the presence of a single independent lineage within the Chlamydomonadales. The genus Sanguina is described, with Sanguina nivaloides as its type. It is distinguishable from other red cysts forming alga by the number of cell wall layers, cell size, cell surface morphology and habitat preference. Sanguina nivaloides is a diverse species containing a total of 18 haplotypes according to nuclear ribosomal DNA internal transcribed spacer 2, with low nucleotide divergence (≤3.5%). Based on molecular data we demonstrate that it has a cosmopolitan distribution with an absence of geographical structuring, indicating an effective dispersal strategy with the cysts being transported all around the globe, including trans-equatorially. Additionally, Sanguina aurantia is described, with small spherical orange cysts often clustered by means of mucilaginous sheaths, and causing orange blooms in snow in subarctic and Arctic regions.

• Keywords
• Chlamydomonas nivalis, haplotype network, red snow, snow algae,
• MeSH
• Chlorophyta classification   genetics   physiology   MeSH
• Ecosystem MeSH
• Phylogeny MeSH
• Phylogeography MeSH
• DNA, Ribosomal Spacer MeSH
• Rhodophyta MeSH
• Snow microbiology   MeSH
• Freezing MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Europe MeSH
• South America MeSH
• North America MeSH
• Names of Substances
• DNA, Ribosomal Spacer MeSH

Long-lasting, slowly melting snowfields in mountainous regions are frequently populated by specialised microalgae whose diversity is still vastly underestimated. Cysts causing sub-surficial green snow were collected in the Austrian Alps, Tyrol, and morphologically accorded to the snow alga Scotiella cryophila sensu Chodat, initially described from Switzerland. The cytology and photobiology of this population were investigated to understand mechanisms of adaptation to the harsh habitat. Cysts of S. cryophila K-1 had secondary cell walls with pronounced rib-like surface structures and contained several small spherical plastids. The cytoplasm was dominated by lipid bodies, which developed reddish secondary pigmentation. Partial life cycle observations showed that daughter cells lacked structured cell walls. Cysts performed active photosynthesis at temperature conditions close to the freezing point and were photoinhibited at irradiances greater than 70 μmol m-2 s-1. This corresponded exactly to habitat conditions 20 to 40 cm below the snow surface. Phylogenetic analyses using 18S rDNA, rbcL and ITS2 rDNA sequences indicated that S. cryophila K-1 is related to Chloromonas, known to contain several snow algae. The taxon forms an independent lineage and is clearly genetically distinct from the type strain of Chloromonas rosae var. psychrophila from North America that is supposed to have morphologically identical cysts. For a taxonomic treatment including a species assignment of S. cryophila K-1 from Europe within Chloromonas, flagellates will have to be cultivated from cysts or from acquired field material for a detailed morphological description. Acquisition and genetic analysis of cysts that resemble S. cryophila from America could elucidate their relationship to European samples.

• Keywords
• Cryoflora, Cryospheric algae, Cysts, Extremophiles, Fluorometry, Ultrastructure,
• Publication type
• Journal Article MeSH