Ecophysiology of Chloromonas hindakii sp. nov. (Chlorophyceae), Causing Orange Snow Blooms at Different Light Conditions

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.


Introduction
For microalgae, snow and ice surfaces are habitats characterized by a multitude of abiotic stresses, including low nutrients, diurnal freeze-thaw cycles, high UV and photosynthetically active radiation (PAR) at the surface, and an overall short growing season. In these ecosystems, diverse algal communities develop distinct snow discolorations and exhibit individual cellular metabolic profiles [1]. The organisms responsible for such a phenomenon mainly belong to green algae [2,3]. Prominent genera thriving in snow are Chloromonas [4] and Sanguina (formerly assigned to Chlamydomonas) [5].
These microalgae are essential primary producers in such an extreme ecosystem (e.g., [6]), where phototrophic life is restricted to a few specialized organisms. For instance, they provide a basic ecosystem, e.g., for snow bacteria [7], fungi [8], and ciliated protozoa [9]. Snow algae microbial communities play an important role in snow food webs and supply nutrients that are delivered throughout the ecosystem (e.g., supraglacial and periglacial environments) [10].
Insight in complex feedbacks between snow and climate were recently done by [11]. Snow in the mid-latitudes appears to be most sensitive to climate change [12], and mid-latitude areas below 1200 m a.s.l. are assumed to suffer complete snow loss by the end of 21st century [13]. Therefore, For sampling, the selection of virtually monospecific spots was done with a field microscope according to [5]. Orange snow was harvested with a sterile shovel, placed in 5 l buckets, 1 l thermos bottles, or 50 mL centrifugation tubes, and transported the same day to the laboratory. Prior to photosynthesis measurements, samples were melted gently at darkness overnight at 4-5 • C. Electrical conductivity (EC) and pH of the meltwater were obtained with WTW Instruments (Cond 340i and Inolab, Germany) or with HANNA (Combo EC, ftb Romania). Snow water equivalent (SWE; referred to as 'snow water content' in the following reference) was carried out as described previously [20]. For sampling, the selection of virtually monospecific spots was done with a field microscope according to [5]. Orange snow was harvested with a sterile shovel, placed in 5 l buckets, 1 l thermos bottles, or 50 mL centrifugation tubes, and transported the same day to the laboratory. Prior to photosynthesis measurements, samples were melted gently at darkness overnight at 4-5 °C. Electrical conductivity (EC) and pH of the meltwater were obtained with WTW Instruments (Cond 340i and Inolab, Germany) or with HANNA (Combo EC, ftb Romania). Snow water equivalent (SWE; referred to as 'snow water content' in the following reference) was carried out as described previously [20].

Strain Isolation
For obtaining a unialgal strain of C. hindakii sp. nov., a subsample of WP129 (containing only sedimented cysts, no flagellates observed) with visible orange coloration was put into sterile 2 mL cryotubes and the meltwater replaced with deionized water. For induction of germination of the cysts, the cells were kept at 1 °C during the day (14 h) resp. −1 °C during the night (10 h) in a Percival LT-36VL (CLF Plant Climatics, Wertingen, Germany). The light intensity generated by fluorescent tubes was approximately 40-70 μmol PAR m −2 s −1 . After several weeks, many cysts developed daughter cells ( Figure S2), and subsequently, green flagellates were present in the supernatant; 10 µ L

Strain Isolation
For obtaining a unialgal strain of C. hindakii sp. nov., a subsample of WP129 (containing only sedimented cysts, no flagellates observed) with visible orange coloration was put into sterile 2 mL cryotubes and the meltwater replaced with deionized water. For induction of germination of the cysts, the cells were kept at 1 • C during the day (14 h) resp. −1 • C during the night (10 h) in a Percival LT-36VL (CLF Plant Climatics, Wertingen, Germany). The light intensity generated by fluorescent tubes was approximately 40-70 µmol PAR m −2 s −1 . After several weeks, many cysts developed daughter  (Figure S2), and subsequently, green flagellates were present in the supernatant; 10 µL aliquots of the latter were transferred into liquid 0.6 N Bold's Basal Medium (BBM) [24] and irradiation was dimmed to 20-30 µmol PAR m −2 s −1 . In the next step, the culture was used for genetics, microscopy, fluorometry, and lipid analysis. This strain was deposited as CCCryo 531-19 at the Culture Collection of Cryophilic Algae in Germany.

Light and Electron Microscopy
Light microscopy (LM) was performed with an Olympus BX43 at 1000× magnification using oil immersion, equipped with Nomarski Contrast and an Olympus DP27 camera or digital camera DXM 1200F (Nikon, Melville, NY, USA), using cellSens Entry Imaging Software. Scanning and transmission electron microscopy (SEM and TEM) were carried out as described previously [20].

Isolation of DNA, PCR, Sequencing
DNA isolation was carried out with a DNeasy Plant Mini Kit (Qiagen, Germany), as in [20]. If less than 20 mg wet biomass was available, DNA was extracted using the Instagene Matrix Kit (Bio-Rad Laboratories, Hercules, CA, USA) according to [25]. The 18S small subunit ribosomal RNA gene (18S rDNA), internal transcribed spacer region 2 (ITS2 rDNA), and ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) marker regions were amplified from DNA isolates by polymerase chain reaction (PCR) using existing primers (Table 2). Amplification reactions were described in [20]. PCR products were purified and sequenced using an Applied Biosystems automated sequencer (ABI 3730xl) at Macrogen Europe (Amsterdam, Netherlands). The obtained sequences of C. hindakii sp. nov. were submitted to NCBI Nucleotide sequence database (accession numbers at Table 3).

ITS2 rRNA Secondary Structure Prediction and Phylogenetic Analysis
The methods of annotation and prediction of the secondary structure of the nuclear rDNA ITS2 region were the same as in [20]. The secondary structure of nuclear rDNA ITS2 of C. hindakii sp. nov. was drawn using VARNA version 3.9 [32]. The 18S rDNA alignment contained 40 sequences (1567 bp) examined in previous studies [4,22], as well as three specimens of field-collected cysts and C. hindakii strain WP129 (= CCCryo 531-19); the rbcL matrix consisted of 37 sequences (924 bp), as well as four specimens of field-collected cysts and C. hindakii strain WP129 (= CCCryo 531-19); the mesophilic species of the genus Chloromonas sensu [33] or the Chloromonadinia clade [34] were selected as the outgroup. The best-fit nucleotide substitution model was estimated by jModeltest 2.0.1 [35]. Based on the Akaike Information Criterion, the GTR+I+G model was selected for 18rDNA. Three partitions were set for rbcL gene sequences and the following substitution models were applied: TIM1+I+G (first codon position), K80+I (second codon position), and GTR+I+G (third codon position). The 18S rDNA and rbcL phylogenetic trees were inferred by Bayesian inference (BI) and maximum likelihood (ML) according to [36], with the minor modification that Markov Chain Monte Carlo runs were carried out for three million generations in BI. Convergence of the two cold chains was checked by the average standard deviation of split frequencies (0.000533 and 0.00105 for 18S rDNA and rbcL dataset, respectively). Bootstrap analyses and Bayesian posterior probabilities were performed as described by [36].

Photosynthesis
In vivo chlorophyll fluorescence parameters were obtained with a pulse-amplitude modulated fluorometer (PAM 2000, Heinz Walz GmbH, Germany) in a 0.6 mL chamber and cooled with an ice bath to approximately 2 • C. To obtain the relative electron transport rates (rETR) and the light saturation point I k , cells were exposed to photon flux densities (PFD) of 5,9,34,67,104,201,366,622,984,1389,1666, and 2018 µmol photons m −2 s −1 for 30 s each. Four independent replicates were measured. For further details, see [20].

Pigment Analysis
Carotenoids and chlorophylls were analyzed by HPLC (Agilent 1200 ChemStation) equipped with a quaternary pump and a diode array detector at 450 nm, using a reversed phase YMC C30 Carotenoids column (YMC Europe, Dinslaken, Germany), ID 300 mm × 4.6 mm at 25 • C. α-tocopherol was simultaneously measured with a fluorescence detector (Em/Ex = 295/325 nm). The flow rate was 1 mL min −1 , analysis time was 20 min following an 8 min post-run prior next injection. The mobiles phases were (A) methanol (gradient grade), (B) methyl tert-butyl ether (MTBE; HPLC grade) with 1.3% deionized water (w/w), and (C) MTBE (HPLC grade). The linear solvent gradient was: From 0 to 4 min at 82% A, 18% B, from 4 to 12 min to 50% A, 0% B, 50% C, from 12 to 20 min to 20% A, 80% C. Post-run was 20% A, 0% B, 80% C from 20 to 23 min, from 23 to 24 min to 82% A, 18% B, and then kept for 4 min. Cells were lyophilized for 48 h and frozen at −25 • C prior use. Extraction was performed with a porcelain mortar and pestle (Z247464 and Z247502, Sigma-Aldrich), using liquid nitrogen for 2 min of precooling and during grinding of cells for 1 min. The cell powder (approximately 10-50 mg) was suspended in approximately 5 mL of chloroform/dichloromethane (2/1) with 1 mM BHT (butylated hydroxytoluene) as antioxidants, the suspension grinded for another minute, and then transferred with a glass pipette into 50 mL plastic tubes for a subsequent post-extraction for at least 12 h at −25 • C. Afterwards, the supernatant was gently evaporated at 30 • C and resuspended in a defined volume of organic solvents (usually 5 mL) composed of 82% mobile phase A and 18% mobile phase B. Prior to injection, the extracts were centrifuged for 10 min at 10,000× g and 1 • C. Peak identification was done by peak retention time and peak spectrum in relation to calibration standards (Sigma-Aldrich, Darmstadt, Germany).

Lipid Extraction and Fatty Acid Methyl Esters Analysis (FAMEs)
The extraction procedure was based on the method of [37], and elution was done from a Sep-Pak Vac Silica cartridge 35cc (Waters; 10 g normal-phase silica) by chloroform (neutral lipids), acetone (glycolipids), and methanol (phospholipids) [38]. All classes of lipids were saponified overnight in 10% KOH in methanol at room temperature. The structures of FAMEs were confirmed by comparison with Gas Chromatography/Mass Spectrometry retention times and fragmentation patterns with those of standard FAMEs (Supelco, Prague) method of [39,40]. Procedures were described in detail by [20].

Habitat Conditions
In the High Tatra (Slovakia, Poland), and Krkonoše and Jeseníky Mountains (Czech Republic), orange snow fields were found in May and June 2017 and 2019 at altitudes from 984 to 2082 m a.s.l. (Figure 2, Table 1). The habitat conditions of these localities are shown in Table 4. The orange blooms occurred at open sites above timberline (snow surface: Samples "WP194", "WP129", "WP130", "WP136"; for "LP06", spots were visible at the surface but the main bloom was harvested at 3-5 cm depth), a semi-shaded site above timberline (close to a boulder, sample "NW"), below dwarf pines ("Rozcestí"). Below timberline, the species was found at open sites (at avalanche slopes, data not shown), semi-shaded (samples "DD2", "Jes19-1"), and full-shaded sites ("Jes19-6"). For the purpose of rapid light curves measurements, a sample from high light conditions (WP194) and samples from low light conditions (LP06 and DD2) were investigated.

Lipid Extraction and Fatty Acid Methyl Esters Analysis (FAMEs)
The extraction procedure was based on the method of [37], and elution was done from a Sep-Pak Vac Silica cartridge 35cc (Waters; 10 g normal-phase silica) by chloroform (neutral lipids), acetone (glycolipids), and methanol (phospholipids) [38]. All classes of lipids were saponified overnight in 10% KOH in methanol at room temperature. The structures of FAMEs were confirmed by comparison with Gas Chromatography/Mass Spectrometry retention times and fragmentation patterns with those of standard FAMEs (Supelco, Prague) method of [39,40]. Procedures were described in detail by [20].

Habitat Conditions
In the High Tatra (Slovakia, Poland), and Krkonoše and Jeseníky Mountains (Czech Republic), orange snow fields were found in May and June 2017 and 2019 at altitudes from 984 to 2082 m a.s.l. ( Figure 2, Table 1). The habitat conditions of these localities are shown in Table 4. The orange blooms occurred at open sites above timberline (snow surface: Samples "WP194", "WP129", "WP130", "WP136"; for "LP06", spots were visible at the surface but the main bloom was harvested at 3-5 cm depth), a semi-shaded site above timberline (close to a boulder, sample "NW"), below dwarf pines ("Rozcestí"). Below timberline, the species was found at open sites (at avalanche slopes, data not shown), semi-shaded (samples "DD2", "Jes19-1"), and full-shaded sites ("Jes19-6"). For the purpose of rapid light curves measurements, a sample from high light conditions (WP194) and samples from low light conditions (LP06 and DD2) were investigated.

Maximal Population Density and Morphology of Field-Collected Cysts
The population densities reached from 19,950 to 79,100 orange cysts mL −1 meltwater (Table 4). Neither green nor orange flagellates were observed in the field material. Cell wall surfaces and intracellular organization were observed by light and electron microscopy ( Figure 3). The fusiform cysts with wall surface structures corresponded to those of the aplanozygote of C. nivalis, as proposed by [41]. Cells were 18.5-34.3 µ m long and 11.9-23.1 µ m wide, with a length to width ratio of 1.2:1.9 ( Figure S3). Typically, there were 7-10 flanges at the equatorial plane. Wall flanges were either

Maximal Population Density and Morphology of Field-Collected Cysts
The population densities reached from 19,950 to 79,100 orange cysts mL −1 meltwater (Table 4). Neither green nor orange flagellates were observed in the field material. Cell wall surfaces and intracellular organization were observed by light and electron microscopy ( Figure 3). The fusiform cysts with wall surface structures corresponded to those of the aplanozygote of C. nivalis, as proposed by [41]. Cells were 18.5-34.3 µm long and 11.9-23.1 µm wide, with a length to width ratio of 1.2:1.9 ( Figure S3). Typically, there were 7-10 flanges at the equatorial plane. Wall flanges were either straight or slightly undulating. SEM showed that four prominent flanges always joined at the apex and antapex of the cell, and these flanges ran from one pole to the other. Other wall flanges were shorter and terminated either isolated in the subapex zone, or less often, fused with another flanges. The cytoplasm was dominated by orange-reddish pigmented compartments. Mature cysts had small chloroplast discs (data not shown). The daughter cells within the cysts were smooth-walled ( Figure 3e).
Microorganisms 2019, 7, x 7 of 22 straight or slightly undulating. SEM showed that four prominent flanges always joined at the apex and antapex of the cell, and these flanges ran from one pole to the other. Other wall flanges were shorter and terminated either isolated in the subapex zone, or less often, fused with another flanges. The cytoplasm was dominated by orange-reddish pigmented compartments. Mature cysts had small chloroplast discs (data not shown). The daughter cells within the cysts were smooth-walled ( Figure  3e).

Phylogeny and Comparative Analysis of Internal Transcribed Spacer 2
The sequences of 18S rDNA and rbcL among the strain WP129 (= CCCryo 531-19) and field-collected cysts were identical. According to phylogenies of 18S rDNA ( Figure 4) and rbcL ( Figure 5), this species is a member of "Chloromonas clade B" sensu [4]. Chloromonas hindakii is related to Chloromonas nivalis Gassan-B (11 bp different out of 1650 bp in 18S rDNA), Chloromonas polyptera DRAnt023 (15 bp different out of 1653 bp in 18S rDNA), Scotiella cryophila K-1 (21 bp out of 1680 bp in 18S rDNA), and Chloromonas nivalis subsp. tatrae LP01 (34 bp out of 1683 bp in 18S rDNA). The new species formed a well supported subclade with other specimens of field cysts identified as Chloromonas nivalis P24/DR4 from the Austrian Alps and Chloromonas nivalis subsp. tatrae from the High Tatra Mountains (Slovakia) in 18S rDNA phylogeny. In contrast, the new species formed a well supported subclade together with Scotiella cryophila K-1 from the Austrian Alps in the rbcL phylogeny. Within all investigated samples (Table 3), the number of nucleotide differences in the entire ITS2 region ranged from 0 to 4 bp, and no compensatory base changes (CBCs) were detected ( Figure 6). Conversely, one CBC was found between strain WP129 (= CCCryo 531-19) and an uncultured environmental clone ALBC6 from Switzerland ( Figure S4), and between strain WP129 (= CCCryo 531-19) and Chloromonas nivalis Gassan-B from Japan ( Figure S5), even near to the 5' apex of III helix, the most conserved part of ITS2 [42].  [4]. Posterior probabilities (0.95 or more) and bootstrap values from maximum likelihood analyses (50% or more) are shown. Full statistical support (1.00/100) is marked with an asterisk. Thick branches represent nodes receiving the highest posterior probability support (1.00). Newly obtained sequences are in bold. Accession numbers, strain, or field sample codes are indicated after each species name.

Morphology of Vegetative Cells of Chloromonas hindakii sp. nov.
The strain consisted of green and solitary cells, bean-shaped with a rounded posterior end, 7-12.5 μm wide and 17-24.5 μm long (Figure 7). Cells had two equal flagella (if present) at the anterior end, a single chloroplast not fully occupying the posterior end of the protoplast (Figure 7a), two contractile vacuoles near the base of the flagella (Figure 7a), and the wall had no prominent anterior papilla (Figure 7b). The compact, elongated to slightly bean-shaped chloroplast had an emargination in the median region where the nucleus is usually located. The plastid was further lacking an eyespot   (Figure 7a), and the wall had no prominent anterior papilla (Figure 7b). The compact, elongated to slightly bean-shaped chloroplast had an emargination in the median region where the nucleus is usually located. The plastid was further lacking an eyespot and pyrenoids. The nucleus was almost spherical, located in the ventral half in the middle of the protoplast (Figure 7b). Asexual reproduction occurred via zoospore (autospore) formation. Generally, two, four, or rarely, eight daughter cells were produced within the parental wall (Figure 7c-e). Sexual reproduction was not observed in the culture, even under nitrogen starvation; nor was the formation of cysts or accumulation of secondary reddish pigments. For testing temperature preferences, cells of C. hindakii were exposed to 15 • C but started to decay after 2 weeks, whereas at 5 • C they grew well long-term, and likewise at 1 • C (data not shown). Figure 7f shows an overview of a flagellate by TEM. The papilla was small and flattened (Figure 7g). Cytokinesis took place in equatorial position of the cell (Figure 7h), resulting either in two spherical cells surrounded by undulating cytoplasmatic membrane only (Figure 7i), or resulting in two elongated cells per sporangium with a prominent rough endoplasmic reticulum (indicating active metabolism). The daughter cells already possessed flagella (see an ultrastructure trait marked with "f") (Transversal section, Figure 7j). Prior the release from the mother cell, the flagellates developed their own cell wall. The majority released flagellates were bean-shaped, but unusual spherical cells were also observed. Older, mature flagellates contained numerous small discoid chloroplasts with starch grains (Figure 7k). and pyrenoids. The nucleus was almost spherical, located in the ventral half in the middle of the protoplast (Figure 7b). Asexual reproduction occurred via zoospore (autospore) formation. Generally, two, four, or rarely, eight daughter cells were produced within the parental wall ( Figure  7c-e). Sexual reproduction was not observed in the culture, even under nitrogen starvation; nor was the formation of cysts or accumulation of secondary reddish pigments. For testing temperature preferences, cells of C. hindakii were exposed to 15 °C but started to decay after 2 weeks, whereas at 5 °C they grew well long-term, and likewise at 1 °C (data not shown). Figure 7f shows an overview of a flagellate by TEM. The papilla was small and flattened ( Figure  7g). Cytokinesis took place in equatorial position of the cell (Figure 7h), resulting either in two spherical cells surrounded by undulating cytoplasmatic membrane only (Figure 7i), or resulting in two elongated cells per sporangium with a prominent rough endoplasmic reticulum (indicating active metabolism). The daughter cells already possessed flagella (see an ultrastructure trait marked with "f") (Transversal section, Figure 7j). Prior the release from the mother cell, the flagellates developed their own cell wall. The majority released flagellates were bean-shaped, but unusual spherical cells were also observed. Older, mature flagellates contained numerous small discoid chloroplasts with starch grains (Figure 7k).

Chloromonas hindakii Procházková & Remias sp. nov. (Figures 3 and 7)
DESCRIPTION: Vegetative cells solitary, having two equal flagella, two contractile vacuoles near the base of the flagella, a single chloroplast, single spherical nucleus positioned in the ventral half at the middle of the cell, without prominent anterior papilla. Cells elongate, kidney-shaped or bean-shaped with rounded posterior end; 7-11.5 µm wide and 17-24.5 µm long. A single chloroplast not occupying the posterior end of the protoplast and lacking eyespot and pyrenoid. Asexual reproduction by formation of two, four, or eight zoospores within the parental cell. Cell aggregates not observed in old cultures. Sexual reproduction unknown. Zygotes or cysts elongate to fusiform, 18.5-34.3 µm long and 11.9-23.1 µm wide, with length to width ratio within a range of 1.2-1.9. Cell walls with rib-like surface structures, such flanges are either straight or slightly undulating. Four prominent flanges always join at the apex and antapex of the cell, and they run entirely from one pole to the other. Further wall flanges are shorter, i.e., terminating either isolated at the subapical zone or less often fusing with another flange, or representing solitary flanges. The cytoplasm of cysts or zygotes usually contains reddish carotenoid droplets. The species differs from any other described representatives of the genus Chloromonas in the nuclear 18S rDNA, ITS rDNA, and plastid rbcL gene sequences (accession numbers: MN251865, MN251865, and MN251877, respectively  (1937-2019), a Slovak phycologist with influential contributions on microscopic algae. He described many new genera and species, including snow algae (e.g., [43,44]), and was one of the pioneers in successfully cultivating snow algae at laboratory conditions [45].

Photosynthesis
The photosynthetic activity of C. hindakii sampled at locations with different light conditions was tested and rapid light curves generated ( Figure 8). Additionally, the performance of field cysts was compared with the laboratorial strain. Generally, the cysts were not dormant in terms of photosynthesis, as indicated by rETR. For the strain, and field cysts from low light conditions (below dwarf pine or 5 cm below the snow surface at a site above timberline), photoinhibition occurred above 200 µmol PAR m −2 s −1 . The strain showed an α value of 0.23, a relative ETRmax of 6.6 ± 2.3, and an I k value of 33 ± 8 µmol PAR m −2 s −1 . Field cysts from low light conditions had one-quarter lower alpha values (0.17 and 0.16) and two-fold higher I k (73 and 78). In contrast, cysts from the snow surface above timberline (i.e., high light conditions) showed later signs of photoinhibition, starting from three times higher irradiances (600 µmol photons m −2 s −1 ); they also showed a two-fold higher ETRmax (14.5 ± 1) and four times higher I k (129 ± 27), but a nearly twice lower α (0.13), when compared to the strain. nov. The effect of increasing photon fluence rates (x-axis) on the relative electron transport rate (rETR; y-axis) of chloroplasts was measured for vegetative flagellates (green diamonds, strain WP129 (= CCCryo 531-19) grown in the lab) and non-motile field-collected cysts (blue trianglessemi-shaded site close to a spruce canopy, sample DD2; orange circles-site above timberline, population located mainly 3-5 cm below snow surface, sample LP06; red boxes-site above timberline, population at snow surface, sample WP194). Values are means of four replicate measurements (n = 4, ± SD). The data points were fitted to the photoinhibition model of [46].

Pigment Composition
The orange pigmentation of cysts of C. hindakii was caused by the secondary (non-plastid) carotenoid astaxanthin. Its abundance and those of other pigments and α-tocopherol was calculated in reference to chlorophyll-a (Table 5). Astaxanthin comprised 19.8% and 22.1% of all pigments, respectively (WP194, LP06). At HPLC, it occurred in a range of several peaks with identical absorption spectra, all of them likely esters with different fatty acids ( Figure S6). Chlorophylls (a and b) comprised 57.7% and 57.6% of all pigments, primary (plastid) carotenoids represented 22.5% and 20.3% of the pigment pool. The overall ratio of cysts for astaxanthin to chl-a was 0.4 and 0.5, respectively. Further details are given in Table 5. In contrast, the laboratorial strain always stayed green (data not shown). Table 5. Relative cellular content of carotenoids, chlorophyll b, and α-tocopherol in relation to chlorophyll a (= 1) in field samples of Chloromonas hindakii sp. nov. from the High Tatras at the Slovak side (sample WP194; high light conditions) and at the Polish side (LP06; low light conditions), determined by HPLC. Abbreviations: n & v, neoxanthin and violaxanthin; lut, lutein; zea, zeaxanthin; chl b, chlorophyll b; β-car, β-carotene; ast, astaxanthin (free/unesterified); ast-E, astaxanthin derivatives (esters); ast-tot, astaxanthin in total (free and derivatives); α-toc; α-tocopherol; n.d., not detected.

Fatty Acid (FA) Composition
The relative content of FAs (percentage of total lipids and percentage of the three major lipid groups) in C. hindakii is given in Table 6. FAs with 14 to 18 C prevailed in field cysts and the strain (WP129). Cells showed high levels of PUFAs (65.8% and 58.1%, respectively, of total lipids), whereas the content of saturated acids (SAFA) did not exceed 23% (mainly palmitic acid, 16:0). The main nov. The effect of increasing photon fluence rates (x-axis) on the relative electron transport rate (rETR; y-axis) of chloroplasts was measured for vegetative flagellates (green diamonds, strain WP129 (= CCCryo 531-19) grown in the lab) and non-motile field-collected cysts (blue triangles-semi-shaded site close to a spruce canopy, sample DD2; orange circles-site above timberline, population located mainly 3-5 cm below snow surface, sample LP06; red boxes-site above timberline, population at snow surface, sample WP194). Values are means of four replicate measurements (n = 4, ± SD). The data points were fitted to the photoinhibition model of [46].

Pigment Composition
The orange pigmentation of cysts of C. hindakii was caused by the secondary (non-plastid) carotenoid astaxanthin. Its abundance and those of other pigments and α-tocopherol was calculated in reference to chlorophyll-a (Table 5). Astaxanthin comprised 19.8% and 22.1% of all pigments, respectively (WP194, LP06). At HPLC, it occurred in a range of several peaks with identical absorption spectra, all of them likely esters with different fatty acids ( Figure S6). Chlorophylls (a and b) comprised 57.7% and 57.6% of all pigments, primary (plastid) carotenoids represented 22.5% and 20.3% of the pigment pool. The overall ratio of cysts for astaxanthin to chl-a was 0.4 and 0.5, respectively. Further details are given in Table 5. In contrast, the laboratorial strain always stayed green (data not shown). Table 5. Relative cellular content of carotenoids, chlorophyll b, and α-tocopherol in relation to chlorophyll a (= 1) in field samples of Chloromonas hindakii sp. nov. from the High Tatras at the Slovak side (sample WP194; high light conditions) and at the Polish side (LP06; low light conditions), determined by HPLC. Abbreviations: n & v, neoxanthin and violaxanthin; lut, lutein; zea, zeaxanthin; chl b, chlorophyll b; β-car, β-carotene; ast, astaxanthin (free/unesterified); ast-E, astaxanthin derivatives (esters); ast-tot, astaxanthin in total (free and derivatives); α-toc; α-tocopherol; n.d., not detected.

Geographic Distribution and Ecology
Based on the discoveries in several mountain ranges of Poland, Slovakia, and the Czech Republic, C. hindakii sp. nov. seems to be a common member of the local cryoflora. Remarkably, it causes orange blooms both below and above timberline, contrary to many other snow algae, which usually thrive either in exposed or shaded habitats. The distribution of the species, at altitudinal gradient, ranged from montane to subalpine and alpine vegetation belt; e.g., for Krkonoše Mountains, see [47] (p. 271). The discoloration may vary; along with lower population densities, the snow was once reported to be pinkish [48]. A further, hitherto unidentified snow alga with eyespot and prominent cellular tail from the same sampling region reported as´C. nivalis´ [48] is not in accordance to the morphology of C. hindakii (compare Figure 2b,c in [48] to Figure 7a,b in this study). However, in the study of [48], cysts morphologically identical to C. hindakii were found in Krkonoše at a wide altitudinal gradient from 740 to 1545 m a.s.l., at shaded (broad leaf and spruce trees) as well as at open sites (assigned to C. nivalis, see Figure 2e-g in [48]). Moreover, C. hindakii may occur also in other mountains in Slovakia [49]. Moreover, it may be found in the Bulgarian Vitosha Mountains, since [50] reported morphologically corresponding cysts in term of cell size, prominent cell apexes, and number and organization of longitudinal cell wall flanges. The overall geographic distribution of C. hindakii, including further regions like the European Alps, is not known yet, but distinct morphologic traits reported in this study and deposited molecular marker sequences should help for a future correct recognition.

Taxonomy and Related Species
To our knowledge, the vegetative strain of C. hindakii is the first available with a "C. nivalis-like" morphology of cysts (fusiform cells with prominent cell wall flanges) within the Chloromonas 'snow clade B' (sensu [4]). In many cases for snow algae, cysts were not made to germinate for generating a flagellate culture. Comparative analysis of the ITS2 rRNA secondary structure showed that C. hindakii is closely genetically related to an uncultured environmental clone, ALBC6, from a glacier forefield in Switzerland (accession number JX435348, [51]) and to C. nivalis Gassan-B from snow in Japan (accession number LC012758, [52]), indicating that the closest relatives also thrive in cold habitats. However, by using solely light microscopy, the cysts of C. hindakii have probably been reported several times as those of C. nivalis (as "Scotiella nivalis") in the Krkonoše [48,[53][54][55][56] and High Tatra Mountains [49,[57][58][59][60]. On the other hand, this is the first report about cryoflora from the Jeseníky Mountains. By the help of electron microscopy and molecular methods, it was possible to describe this new species out of the "collective" taxon C. nivalis. This goes along with the description of further new snow algae like C. hoshawii or C. remiasii, which were formerly assigned to C. nivalis as well [61].

Morphology of the Field Cysts
Field-collected cysts in snow identifiable by LM as the cosmopolitan taxon C. nivalis were recently shown to represent multiple species [61]. Detailed morphological comparisons of the cyst (i.e., cell wall flanges distribution and characterization [20]) and flagellates [61] is crucial for classical species determination. Morphologically, cysts of C. hindakii differ from its close molecular relative Scotiella cryophila K-1 in having significantly less elongated cysts (the latter has length to width ratios from 2.1 to 4.7 [22]). Moreover, C. hindakii cysts differ from genetically distinct C. nivalis in the Austrian Alps [18], and C. nivalis subsp. tatrae [20] in the High Tatras in their cell size (larger cysts) and more prominent cell wall flanges. The number of cell wall flanges when observed at equatorial position is lower for the new species compared to C. nivalis subsp. tatrae (the latter has (9)10 to 12 (14) flanges). These two species co-occurred occasionally in the High Tatra Mountains (own observations). Aplanozygotes of C. nivalis from North America [41] possess a lower number of flanges at cross-section (six to eight), are similarly large (16-37 µm × 10-27 µm), but lack fusing/diverting flanges apart from the apex; however, here are no sequences available for these cysts yet. In addition, the identity of several vegetative Chloromonas strains isolated from the collection sites of C. nivalis in North America [41] is still unknown, and morphological details of their source material are missing (i.e., UTEX SNO16, UTEX SNO17, UTEX SNO18, UTEX SNO19, and UTEX SNO21). Matsuzaki et al. [4] reported for C. muramotoi a regular cell wall flange organization (i.e., four long flanges reached to both poles of the cell) similar to C. hindakii; however, C. muramotoi can be discriminated by its markedly smaller cell sizes (9.1-13.4 µm wide and 15.6-22.4 µm long). Moreover, a bifurcation of wall flanges was not observed for C. muramotoi and shorter flanges were only medially located (i.e., not reaching subapical cell regions). In this aspect, the new species is somehow similar to another snow alga, C. miwae, which, however, is smaller (10-16 µm wide and 20-26 µm long) and lacks visible accumulation of secondary carotenoids [52]. Mature cysts of C. hindakii have small chloroplast discs like all other cysts of Chloromonas species dwelling in snow (e.g., see chloroplast shape in Figure 22 in [20]). To conclude, it is generally possible to distinguish the cysts of C. hindakii morphologically from cysts of other snow algae.

Morphology of the Strain and Context to C. nivalis
The vegetative flagellates of C. hindakii differ from the five other most similar looking elongate to bean-shaped Chloromonas species isolated from snow as follows: The chloroplast of C. hindakii does not occupy the posterior ending of the protoplast, in contrast to C. krienitzii [52]. C. hindakii is larger in size than C. fukushimae when kept at the same cultivation conditions (4-9 µm × 15-23 µm; [62]) and develops only rarely eight zoospores in zoosporangia. Large cell aggregates were not observed in old cultures of Chloromonas hindakii in opposition to Chloromonas polyptera, where aggregates are made up of more than 16 cells [63]. Chloromonas hindakii differs in chloroplast morphology from C. rostafinskii-the latter has a cup-shaped chloroplast [64]. Chloromonas hindakii differs from Chloromonas rubroleosa in papilla shape and number of contractile vacuoles near the base of flagella-the latter has prominent bimamillate papilla and four contractile vacuoles [65]. Additionally, the vegetative morphology of C. hindakii is different from species not possessing bean-shaped flagellates, such as the North American strain UTEX SNO71 [4], which was recently proposed to represent the 'true' C. nivalis [4,41]. The latter has tear drop-shaped flagellates with a prominent posterior end. However, Robert Chodat´s type locality of C. nivalis in France close to Mont Blanc has not yet been resurveyed for revealing the molecular identity of its population. Moreover, in the original description the flagellate morphology is missing [66]. Recent molecular data suggest that species with a C. nivalis-like morphology of cysts evolved several times in the history of snow-thriving Chloromonas species [4,52,61].

Photobiology of Field Cysts vs. Laboratorial Strain
The rapid light curves from different habitats showed a high intraspecific flexibility of C. hindakii in terms of light acclimation, reflecting the occurrence in semi-shaded locations close to spruce canopy, below dwarf pine vegetation, as well as in exposed sites above timberline. This likely indicates the capabilities of the photosynthetic apparatus in regard to changeable short-and long-term incident irradiations, such as (a) diurnal changes due to topographic or/and vegetation shadings [48], (b) the distance of the cells from the snow surface during melting season [16], and (c) actual weather situation (e.g., sunny vs. cloudy day; [67]).
The fluorometric measurements of cysts showed that the photosystem II was well adapted to medium-to-high levels of irradiation. The cysts from the high light conditions (red graph in Figure 8) became photoinhibited at much higher irradiances than cysts from low light conditions (blue and orange graphs in Figure 8) or the laboratory strain, which was grown at low-light conditions (green graph in Figure 8). At open sites, at a snow depth of 20 cm, usually less than 5% of incident irradiation may be available [15]. Thus, in case of this study, the cysts found at an alpine location 5 cm below snow surface (sample LP06) perceived similarly low PAR like the cells at snow surface below spruce canopy (sample DD2). As a consequence, the characteristics of the rapid light curves (i.e., alpha, I k and ETR values) of cells from these two different shaded sites are similar, which indicates that acclimation of the photosynthesis to certain snow depths is similar for C. hindakii with acclimation to a snow surface at semi-shaded site. Semi-shaded sites below timberline differ from open sites above timberline in the amplitude of PAR; for the former in the Krkonoše Mountains it reaches up to~1300 µmol photons m 2 s −1 [48]. In contrast, the cysts from open alpine sites (sample WP194) in the High Tatra Mountains were exposed to PAR exceeding 2000 µmol photon m 2 s −1 (see Supplementary Figure S27 in [16]). Likewise, other species were still able to perform positively at such high irradiances, and the light saturation (optimal) irradiance (i.e., I k ) of C. hindakii was nearly as high as those for cysts of C. nivalis and C. nivalis subsp. tatrae, causing reddish/brownish snow [18,20]. In contrast, the strain of C. hindakii exhibited a similarly low I k and high light utilization efficiency (alpha value) to those recorded for cysts of the snow alga Scotiella cryophila caused green subsurface stripes at a depth of 20 cm below the snow surface [22]. In other words, photosynthesis was adapted to low light conditions. The high intraspecific flexibility of C. hindakii's response to different light conditions allows this species to colonize, respectively survive at changeable conditions in snow.

Pigments and Fatty Acid Composition
The average levels of astaxanthin that cause the reddish coloration of C. hindakii are comparable to mature cysts of C. nivalis from the Austrian Alps [18]. Still, the astaxanthin to chlorophyll a ratio is three times lower than for C. nivalis subsp. tatrae from exposed sites at the High Tatras [20]. The main role of astaxanthin is likely to act as sunscreen against excessive visible and harmful UV irradiation.
On the other hand, too much astaxanthin would shade the plastids too much, thus decreasing the photosynthetic performance at sites with lower irradiation levels. Apparently, this species has developed a compromise. Probably, the accumulation of astaxanthin reflects the extent of endogenous cyst maturation, regardless of whether the cells are found at high light or at low light conditions (i.e., WP194 had similar astaxanthin to chl-a ratio like LP06). The laboratorial strain of C. hindakii produces no astaxanthin (data not shown), different to other microalgae feasible for biotechnology [68].
With the same protocol as for pigments, the plastid antioxidant α-tocopherol was quantified. The cysts of C. hindakii showed one magnitude higher concentrations than C. nivalis from the Austrian Alps [18], but the quantities were similarly low to cells that cause red snow (Sanguina nivaloides, referred as Chlamydomonas nivalis in [23]). The accumulation of vitamin E was reported to increase during the maturation process of Chlainomonas sp. cysts under harsh environmental conditions in the late melting season [16].
In both stages, the strain and the field cysts of C. hindakii had a high level of PUFAs, which is regarded as an adaptation to cold conditions [69]. High levels of PUFAs are common also for other snow-dwelling Chloromonas species (e.g., see FAs composition in Table 5 in [20]) and algae living in other cold habitats such as lake ice [70]. Hexadecatetraenoic acid was present in flagellates of C. hindakii at relatively high levels of total FAs (~30%); its production in C. hindakii was the same as in a strain of C. remiasii CCCryo 005-99 isolated from snow in the Arctic and kept under nitrogen deficient conditions [71]. A likewise high accumulation of hexadecatetraenoic acid was found in edible marine macroalgae Undularia pinnatifida and Ulva pertusa [72]. This fatty acid extracted from algae was shown to have physiological effects on the human health [73]; it inhibited production of leukotrienes involved in several inflammatory and allergic reactions (e.g., [74]). What is more, alpine and polar microalgal strains are promising candidates for biotechnology [75], also in outdoor bioreactors at low temperature conditions [76]. Higher levels of saturated palmitic acid and monounsaturated oleic acid in C. hindakii correspond to similar amounts found in other strains of genus Chloromonas [77].

Conclusions
Molecular phylogenetic assays, in combination with morphological observation of cysts (likely zygotes) and vegetative flagellates, demonstrated that C. hindakii is a new species within a group of morphologically and taxonomically closely related cryoflora phytoflagellates, and most of them were formerly associated with the collective taxon C. nivalis. An unialgal vegetative strain with motile flagellates was established from field-collected cysts under suitable laboratorial conditions simulating the temperature of melting snow. For further detail in physiological, cytological, or closer molecular investigations, a clonal strain should be derived from a single cell. C. hindakii occurred at very different altitudes from the montane belt to subalpine and alpine locations of mid-latitude mountains, and it exhibited an infraspecific photosynthetic flexibility accordingly. This study shows for the first time for a snow alga that populations from high light conditions get photoinhibited at higher irradiances than those harvested from low light conditions. The physiologic light preferences reflected the light conditions in the original habitat. PUFAs prevailed in the lipids of C. hindakii, and astaxanthin was the pigment responsible for the orange coloration of the snow bloom. Generally, the response of a cold-adapted alga exposed to different light conditions can be investigated, along with screening which responses are activated (e.g., effect on pigments, proteins), as done for Koliella antarctica [78]. In future, the intraspecific variability among populations of other snow algae and its relation to the incident irradiation may be tested. Funding: This research was funded by the Czech Science Foundation (GACR) projects 18-02634S granted to L.N. and L.P., 17-00027S granted to T.Ř., and by the Austrian Science Fund (FWF) project P29959 granted to D.R.