Melting glacier surfaces are increasingly affected by blooms of psychrophilic microalgae, which darken the ice and lower its albedo, accelerating melting. These microalgae contain distinct vacuoles filled with brownish pigments that were earlier described as the unusual plant phenol purpurogallin. Recently, we discovered so far unreported, large amounts of iron dissolved in aqueous extracts of the glacier ice algae Ancylonema alaskanum. Since the vacuole content was very dark but the chromatographically isolated, aforementioned phenol was only yellowish, a putative complexation of iron with purpurogallin was assumed to be the reason. Application of several protocols, including Raman microscopy on both living cells and extracts, provided strong evidence that this microalga sequesters iron and forms organic metal complexes. Consequently, substantial amounts of so far uncharacterised Fe-complexes of purpurogallin are inferred to be present in Ancylonema, and that putative polymerisation of this compound impeded an earlier analytical discovery. This finding holds significant ecological implications for cold regions. The pigmentation not only enhances the tolerance of glacier ice algae to excessive UV and visible radiation but also influences our current understanding of the biochemical iron cycle in cryosphere-dominated polar and alpine regions. Further downstream consequences of this biological iron source remain to be elucidated.

• Keywords
• Raman microscopy, cryoflora, glaciers, polyphenols, secondary pigmentation,
• MeSH
• Phenols * metabolism   chemistry   MeSH
• Ice Cover * microbiology   MeSH
• Microalgae * radiation effects   chemistry   metabolism   MeSH
• Light MeSH
• Ultraviolet Rays * MeSH
• Iron * metabolism   chemistry   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Phenols * MeSH
• Iron * MeSH

Filamentous green algae of the genus Zygnema are an essential part of hydro-terrestrial ecosystems. Despite several studies on their resistance to natural stresses, little is known about the composition of their assemblages and the changes they undergo over time. Two sites at altitudes above 2200 m a.s.l. in the Austrian Alps were selected for a 2-year observation period and sampled five times. Molecular phylogenetic analysis of the 152 isolated strains of Zygnema sp. was performed based on the rbcL and trnG sequences. Seven genotypes were found at these sites during the samplings, but their proportion varied throughout the seasons. The site with a more stable water regime also had a more stable representation of genotypes, in contrast to the site with fluctuating water availability. The mats formed resistant pre-akinetes at the end of the season with reduced photosynthetic activity. Contrary to expectations, the mats were not exposed to extremely cold temperatures in winter due to snow cover. Some genotypes have been previously observed at this site, indicating that the population composition is stable. This work highlights the importance of resistant pre-akinetes in surviving winter conditions, the ability of algae to re-establish mats, and the need to address the hidden diversity of the genus Zygnema.

• Keywords
• Chlorophyll fluorescence, Cryptic diversity, Freezing, Hidden diversity, Overwintering,
• MeSH
• Ecosystem * MeSH
• Phylogeny MeSH
• Seasons MeSH
• Streptophyta * MeSH
• Water MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Austria MeSH
• Names of Substances
• Water MeSH

Green algae of the genus Zygnema form extensive mats and produce large amounts of biomass in shallow freshwater habitats. Environmental stresses including freezing may perturb these mats, which usually have only annual character. To estimate the limits of survival at subzero temperatures, freezing resistance of young Zygnema sp. (strain MP2011Skan) cells and pre-akinetes was investigated. Young, 2-week-old cultures were exposed to temperatures of 0 to - 14 °C at 2-K steps, whereas 8-month-old cultures were frozen from - 10 to - 70 °C at 10-K intervals. Cell viability after freezing was determined by 0.1% auramine O vital fluorescence staining and measurements of the effective quantum yield of photosystem II (ФPSII). At - 8 °C, the young vegetative cells were unable to recover from severe frost damage. But temperatures even slightly below zero (- 2 °C) negatively affected the cells' physiology. Single pre-akinetes could survive even at - 70 °C, but their LT50 value was - 26.2 °C. Severe freezing cytorrhysis was observed via cryo-microscopy at - 10 °C, a temperature found to be lethal for young cells. The ultrastructure of young cells appeared unchanged at - 2 °C, but severe damage to biomembranes and formation of small foamy vacuoles was observed at - 10 °C. Pre-akinetes did not show ultrastructural changes at - 20 °C; however, vacuolization increased, and gas bubbles appeared at - 70 °C. Our results demonstrate that the formation of pre-akinetes increases freezing resistance. This adaptation is crucial for surviving the harsh temperature conditions prevailing in the High Arctic in winter and a key feature in seasonal dynamics of Zygnema sp.

• Keywords
• Auramine O, Chlorophyll fluorescence, Freezing, Ice, Live cell staining, Ultrastructure,
• MeSH
• Chlorophyll chemistry   MeSH
• Freezing MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Arctic Regions MeSH
• Names of Substances
• Chlorophyll MeSH

Within streptophyte green algae Zygnematophyceae are the sister group to the land plants that inherited several traits conferring stress protection. Zygnema sp., a mat-forming alga thriving in extreme habitats, was collected from a field site in Svalbard, where the bottom layers are protected by the top layers. The two layers were investigated by a metatranscriptomic approach and GC-MS-based metabolite profiling. In the top layer, 6569 genes were significantly upregulated and 149 were downregulated. Upregulated genes coded for components of the photosynthetic apparatus, chlorophyll synthesis, early light-inducible proteins, cell wall and carbohydrate metabolism, including starch-degrading enzymes. An increase in maltose in the top layer and degraded starch grains at the ultrastructural levels corroborated these findings. Genes involved in amino acid, redox metabolism and DNA repair were upregulated. A total of 29 differentially accumulated metabolites (out of 173 identified ones) confirmed higher metabolic turnover in the top layer. For several of these metabolites, differential accumulation matched the transcriptional changes of enzymes involved in associated pathways. In summary, the findings support the hypothesis that in a Zygnema mat the top layer shields the bottom layers from abiotic stress factors such as excessive irradiation.

• MeSH
• Chlorophyta genetics   metabolism   MeSH
• Ecosystem MeSH
• Photosynthesis genetics   MeSH
• Stress, Physiological MeSH
• Metabolome MeSH
• Streptophyta genetics   metabolism   MeSH
• Transcriptome MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Arctic Regions MeSH
• Svalbard MeSH

Filamentous conjugating green microalgae (Zygnematophyceae, Streptophyta) belong to the most common primary producers in polar hydro-terrestrial environments such as meltwater streamlets and shallow pools. The mats formed by these organisms are mostly composed of sterile filaments with Zygnema morphology, but the extent of their diversity remains unknown. Traditional taxonomy of this group is based on reproductive morphology, but sexual reproduction (conjugation and formation of resistant zygospores) is very rare in extreme conditions. In the present study we gave the first record of zygospore formation in Svalbard field samples, and identified conjugating filaments as Zygnemopsis lamellata and Zygnema cf. calosporum. We applied molecular phylogeny to study genetic diversity of sterile Zygnema filaments from Svalbard in the High Arctic. Based on analysis of 143 rbcL sequences, we revealed a surprisingly high molecular diversity: 12 Arctic Zygnema genotypes and one Zygnemopsis genotype were found. In addition, we characterized individual Arctic genotypes based on cell width and chloroplast morphology using light and confocal laser scanning microscopy. Our findings highlight the importance of a molecular approach when working with sterile filamentous Zygnematophyceae, as hidden diversity might be very beneficial for adaptation to harsh environmental conditions, and experimental results could be misinterpreted when hidden diversity is neglected.

• Keywords
• Arctic, Svalbard, Zygnema, Zygnemopsis, chloroplast shape, cryptic diversity, microscopy, molecular phylogeny, rbcL,
• Publication type
• Journal Article MeSH

Species of Zygnema form macroscopically visible mats in polar and temperate terrestrial habitats, where they are exposed to environmental stresses. Three previously characterized isolates (Arctic Zygnema sp. B, Antarctic Zygnema sp. C, and temperate Zygnema sp. S) were tested for their tolerance to experimental UV radiation. Samples of young vegetative cells (1 month old) and pre-akinetes (6 months old) were exposed to photosynthetically active radiation (PAR, 400-700 nm, 400 μmol photons m-2 s-1) in combination with experimental UV-A (315-400 nm, 5.7 W m-2, no UV-B), designated as PA, or UV-A (10.1 W m-2) + UV-B (280-315 nm, 1.0 W m-2), designated as PAB. The experimental period lasted for 74 h; the radiation period was 16 h PAR/UV-A per day, or with additional UV-B for 14 h per day. The effective quantum yield, generally lower in pre-akinetes, was mostly reduced during the UV treatment, and recovery was significantly higher in young vegetative cells vs. pre-akinetes during the experiment. Analysis of the deepoxidation state of the xanthophyll-cycle pigments revealed a statistically significant (p < 0.05) increase in Zygnema spp. C and S. The content of UV-absorbing phenolic compounds was significantly higher (p < 0.05) in young vegetative cells compared to pre-akinetes. In young vegetative Zygnema sp. S, these phenolic compounds significantly increased (p < 0.05) upon PA and PAB. Transmission electron microscopy showed an intact ultrastructure with massive starch accumulations at the pyrenoids under PA and PAB. A possible increase in electron-dense bodies in PAB-treated cells and the occurrence of cubic membranes in the chloroplasts are likely protection strategies. Metabolite profiling by non-targeted RP-UHPLC-qToF-MS allowed a clear separation of the strains, but could not detect changes due to the PA and PAB treatments. Six hundred seventeen distinct molecular masses were detected, of which around 200 could be annotated from databases. These results indicate that young vegetative cells can adapt better to the experimental UV-B stress than pre-akinetes.

• Keywords
• Green algae, Metabolomics, UV simulation, UV-A, UV-B, Ultrastructure,
• MeSH
• Chlorophyta metabolism   MeSH
• Photosynthesis physiology   MeSH
• Ultraviolet Rays adverse effects   MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Antarctic Regions MeSH
• Arctic Regions MeSH

Research in algae usually focuses on the description and characterization of morpho-and phenotype as a result of adaptation to a particular habitat and its conditions. To better understand the evolution of lineages we characterized responses of filamentous streptophyte green algae of the genera Klebsormidium and Zygnema, and of a land plant-the moss Physcomitrellapatens-to genotoxic stress that might be relevant to their environment. We studied the induction and repair of DNA double strand breaks (DSBs) elicited by the radiomimetic drug bleomycin, DNA single strand breaks (SSB) as consequence of base modification by the alkylation agent methyl methanesulfonate (MMS) and of ultra violet (UV)-induced photo-dimers, because the mode of action of these three genotoxic agents is well understood. We show that the Klebsormidium and Physcomitrella are similarly sensitive to introduced DNA lesions and have similar rates of DSBs repair. In contrast, less DNA damage and higher repair rate of DSBs was detected in Zygnema, suggesting different mechanisms of maintaining genome integrity in response to genotoxic stress. Nevertheless, contrary to fewer detected lesions is Zygnema more sensitive to genotoxic treatment than Klebsormidium and Physcomitrella.

• Keywords
• DNA damage and repair, Klebsormidium, Physcomitrella patens, Zygnema, bleomycin, methyl methanesulfonate, ultraviolet light,
• Publication type
• Journal Article MeSH

The basal streptophyte Klebsormidium and the advanced Zygnema show adaptation to terrestrialization. Differences are found in photoprotection and resistance to short-term light changes, but not in CO 2 acquisition. Streptophyte green algae colonized land about 450-500 million years ago giving origin to terrestrial plants. We aim to understand how their physiological adaptations are linked to the ecological conditions (light, water and CO2) characterizing modern terrestrial habitats. A new Klebsormidium isolate from a strongly acidic environment of a former copper mine (Schwarzwand, Austria) is investigated, in comparison to Klebsormidium cf. flaccidum and Zygnema sp. We show that these genera possess different photosynthetic traits and water requirements. Particularly, the Klebsormidium species displayed a higher photoprotection capacity, concluded from non-photochemical quenching (NPQ) and higher tolerance to high light intensity than Zygnema. However, Klebsormidium suffered from photoinhibition when the light intensity in the environment increased rapidly, indicating that NPQ is involved in photoprotection against strong and stable irradiance. Klebsormidium was also highly resistant to cellular water loss (dehydration) under low light. On the other hand, exposure to relatively high light intensity during dehydration caused a harmful over-reduction of the electron transport chain, leading to PSII damages and impairing the ability to recover after rehydration. Thus, we suggest that dehydration is a selective force shaping the adaptation of this species towards low light. Contrary to the photosynthetic characteristics, the inorganic carbon (C i ) acquisition was equivalent between Klebsormidium and Zygnema. Despite their different habitats and restriction to hydro-terrestrial environment, the three organisms showed similar use of CO2 and HCO3- as source of Ci for photosynthesis, pointing out a similar adaptation of their CO2-concentrating mechanisms to terrestrial life.

• Keywords
• Desiccation, Green algae, Light, Photosynthesis,
• MeSH
• Dehydration MeSH
• Species Specificity MeSH
• Ecology MeSH
• Ecosystem MeSH
• Phenotype MeSH
• Photosynthesis physiology   radiation effects   MeSH
• Adaptation, Physiological * MeSH
• Carbon Dioxide metabolism   MeSH
• Charophyceae physiology   radiation effects   MeSH
• Light MeSH
• Water physiology   MeSH
• Desiccation MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• Carbon Dioxide MeSH
• Water MeSH

Filamentous green algae of the genus Zygnema (Zygnematophyceae, Streptophyta) are key components of polar hydro-terrestrial mats where they face various stressors including UV irradiation, freezing, desiccation and osmotic stress. Their vegetative cells can develop into pre-akinetes, i.e. reserve-rich, mature cells. We investigated lipid accumulation and fatty acid (FA) composition upon pre-akinete formation in an Arctic and an Antarctic Zygnema strain using transmission electron microscopy and gas chromatography coupled with mass spectrometry. Pre-akinetes formed after 9 weeks of cultivation in nitrogen-free medium, which was accompanied by massive accumulation of lipid bodies. The composition of FAs was similar in both strains, and α-linolenic acid (C18:3) dominated in young vegetative cells. Pre-akinete formation coincided with a significant change in FA composition. Oleic (C18:1) and linoleic (C18:2) acid increased the most (up to 17- and 8-fold, respectively). Small amounts of long-chain polyunsaturated FAs were also detected, e.g. arachidonic (C20:4) and eicosapentaenoic (C20:5) acid. Pre-akinetes exposed to desiccation at 86% relative humidity were able to recover maximum quantum yield of photosystem II, but desiccation had no major effect on FA composition. The results are discussed with regard to the capability of Zygnema spp. to thrive in extreme conditions.

• Keywords
• desiccation stress, fatty acid methyl ester, lipids, nitrogen starvation, polar green microalgae,
• MeSH
• Nitrogen metabolism   MeSH
• Photosystem II Protein Complex genetics   metabolism   MeSH
• Lipid Droplets metabolism   MeSH
• Fatty Acids chemistry   metabolism   MeSH
• Osmotic Pressure MeSH
• Streptophyta chemistry   genetics   metabolism   radiation effects   MeSH
• Ultraviolet Rays MeSH
• Desiccation MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Antarctic Regions MeSH
• Arctic Regions MeSH
• Names of Substances
• Nitrogen MeSH
• Photosystem II Protein Complex MeSH
• Fatty Acids MeSH

BACKGROUND: Filamentous Zygnematophyceae are typical components of algal mats in the polar hydro-terrestrial environment. Under field conditions, they form senescent vegetative cells, designated as pre-akinetes, which are tolerant to desiccation and osmotic stress. KEY FINDINGS: Pre-akinete formation and desiccation tolerance was investigated experimentally under monitored laboratory conditions in four strains of Arctic and Antarctic isolates with vegetative Zygnema sp. morphology. Phylogenetic analyses of rbcL sequences revealed one Arctic strain as genus Zygnemopsis, phylogenetically distant from the closely related Zygnema strains. Algae were cultivated in liquid or on solidified medium (9 weeks), supplemented with or lacking nitrogen. Nitrogen-free cultures (liquid as well as solidified) consisted of well-developed pre-akinetes after this period. Desiccation experiments were performed at three different drying rates (rapid: 10% relative humidity, slow: 86% rh and very slow); viability, effective quantum yield of PS II, visual and ultrastructural changes were monitored. Recovery and viability of pre-akinetes were clearly dependent on the drying rate: slower desiccation led to higher levels of survival. Pre-akinetes survived rapid drying after acclimation by very slow desiccation. CONCLUSIONS: The formation of pre-akinetes in polar Zygnema spp. and Zygnemopsis sp. is induced by nitrogen limitation. Pre-akinetes, modified vegetative cells, rather than specialized stages of the life cycle, can be hardened by mild desiccation stress to survive rapid drying. Naturally hardened pre-akinetes play a key role in stress tolerance and dispersal under the extreme conditions of polar regions, where sexual reproduction and production of dormant stages is largely suppressed.

• MeSH
• DNA, Plant analysis   MeSH
• Nitrogen metabolism   MeSH
• Ecosystem MeSH
• Photosystem II Protein Complex chemistry   metabolism   MeSH
• Phylogeny MeSH
• Molecular Sequence Data MeSH
• Osmotic Pressure MeSH
• Polymerase Chain Reaction MeSH
• Base Sequence MeSH
• Streptophyta classification   metabolism   ultrastructure   MeSH
• Microscopy, Electron, Transmission MeSH
• Desiccation MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Antarctic Regions MeSH
• Arctic Regions MeSH
• Names of Substances
• DNA, Plant MeSH
• Nitrogen MeSH
• Photosystem II Protein Complex MeSH