In polar regions, melting snow fields can be occupied by striking blooms of chrysophycean algae, which cause yellowish slush during summer. Samples were harvested at King George Island (South Shetland Islands, Maritime Antarctica) and at Spitsbergen (Svalbard archipelago, High Arctic). The populations live in an ecological niche, where water-logged snow provides a cold and ephemeral ecosystem, possibly securing the survival of psychrophilic populations through the summer. A physiological adaptation to low temperatures was shown by photosynthesis measurements. The analysis of soluble carbohydrates showed the occurrence of glycerol and sugars, which may play a role in protection against intracellular freezing. Although both populations were made of unicells with Ochromonas-alike morphology, investigation by molecular methods (18S rDNA sequencing) revealed unexpectedly a very close relationship to the mountain-river dwelling Hydrurus foetidus (Villars) Trevisan. However, macroscopic thalli typical for the latter species were never found in snow, but are known from nearby localities, and harvested samples of snow algae exposed to dryness evolved a similar pervading, ‘fishy’ smell. Moreover, in both habitats tetrahedal zoospores with four elongate spikes were found, similar to what is known from Hydrurus. Our molecular results go along with earlier reports, where chrysophycean sequences of the same taxonomic affiliation were isolated from snow. This points to a distinct group of photoautotrophic, Hydrurus-related chrysophytes, which are characteristic for long-lasting, slowly melting snow packs in certain cold regions of the world.
Blooms of chrysophycean algae occurring in ‘conventional’ marine and freshwater ecosystems have been known for a long time (Nicholls 1995), but mass accumulations thriving in thawing snow fields, thus causing striking yellowish colorations, have been rarely recognized. In contrast, psychrophilic green algae like Chlamydomonas nivalis (Bauer) Wille, which cause red snow in polar and alpine regions, seem to be more frequent and are better known in means of ecology, taxonomy and physiology (Kol 1968; Hoham & Duval 2001; Komárek & Nedbalová 2007). Reports about Chrysophyta living in snow are sparse, and the best described habitats were at North America (Stein 1963; Hoham et al. 1993) and the Japanese Alps (Tanabe et al. 2011). Novis (2002) found two species in snow of the Alps of New Zealand, but yellow snow caused by chrysophytes has not been found in the European Alps so far (Remias 2012). By means of microscopy, the causes of such phenomena were frequently assigned to the unicellular genera Ochromonas smithii Fukushima or Chromulina chionophila Stein. Although Chrysophyta are generally regarded as specialists of cold stenotherm, oligotrophic habitats (Kristiansen 2009); however, the mechanisms causing seasonal colored snow are still unknown. Recently, Tanabe et al. (2011) showed that O. smithii and O. itoi Fukushima, isolated from snow, possess a highly active xanthophyll cycle so that the chloroplast can cope with elevated levels of solar irradiation typical for snow surfaces by the help of these carotenoids. Generally, excessive irradiation is not the sole stress-factor, because melting snow can also be harsh in terms of the frequently occurring freeze-thaw-cycles.
The taxonomic affiliation of snow dwelling chrysophytes remains largely unclear. Isolates that were earlier associated with the genus Ochromonas by light microscopy (LM) recently showed a close molecular relationship to Hydrurus Agardh (Klaveness et al. 2011). This was surprising, taking a very different morphology of the latter into consideration, typically causing macroscopic thalli in periglacial and high alpine rivers (Rott et al. 2006). Klaveness et al. (2011) showed that chrysophycean sequences, isolated from snow and sampled at many different locations worldwide, almost exclusively belong to the Hydrurus-clade. However, the ecology of these snow habitats and the physiology of cells living there have not been described closer yet. The aim of this work was to track down the morphological and molecular affiliation of the chrysophytes causing the striking natural populations of yellow snow in Arctic and Antarctic regions, and to report ecophysiological data about photosynthesis and soluble carbohydrates to survey the level of cellular adaptation to this special habitat.
Materials and Methods
Field work and sample preparation
Antarctic samples were taken from the edge of a seasonal snowdrift at Potter Peninsula, next to the south coast of King George Island (South Shetland Islands, Maritime Antarctica) approximately 0.5 km west of the Argentinean ‘Jubany’ station, S62°14.48 W58°40.85, 1 m a.s.l. at 6 January 2009 (sample Ant_26a). Arctic samples were taken from surface slush of Larsbreen, a glacier at Spitsbergen (Svalbard Archipelago), 3 km south of Longyearbyen, N78°11.21 E15°33.87, 401 m a.s.l. at 12 July 2010 (sample Sva_10_3). Field samples were directly observed with a portable microscope (Evolution, Pyser-SGI, Kent, UK). After harvest, photomicrographs were taken with a Nikon Coolpix 8400 at a Zeiss Axiolab microscope. Some cells were fixed in 2.5% glutaraldehyde immediately after collection for later light microscopy observation.
DNA extraction and PCR amplification
Samples from chrysophycean blooms were filtered onto Whatman GF/C filters and genomic DNA was extracted using the DNeasy Tissue kit (Qiagen, Hilden, Germany). Filters were transferred to a 2 mL tube and incubated for 1–3 h in buffer animal tissue lysis supplemented with Proteinase K. DNA was subsequently extracted following the instructions of the supplier.
Polymerase chain reaction (PCR) was carried out using universal primers for the ribosomal small subunit (18S rDNA) (Medlin et al. 1988, slightly modified by Preisfeld et al. 2000) 18SF1 (5′- AAT CTG GTT GAT CCT GCC AG -3′) and 18SR1 (5′-TGA TCC TTC TGC AGG TTC ACC TAC-3′). The cycling profile consisted of 3 min denaturation at 95°C, followed by 35 PCR cycles (95°C for 45 s, 55°C for 45 s and 72°C for 2 min) with a final extension step of 7 min at 72°C. The PCR products were checked on a 1% agarose gel, quantified using a Spectrophotometer Nano Drop ND-2000 (NanoDrop Technologies, Wilmington, DE, USA), and commercially sequenced. The sequences were assembled and submitted to the BLAST search program of the National Center for Biotechnology Information to find closely related sequences. Sequence alignment of 64 sequences selected from the publication of Klaveness et al. 2011 and our own isolates was constructed with the alignment program MAFFT (Katoh et al. 2002) and subsequently processed manually and corrected where necessary. The resultant alignment dataset of 1618 characters was used for tree construction. The molecular data of the three samples were deposed at EMBL Nucleotide Sequence Database (Accession numbers HE820739-HE820741).
Phylogenetic inference was based on maximum likelihood (ML), maximum parsimony (MP), distance (neighbor joining; NJ), using maximum likelihood–corrected distances based on the GTR + I + G model, and Bayesian inference (BI). The tree was rooted with Leukarachnion sp. (FJ356265) as outgroup, following Klaveness et al. (2011). ML, MP and NJ were calculated using PAUP* (version 4.0b10; Swofford 2003). For MP analysis, the heuristic search settings were based on simple taxon addition, tree-bisection-reconnection branch swapping algorithm and Multrees option enabled. For the ML analysis, Modeltest 3.7 (Posada & Crandall 1998; Posada & Buckley 2004) was used to select the model of sequence evolution best fitting the dataset. The ML tree (Fig. 3) was inferred using the GTR + I + G model, chosen by Modeltest 3.7 as the best model according to the Akaike information criterion with base frequencies A 0.2490, C 0.1952, G 0.2596, T 0.2962; rate matrix A-C 1.0690, A-G 3.2867, A-T 1.4076, C-G 0.2657, C-T 5.2620, G-T 1.000; proportion of invariable sites (I = 0.6183); and gamma distribution shape parameter (G = 0.4768). To test the confidence of the tree topology, bootstrap (BS) analyses were performed using distance (NJ, 1000 replicates), parsimony (MP; 1000 replicates) and maximum likelihood (ML; using the GTR+I+G model; 100 replicates) criteria. Bayesian analyses were performed using MrBayes version 3.2 (Huelsenbeck & Ronquist 2001; Ronquist & Huelsenbeck 2003). Two runs with four Monte Carlo–Markov chains iterations with the covarion setting were performed for 2 million generations. The first 25% of the generations was discarded as burn-in. A 50% majority-rule consensus tree was constructed with PAUP* to calculate the posterior probabilities (PP).
Phenolics and carbohydrate analysis
Field samples were cleaned by using 400-, 200- and 100-μm stainless steel sieves (Retsch, Germany). Subsequently, the cells were accumulated onto Whatman GF/C filters, freeze-dried for 48 h and afterwards broken for 4 min at 1800 bpm with a grinding mill (Dismembrator S, Sartorius, Göttingen, Germany) with 10 mm quartz balls and Teflon jars precooled in liquid nitrogen prior to use. The samples were extracted in 80% ethanol in an ultrasonic bath for 2 h. Analysis of soluble carbohydrates was performed with an Agilent ChemStation 1100 high performance liquid chromatography (HPLC) equipped with diode array detector and refractive index detector. Soluble carbohydrates were separated with a Phenomenex Rezex RCM – Monosaccharide Ca2+ column (300 × 8 mm) thermostated to 80°C and at isocratic aqueous conditions (0.6 mL min−1). Prior to injection, the samples were evaporated and redissolved in Millipore water, centrifuged and filtered through regenerated 0.45 μm cellulose filters. The compounds were identified and quantified considering the following calibration standards (Sigma Aldrich, Vienna, Austria). Aldoses and ketoses: sucrose, galactose, glucose, mannose, trehalose, maltose, cellubiose, raffinose, xylose, rhamnose, fucose, fructose, verbascose, stachyose, 1-kestose and ribose. Sugaralcohols: galactinol, meso-erythritol, mannitol, arabitol, dulcitol, xylitol, sorbitol, adonitol and glycerol. The presence of phenolic compounds in 20% methanolic extracts was analyzed with a different HPLC RP C18 method described in Remias et al. (2012b).
Light- and temperature-dependent photosynthesis and respiration of Antarctic samples were measured with a Fibox 3 optode (PreSens, Regensburg, Germany) in a 3-mL polyacrylic chamber with a magnetic stirrer at 1, 10 and 20°C and from 0 to 1362 μmol photosynthetic active irradiance (PAR) m−2 s−1. Two milliliters of algal suspension was mixed with 1 mL 0.1 M HCO3− source before measurement. Light levels were calibrated with a QRT1 sensor (Hansatech, King's Lynn, UK) and arranged with Hansatech A5 neutral filters. Oxygen turnover was normalized to the amount of chlorophyll a and c per sample, which was determined after cell extraction with 90% acetone, stabilized with 50 mM Hepes puffer (pH 7.6), by spectrophotometer according to Porra (2002). The oxygen turnover rates were obtained using the ‘trend’ function of Excel (Microsoft Office), processing the raw data points (12 per min) of the Fibox. Then, results of each irradiation level were analyzed with single factor analysis of variance (ANOVA) to test for significant differences (P < 0.05) in rates of photosynthesis and dark respiration. A post-hoc test was applied with SPSS (IBM Corporation, Armonk, NY, USA) using the Bonferroni method to find significant differences between different temperature levels.
Field parameters and cell morphology
The Antarctic samples at King George Island thrived in a semi-permanent snowdrift next to the coast of the Potter Cove, where they caused a marginal yellow to brownish bloom, partially shaded by algal-free snow above (Fig. 1a). The coloration occurred as a stripe several centimeters thick, and it lasted more than 10 cm into the interior. The snow pack was isolated against the permafrost ground by a basal ice sheet, which evidently delayed the water outflow from the snow above and supported the continuous presence of meltwater (Fig. 1b). The pH was 5.7, and the electrical conductivity 26.5 μS cm−1. Similar yellow snow was found at slopes of other parts of the island, for example, at Barton Peninsula.
The Arctic population on Larsbreen, Spitsbergen grew at exposed, unshaded sites directly at the glacier surface, causing a yellowish slush, which was very soaked due to strong underground water-flow (Fig. 1c). The visible coloration reached more than 20 cm in depth, sometimes becoming more yellowish-green (Fig. 1d,e). Here, the meltwater pH was 5.9, and the electrical conductivity reached 3.8 μS cm−1. We found comparable populations also in seasonal snowfields without glacial underground elsewhere in the region, for example, south of Svalbard Airport (sample Sva_10_9).
The cells of both sites were difficult to observe by LM, because they quickly burst when exposed under a coverslip, most likely because of temperature stress. The common morphological characteristics of both populations were elongate ovoid to roundish shapes and a smooth cell membrane. Motility was provided by one longer distant and one much shorter adjacent flagella. In the interior, one or two disc-like chloroplasts and several roundish organelles (‘chrysolaminarin bodies’) were observed (Fig. 2a). The cells of the Arctic population were fixed with glutaraldehyde to measure an average cell diameter of 4.2 ± 0.3 μm. Hours after collection, some immotile and more globular cells occurred in loose groups, embedded in extracellular matrix (Fig. 2b). In each habitat, a smaller portion was made of characteristic tetrahedal zoospores with four elongate cell spikes (Fig. 2c). Resting stages with thickened cell walls (‘stomatocysts’) were never observed. Generally, cells were very sensitive to higher temperatures, first losing flagella, then getting a more roundish shape and finally disintegrating after some hours.
Freshly collected field specimens were practically scentless, but cells dried on glass fiber filters evolved a strong odor similar to Hydrurus foetidus. The yellow snow contained virtually no other algae than chrysophytes; however, several blackish springtails of 2–3 mm length were observed in the Arctic as well as Antarctic samples.
The 18S phylogeny of selected 64 chrysophyte taxa (including the isolates from this study) revealed the placement of the Arctic as well as the Antarctic isolates within the Hydrurus-clade (Klaveness et al. 2011) (Fig. 3). Our sequences clustered together with Hydrurus foetidus and the uncultured eukaryote clone JFJ-ICE-Uni10 (isolated from snow in the Bernese Alps) in the abovementioned clade (0.99 PP/87% BS in the ML tree). The Antarctic isolate Ant26a was placed at the base of this subcluster, whereas the 18S rDNA sequences of the arctic isolates Sva_10_3 and Sva_10_9 were 100% identical to each other and the nearest sequence to these isolates was Hydrurus foetidus (0.99 PP/89% BS in the ML tree).
Carbohydrates and photosynthesis
The sum of soluble carbohydrates in the Antarctic chrysophyte amounted for approximately 2.0 mg g−1 algal dry weight. Glycerol was the main constituent with 1.2 mg g−1 dry weight, followed by minor amounts of galactose, saccharose, xylitol and two unknown compounds (not identified by refractive index detector) with less than 0.3 mg g−1 dry weight each. Furthermore, algae from both sites were screened by HPLC with a detection wavelength of 280 nm for phenolic compounds (e.g. as putative UV-screening compounds); however, the results were negative.
Net photosynthesis and respiration of the Antarctic yellow snow was measured under different light- and temperature levels from 0 to 1362 μmol PAR m−2 s−1 (Fig. 4). The golden alga clearly showed a photoautotrophic metabolism. The best values for oxygen production were at 10°C; however, this was only statistically significant from the second light level on. Nonetheless, oxygen production was generally lower at 1 and 20°C. Significant differences between the similar performance curves of these two temperature levels existed only during dark respiration, the latter rose almost fourfold from 1 to 20°C. At 20°C, photosynthesis was still negative even at low light conditions (48 μmol PAR m−2 s−1).
Snow algal blooms can be formed by members of different taxons (Hoham & Duval 2001). Most common are Chlorophyceae, which cause red or green snow, followed by grey snow caused by Zygnematophyceae living on bare glacier surfaces (Remias 2012; Remias et al. 2012a). Other classes which generally dominate psychrophilic habitats, like Cyanoprocaryota or Chrysophyceae, have been reported less frequently from snow. Probably the lack of reports about chrysophycean yellow snow has been caused by the remoteness of such habitats and the sensitivity of cells to higher temperatures after harvest. Even Kol (1968) in her classical compendium about snow algae missed a description of any representatives of this group. However, blooms of chrysophytes in long-lasting snow fields seem not to be a strictly polar phenomenon, likely they occur cosmopolitic in many cold regions of the world considering reports from North America (Stein 1963) or Japan (Tanabe et al. 2011).
The higher electrical conductivity of the meltwater at King George Island may be caused by ionic or nutrient input by seaspray or coastal animals like penguins. However, the huge population of yellow snow on Larsbreen at Spitsbergen, covering several square meters far away from the coast and from animal colonies shows that this kind of nutrient input is not obligate for causing a bloom. A fact encountered at all sampling sites was a constant water flow in the snowpack during summer. This may be the joining aspect when taking the close taxonomic relationship of yellow snow chrysophytes to the cold river dwelling H. foetidus into consideration. Accordingly, H. foetidus is known from Antarctica and has been reported from streamlets at King George Island not far away from our collection sites (Komárek & Komárek 1999). There, it occurs from 4°C on and is the dominating alga at 5 to 6°C. H. foetidus has also been reported for Svalbard (Borge 1911). It is unknown whether such thalli are taxonomically identical to the species causing yellow snow, or at least another ecotype. In any case, a very close molecular relationship is evident as demonstrated by Klaveness et al. 2011 with 18S rDNA sequences and in this work, accordingly. When the yellow snow populations were related to H. foetidus, a formation of macroscopic thalli could be hindered perhaps by low temperatures in the habitat (typically around the freezing point) or by the lack of a suitable substratum for attachment and forming of multicellular stages.
The role of smelling volatiles characteristic for chrysophytes when exposed to desiccation remains unknown. One of the few studies exploring the nature of these released compounds (Jüttner 1981) reported a variety of aldehydes or ketons, for example, alkenols.
The absence of other common snow algae like the green alga Chlamydomonas cf. nivalis in these habitats was remarkable. We found red snow caused by the latter, collective species not far away from the chrysophytes in both the Arctic and Antarctic locations. However, those snow fields were never influenced by strong meltwater streams. Since red snow species are immotile cysts for most of the growing season (Remias 2012), they probably could not establish at locations with heavy water drain like the chrysophycean flagellates do. Accordingly, the latter may not survive in more dry snow where the robust spores of chloromonads are typically present.
Our bipolar sampling of chrysophyte algae inhabiting snowpacks showed a close molecular relationship between arctic and Antarctic psychrophilic organisms and therefore supports the findings of Klaveness et al. (2011) that sequences, closely related to the Hydrurus-clade, originate from high-altitude watersheds, high mountain snow communities and other cold environments. Generally, this result supports a theory of ubiquity of polar microorganisms (Rybalka et al. 2009). On the other hand, studies like De Wever et al. (2009) emphasize a high level of endemic Antarctic lineages of microalgae. Concerning Hydrurus, it is likely, that a number of geographically widespread Hydrurus-related species exist not only in polar regions, since the closely related strain JFJ-ICE-Uni10 was isolated from snow in the Swiss Alps.
The polyphyly of the genus Ochromonas has been shown several times (Andersen et al. , Klaveness et al. 2011). It can be split into ‘true’ representatives of Ochromonas and at least another clade, which aligns with H. foetidus. Therefore, the causers of yellow snow in the Japanese Alps, O. itoi and O. smithii (Tanabe et al. 2011) could eventually represent the same single, Hydrurus-related organism studied in this work: O. itoi with oval shape and smooth cell membranes as vegetative and O. smithii with tetrahedal shape and four elongate spines probably as zygote-like stages. The striking morphology of the latter was observed in detail by Hoffman et al. (1986), who found out that in H. foetidus, the spines are formed by the help of a complex arrangement of microtubuli. In addition, similar zoospores have been reported from the genera Celloniella and Chrysonebula (Hibberd 1977). The molecular tree of this study includes Chrysonebula flava Starmach; however, it is not directly associated with Hydrurus.
Our photosynthesis measurements showed that the cells had the highest activity at 10°C, but still a good performance at a temperature close to ambient snow conditions (1°C). However, the latter values showed no significant difference to 20°C except for dark respiration. Still, we believe that the yellow snow chrysophyte is tentatively psychrophilic: on the one hand, cells exposed to room temperatures evidently disintegrated after a few hours. On the other hand, the short-term oxygen turnover assays that we used may not reflect the best long-term growth conditions for the alga, which we suppose to be below 10°C. The strong rise of dark-respiration from 1 to 10°C (more than threefold) during our measurements indicated a statistical significant temperature stress. This was even worse at 20°C, where the cells were not able to perform positively anymore at lower light conditions (48 μmol PAR m−2 s−1). On the other hand, the chrysophytes showed a tolerance against high light conditions, because no photoinhibition was noticed up to 1362 μmol PAR m−2 s−1. Such a high irradiance is hardly seen in polar habitats, because the maximum Arctic VIS irradiation at noon in July is 1104 μmol PAR m−2 s−1 at a neighboring glacier to the sampling site of Sva_10_3 (Remias et al. 2012a).
Although the amount of soluble carbohydrates was relatively low compared to other Antarctic photoautotrophs, for example, lichens or Prasiola crispa (Lightfoot) Kützing (Roser et al. 1992; Chapman et al. 1994), it is remarkable that glycerol is the most abundant compound in yellow snow chrysophytes (0.12 % of total dry weight). Similar concentrations of glycerol (0.09%) were found in another Antarctic snow alga, Chloromonas polyptera (Fritsch) R.W. Hoham, J.E. Mullet & S.C. Roemer (unpubl. data, 2010). Glycerol can be regarded as secondary metabolite, and it is known for its ability to stabilize biomembranes, thus delimiting damage caused by intracellular water stress, which would be the case when the surrounding habitat became frozen (Thomas et al. 2008).
Putative UV-shielding phenolics were not found in the samples used in this study. Probably Chrysophyta cannot synthesize such metabolites; however, they were found in another psychrophilic alga living on glaciers, Mesotaenium berggrenii (Wittrock) Lagerheim (Remias et al. 2012b). Chlorophyta living in snow use secondary carotenoids like astaxanthin for protection against excessive irradiance (Lemoine & Schoefs 2010). Obviously Chrysophyta do not accumulate such compounds but rely either on a high resistance against excessive irradiation by xanthophyll cycle activities or can prevent harmful exposure on the snow surface by actively swimming into deeper places in the snowpack.
In order to assess the diversity of cold adapted chrysophyte algae and elucidate the phylogenetic relationship, especially of Hydrurus-related species with tetragonal zoospores, studies on plastid pigment physiology and further molecular investigations, like sequencing of more variable loci (e.g. the internal transcribed spacer region), are needed. For these molecular surveys, as well as the description of life cycles and ecophysiological analyses, the establishment of monoclonal strain cultures of these organisms is crucial for further investigations.
This work was supported by the Austrian Science Fund (FWF), P20081, to C.L and by the German Research Foundation (DFG), BO3245/2-1, to J.B. We thank the German AWI (Bremerhaven) for hosting and providing research facilities in the Dallmann Laboratory at the Argentinean Jubany station and Siegfried Aigner for valuable help during the collections at Svalbard.