Lundiana International Journal of Biodiversity 12(1): 1-10. 2016. ISSN 1676-6180 © 2016 Instituto de Ciências Biológicas - UFMG

Review Cold-Adapted Yeasts from Virginia de Garcia1* & Maria Rosa van Broock1

1- Laboratorio de Microbiología Aplicada y Biotecnología, Centro Regional Universitario , Universidad Nacional del Comahue, INIBIOMA (CONICET-UNCo), Argentina.

*Corresponding author: [email protected]

Abstract The Patagonian possess a great variety of , large temperate ice masses, and glacially formed water bodies. Most of these environments are included in protected areas characterized by low anthropogenic impact and minimal atmospheric pollution. Studies on occurrence of psychrotolerant (cold-adapted) and psychrophilic yeasts in these natural environments have allowed the characterization of native cold- adapted yeast communities, the description of six novel species, as well as description of the capability of these yeasts to produce extracellular enzymes, and other metabolites (carotenoids and mycosporines). Cold-adapted yeasts from Patagonian glaciers were able to hydrolyze natural polymers (casein, lipids, starch, pectin and carboxymethylcellulose) at low temperatures and some were able to produce photo- protective compounds. These results suggest a significant ecological role of these organisms as organic matter decomposers and nutrient cyclers, also reveal that some of these yeasts could be used for further metabolic, ecological and biotechnological studies. The objective of this review is to summarize all the findings related to cold adapted yeasts from glacial environments of the South Hemisphere (outside Antarctica), also to highlight the importance of continental ice masses from the microbiological perspective.

Keywords: yeasts, psychrophilic, glaciers, extracellular enzymes.

Glaciers: general characteristics is the largest reservoir of freshwater on Earth Snow and glacial ice are interrelated (Bahr & Radic 2012; Brown et al. 2010). ecosystems, ice is formed from snow as a result of gradual compression and burial for The climate in Patagonia is described as hundreds of thousands of years (Miteva 2008). temperate or cold-temperate. Southern Patagonia has the influence increased of polar A glacier can change its size winds, so that the summer is cooler and wetter by accumulation, which occurs by addition of than further north and snowfall is a common snowfall, compaction and recrystallization, or phenomenon in this region (Correa 1998). by ablation, which is the loss of mass resulting from melting. Many glaciers from temperate, In Argentina there exists a great variety of alpine and seasonal polar climates, store water glaciers and glacially formed water bodies (Ruiz as ice during the colder seasons and release it et al. 2012). Monte is an old later in the form of melt water. Today glacial ice that was degraded by intensive glacial erosion. Its main summit is 3,478 meters above sea level

1 de Garcia & van Broock and the upper most part of the covered by a Glaciers as microbial reservoirs thick permanent cap of ice that is the source- Glacial ice is a substrate that can act as area for 10 glaciers. Four of these are located in long-term, chronological repository of viable Argentina (Río Manso, Castaño Overo, Alerce, microorganisms (Butinar et al. 2007). Due to Frías, in Nahuel Huapi National Park), the harsh conditions (high pressure, darkness, remaining six are in (Rabassa et al. 1978; acidity and low water activity), these Villarosa et al. 2008). Los Glaciares National environments can be considered extreme Park (Argentina) and Patagonian icefields habitats (Price 2007). In temperate glaciers, (Hielos Patagónicos) are the largest temperate habitats include crevasses and water channels ice masses in the Southern Hemisphere and can act as growth substrates for microbial account for more than 60 % of the Southern communities while, at the same time, convey Hemisphere’s glacial area outside Antarctica. water, nutrients, atmospheric gases, and is located within these microbiota into the glacier (Price 2007). icefields (Skvarca et al. 2003; Stuefer et al. 2007). Psychrotolerant and psychrophilic microorganisms have been shown to inhabit Glaciers in many mountainous regions of different types of ice (i.e. snow, sea ice, glacial the world have shown a generalized pattern of ice; Margesin et al. 2007; Turchetti et al. 2013; recession during the 20th century. The recent Zalar & Gunde-Cimerman 2013). These pattern of glacier mass losses is concurrent with microorganisms can use complex biopolymers a period of increasing temperatures in most as energy sources, by synthesizing extracellular regions, supporting the notion that glaciers can enzymes active at low temperatures (Frisvad be used as key indicators of regional and larger- 2007; Margesin et al. 2007). scale climatic changes (Delgado Granados et al. 2007). The glaciers of Patagonia Argentina are Cold-adapted microorganisms not an exception. Monte Tronador Patagonia Microorganisms inhabiting cold Argentina glaciers are a clear example of this environments can be divided into obligate and situation, having experienced a constant retreat facultative psychrophiles, and are defined by for the past 100 years (Villarosa et al. 2008). their growth temperature. Microorganisms can Perito Moreno glacier, however, is one of the grow at 0 - 5 °c with an optimal growth few glaciers that remains stable, i.e., it is neither temperature of 20–30 °c are designated advancing nor retreating (Stuefer et al. 2007). facultative psychrophiles (psychrotolerant or cold-adapted). In contrast, obligate Glacier/climate interactions in the psychrophiles exhibit minimal growth Patagonian region are of relevance to temperature at 0 °c or lower, an optimal understanding the global climate change temperature ~ 15 °c and a maximum growth pattern. In addition, icefields and periglacial temperature of 20 °c (buzzini et al. 2012). areas hold valuable information for Quaternary paleo-environments (Stuefer et al. 2007). Lakes, Cold-adapted microorganisms are generally rivers and peat-lands are the main landforms predominant in ecosystems periodically that create conditions for the study of past and exposed to low temperatures, whereas present environmental variations in the region psychrophilic microorganisms dominate in (Villarosa et al. 2008) and there is also dense permanent cold habitats. Both are believed to forest-cover associated with recent glacial play key roles in the biodegradation of organic deposits. The relevance of studies on the effects matter and the cycling of essential nutrients at of climatic and environmental changes on the cryosphere level (Buzzini et al. 2012). continental glaciers has been shown elsewhere, especially in the northern hemisphere (Branda Studying microbial diversity in extreme et al. 2010; Villarosa et al. 2008). environments, such as ice environments is interesting because these microorganisms exhibit metabolic adaptations to extreme

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Cold-Adapted Yeasts from Patagonia conditions, and may provide insight to potential morphological and biochemical characterization biotechnological applications. Furthermore, of the isolated yeast strains was carried out psychrophilic and psychrotolerant according to the techniques described by microorganisms may be used as bio-indicators Kurtzman et al. (2011). Molecular identification in studies monitoring global warming. In was carried out by sequencing D1/D2 domain addition, changes in the microbial community of the LSU rRNA gene and the internal may occur as these microorganisms are released transcribed spacer region (ITS1, 5.8S and ITS2) into soil, rivers and oceans (Butinar et al. 2007; (de Garcia et al. 2012a). de Garcia et al. 2012a; Kuhn 2007). Obtained results have reported average Yeast from glacial meltwaters and glacial counts from 1 to 3 x 102 colony forming units ice (CFU) L-1 in melt-water rivers (de Garcia et al. Yeasts exhibit excellent survival in water, 2007) from 1 to 5 x 103 CFU L- 1 for continental they occur in a variety of freshwater systems, glacial ice (de Garcia et al. 2012a). Values including eutrophic to ultra-oligotrophic lakes, obtained were similar to those from aquatic lagoons, rivers, groundwater, glaciers and glacial Patagonian environments (freshwater mountain melt-water (Starmer & Lachance 2011). The lakes) (1 x 102 to 1 x 103 CFU) L-1 (Brandao et yeast diversity in these ecosystems is highly al. 2011; Libkind et al. 2003; 2009), and for affected by a variety of abiotic and biotic Italian Alps glacial environments of 1 x 101 and factors, such as temperature, pressure, UV 4 x 103 CFU L-1 (Branda et al. 2010; Buzzini et radiation (UVR), salinity, fauna, flora, soil run- al. 2005; Turchetti et al. 2008). off and anthropogenic effluents (Medeiros et al. 2011). When yeast diversity from Patagonian Glaciers was analyze with the Shannon-Weaver Ice has long been considered only as a index, diversity obtained showed higher values refugia for microorganisms that have been for ice samples from Frias glacier and randomly deposited on its surface. However, it meltwaters from Monte Tronador (H = 2.23 is now known that different types of ice and H = 2.52, respectively), than for ice provide biomes that can support active samples from Perito Moreno (H = 1.72), microbial growth and reproduction (Gostinčar although there were no significant differences et al. 2010). Initially, the microbial presence was in Shannon–Weaver diversity indices among all investigated only at the prokaryotic level. compared communities (P > 0.05). Other However, more recent studies have shown that community similarity analysis (Jaccard index) fungi, primarily basidiomycetous yeasts, also showed that yeast communities from meltwater represent an important part of glacial microbial were the most similar with communities from communities in both polar and mountainous ice from the Frias glacier and Perito Moreno (J glacial environments worldwide (Bergauer et al. = 0.15 and J = 0.13, respectively), however no 2005; Branda et al. 2010; Buttinar et al. 2007; similarity was found when ice yeasts Connell et al. 2008; de Garcia et al. 2012a; communities from Perito Moreno and Frias Turchetti et al. 2008; Vaz et al. 2011). were compared (de Garcia et al. 2012a). Richness values were similar to those reported Yeasts from Patagonian glaciers by Brandao et al. (2011) from water samples Biodiversity, ecology, and taxonomy from Nahuel Huapi Lake (coast sites H= 2.2 Yeast studies in glacial environments of and pelagic sites H= 2.8). Patagonia were isolated filtering 100 and 150 mL of water or melted ice through Millipore® Basidiomycetous yeasts were the nitrocellulose membranes, using a sterilized predominant group (90 %) in glacial Nalgene® device, membranes were then placed environments from Patagonia (de Garcia et al. in general media for yeasts (MYP and YM), 2007; 2012a), this is in agreement with results incubated at 5°C for up to one month (de from similar environments of the world Garcia et al. 2007). Physiological, (Antarctica, Alpine glaciers, Alaska, and Arctic;

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Buzzini et al. 2012; Uetake et al. 2011; Vaz et al. isolates) and was shown to be identical to 2011; Zalar & Gunde-Cimerman 2013). Also it isolates cultured from glacial ice in Alaska has been suggested that the predominance of (Basidiomycota sp. GU 74, Uetake et al. 2011). Basidiomycetous yeasts in these cold extreme It is also related to a strain isolated from environments is due to a more nutritionally Antarctica (CBS 8941), forming a new clade versatile and a higher tolerance to extreme closely related with the Glaciozyma (de Garcia et environmental conditions than other groups of al. 2012a). yeasts (Frisvad 2007; Sampaio 2004; Zalar & Gunde-Cimerman 2013). In this regard, species Several strains of the teleomorphic genus occurring in several phylogenetic lineages of the Mrakia and the anamorphic related genus Agaricomycotina, appear to be predominant Mrakiella were isolated (de Garcia et al. 2012a). (isolated in the highest numbers and are among Production of mycelium and teliospores like the most frequently isolated species) in these structures was observed in Mrakiella. In environments (Connell et al. 2008; Turchetti et addition in 2 strains (Mrakiella sp. CRUB 1272 al. 2008; Zalar & Gunde-Cimerman 2013). and Mrakiella aquatica CRUB 1209) germination of these structures was observed. Hyphae and Up to 48 different yeasts species, from teliospore-like structures developed directly these 42.5 % (17 species) represent possible from a single cell, without mating, and clamp new species were isolated from Patagonian connections were not found. Germinating meltwaters and glacial ice (Tab. 1). These results structures of strain CRUB 1272 produced are in agreement with studies in Antarctic soils three- to five- single celled structures, a septate (South Victoria Land), were approximately 43% structure was observed (de Garcia et al. 2012a). of isolates corresponded to possible previously Results obtained from Patagonian strains of undescribed species (Connell et al. 2008). Mrakiella did not establish if these structures were from asexual or sexual origin. In Mrakia Fewer Ascomycetous yeast species were species germination of sexual teliospores is not identified, with 12 yeasts species of total common and in some species it has rarely been isolates, from these 7 species represent observed (Mrakia frigida and M. gelida; Fell potentially previously unidentified species (Tab. 2011). However, teliospore production has 1, de Garcia et al. 2010b; 2012a). been observed in almost all psychrophilic genera previously described (Mrakia and Basidiomycetous species from Tremellales, Glaciozyma, Thomas-Hall et al. 2010; Fell 2011; have been recently described from cold extreme Turchetti et al. 2011). The presence of this environments of Patagonia. Monte Tronador structure could be related to enhance survival in meltwaters have yielded Cryptococcus a diverse array of harsh environmental spencermartinsiae, Cryptococcus tronadorensis, and Cr. conditions, including cold habitats (de Garcia et frias (de Garcia et al. 2010a; 2012b). Cryptococcus al. 2012a). fonsecae was isolated from the Austral Sea in Patagonia Argentina, subglacial ice of Svalbard, Most yeasts from cold (extreme) habitats and from geographically diverse sea-waters and have metabolic adaptations to low Cr. psychrotolerans has been isolated from sea- temperatures, these microorganisms have the water off of Cape Horn Meridian Argentinian ability to degrade organic macromolecules by Sea and from other cold marine environments secreting extracellular hydrolytic enzymes active (de Garcia 2012b). to low temperatures, this suggest that these yeasts may participate in organic matter Additional interesting result was the decomposition and nutrient cycling in glacial isolation and study of psychrophilic yeasts, environments (de Garcia et al. 2012a; Turchetti particularly Mrakia and Mrakiella genera, and a et al. 2013). This role is also supported by the new set of yeast identified as Psychrophilic yeasts presence of organic carbon and organic and sp. The latter was the most frequently inorganic nitrogen in glacial melt-water and ice recovered from Perito Moreno glacier ice (19 (Margesin et al. 2007).

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activities in cell-free supernatants, of Cold-adapted yeasts: potential psychrotolerant yeasts isolated from aquatic biotechnological applications cold environments of Patagonia Argentina and Cold-active enzymes mesophilic yeasts from Brazilian rain forests. Cold adapted yeasts possess specific Protease and lipase activities from psychrophilic adaptation mechanisms (reproduction, selected strains had significantly higher values metabolic activities, survival and protection) (P < 0.05) at 4 °C than at higher temperatures that enable them to be active at low and even at (10, 15, 20, 25 °C); while opposite results were subzero temperatures (Margesin & Miteva observed for mesophilic strains where activity 2011). Among these adaptations are: was affected by the drop of temperature, modification in lipid membranes, extracellular limited (generally) to 20 and 25 °C (Brizzio et polysaccharides, compatible solutes, antifreeze al. 2007; de Garcia et al. 2013). proteins, protection against reactive oxygen Cold-adapted enzymes are beneficial for species and cold active extracellular enzymes. their enhanced selectivity and high catalytic Enzymes are highly specific in their catalytic activity at low and moderate temperatures, in activity, exhibit fast reaction rates, operate addition to their structural liability that can be under mild conditions of temperature and pH, exploited in multi-step processes requiring rapid are derived from renewable resources, and are and mild inactivation treatments (Huston 2007; biodegradable (Huston 2007). Cold-active Gerday 2013). Enzymatic catalysts derived from enzymes can be exploited in a variety of meso- and thermophilic organisms have industrial applications. For example, they can replaced many harsh conventional processes to catalyze numerous reactions at low and great economic benefit (Margesin et al. 2007). moderate temperatures (< 40°C) more Similarly, cold-adapted enzymes could be efficiently and with fewer undesired chemical exploited to further decrease industrial energy reactions than those that may occur at high expenditures and costs. temperatures (Huston 2007). Thus, cold extreme environments are a potential source of Photo-protective metabolites new yeast taxa with desirable biological Carotenoid pigments and mycosporines are characteristics that could be exploited for the major groups of photo-protective biotechnology research and application (Brizzio compounds in cold-adapted yeasts functioning et al. 2007; de Garcia et al. 2012a; Lee et al. by minimize the damage caused by UVR 2010; Libkind et al. 2005; Margesin & Miteva (Moliné et al. 2009; 2013). Carotenoid pigments 2011; Moliné et al. 2013). are potent antioxidant compounds that mainly afford photo-protection by quenching Production of extracellular enzymatic intracellular reactive oxygen species (ROS) activity (protease, esterase, amylase, pectinase generated by UV radiation (indirect damage), and cellulase) at 5 and 18 °C was studied in these pigments are present in a few lineages over 200 yeast strains isolated from melt-waters primarily of basidiomycetous yeasts of which and glacial ice from glaciers of Patagonia (de many include cold-adapted species and genera Garcia et al. 2007; 2012a). Results indicate a (i.e., Rhodotorula, Rhodosporidium, Sporobolomyces, statistically significant association between Dioszegia, Cystofilobasidium, Phaffia, etc.; Libkind basidiomycetous yeast genera and production et al. 2008; Moliné et al. 2010; 2011; 2013). of extracellular enzymes. This association could facilitate a directed search for genera of interest, Mycosporines (sensu-stricto) are water- both in culture collections and also in the soluble molecules formed by a distinctive environment, with the objective of finding cyclohexenone unit and an amino acid or amino strains with possible biotechnological alcohol conjugated subunit. In yeast there is applications (de Garcia et al. 2012a). only one mycosporine described so far: mycosporine-glutaminol-glucoside (MGG). Brizzio et al. (2007) performed quantitative analysis of extracellular protease and lipase

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Table 1: Yeasts species isolated from glaciers of Patagonia Argentina. In bold are described species and possible new ones found in this environment. CRUB, Regional University Center of Bariloche (Centro Regional Universitario Bariloche), Argentina. Basidiomycetous Total N° Origin of the species References of isolates Bensingtonia yamatoana 1 Ice from Frias Glacier of Mount Tronador 1 Cryptococcus adeliensis 1 Melt waters of Rio Manso Glacier of Mount Tronador 2 Cryptococcus cylindricus 1 Melt water river from Frias Glacier of Mount Tronador 1 Cryptococcus fonsecae 18 Sea water, Cape Horn Meridian Argentinian Sea 3 Cryptococcus frias 2 Melt water river from Frias Glacier of Mount Tronador 3 Cryptococcus psychrotolerans 2 Sea water, Cape Horn Meridian Argentinian Sea 3 Cryptococcus sp. CRUB 1267 1 Melt water river from Frias Glacier of Mount Tronador 2 Cryptococcus spencermartinsiae 3 Melt water river from Frias Glacier of Mount Tronador 4 Cryptococcus stepossus 24 Melt water river from Frias Glacier of Mount Tronador 2 Cryptococcus tronadorensis 2 Melt water from Garganta del Diablo waterfall (Río 3 Manso Glacier) of Mount Tronador Cryptococcus victoriae 9 Ice and melt waters from glaciers of Patagonia Argentina 1 Cryptococcus wieringae 1 Melt waters of Castaño Overo glacier of Mount Tronador 2 Dioszegia butyracea 3 Ice from Frias Glacier of Mount Tronador 1 Dioszegia crocea 25 Ice and melt waters from glaciers of Patagonia Argentina 1; 2 Dioszegia fristingensis 13 Ice and melt waters from glaciers of Patagonia Argentina 2 Guehomyces pullulans 1 Ice from Perito Moreno glacier 1 Holtermanniella festucosa 5 Ice and melt waters from glaciers of Patagonia Argentina 1; 2 Holtermanniella sp. CRUB 1256 1 Melt water river from Frias Glacier of Mount Tronador 1 Leucosporidium creatinivorum* 2 Melt water river from Frias Glacier of Mount Tronador 2; 5 Leucosporidium fragarium* 16 Melt water river from Frias Glacier of Mount Tronador 2; 5 Leucosporidium intermedium 1 Ice from Frias Glacier of Mount Tronador 1; 5 Mrakia frigida 3 Melt water from Garganta del Diablo waterfall (Río 2 Manso Glacier) of Mount Tronador Mrakia robertii 4 Ice from Frias Glacier of Mount Tronador 1 Mrakia sp. 1 CRUB 1707 1 Ice from Frias Glacier of Mount Tronador 1 Mrakia sp. 2 CRUB 1706 1 Melt water from Frias glacier, Mount Tronador 1 Mrakiella aquatica 4 Ice and melt waters from Frias glacier of Mount Tronador 1 Mrakiella sp. CRUB 1272 1 Melt water from Garganta del Diablo waterfall (Río 2 Manso Glacier) of Mount Tronador Psychrophilic strain sp. 1 16 Ice from Perito Moreno Glacier 1 Rhodotorula mucilaginosa 6 Ice and melt waters from glaciers of Patagonia Argentina 1; 2 Sporobolomyces ruberrimus 18 Ice and melt waters from Frias glacier of Mount Tronador 1; 2 Udeniomyces megalosporus 3 Ice from Perito Moreno Glacier 1 Udeniomyces pannonicus 11 Ice and melt waters from Frias glacier of Mount Tronador 1; 2 Udeniomyces pyricola 5 Ice and melt waters from Frias glacier of Mount Tronador 1 Udeniomyces sp. CRUB 1695 1 Melt water from Frias glacier, Mount Tronador 1 Ascomycetous Candida marítima 1 Melt water of Castaño Overo glacier of Mount Tronador 1 Candida mesenterica 4 Ice from Perito Moreno Glacier 1 Candida sp. 1 3 Melt water of Castaño Overo glacier of Mount Tronador 1 Candida sp. 2 CRUB 1719 1 Melt water of Castaño Overo glacier of Mount Tronador 1 Candida sp. 3 CRUB 1220 1 Melt water of Castaño Overo glacier of Mount Tronador 1 Candida sp. 4 CRUB 1295 1 Melt water of Castaño Overo glacier of Mount Tronador 1 Debaryomyces hansenii 2 Ice from Frias Glacier of Mount Tronador 1 Wickerhamomyces patagonicus 1 Melt water of Castaño Overo glacier of Mount Tronador 4 Dimorphic fungi Aureobasidium pullulans 3 Ice and melt waters from Frias and Río Manso glacier of 1; 2 Mount Tronador Hyalodendriella sp. CRUB 1755 1 Ice from Perito Moreno Glacier 1 Phaeococcomyces sp. CRUB 1760 1 Ice from Perito Moreno Glacier 1 1- de Garcia et al. (2012a) ; 2- de Garcia et al. (2007); 3- de Garcia et al. (2012b); 4- de Garcia et al. (2010a); 5- de Garcia et al. (2015). * Leucosporidium species complex

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The production of MGG was studied biodiversity, to general yeasts taxonomy and in different Cryptococcus stepposus strains (isolated also to biogeography analysis of a few from melt-waters from Frias glacier, Monte biotechnologically important species. Studies of Tronador) under light and dark conditions and cold-adapted yeasts from glacial melt-water and the relationship between concentration of ice mentioned here were the first surveys from MGG, UVB-radiation survival, and DNA continental glaciers in the Southern damage was measured (Moliné et al. 2011). Hemisphere (outside Antarctica). It has been When strains were cultured under clearly established that Patagonian glaciers are photosynthetically active irradiation a larger cold-adapted yeast reservoirs with potential accumulation of MGG occurs, and a positive biotechnological applications. relation was observed for MGG production and UVB-radiation survival. This positive References MGG accumulation under light supports the Bahr, D.B. & Radic, V. 2012. Significant total idea of a photoprotective function of this mass contained in small glaciers. The molecule. Additionally, small changes in MGG Cryosphere Dicussions. 6: 737-758. accumulation clearly affected the survival of Cr. Bergauer, P.; Fonteyne, P.A.; Nolard, N.; stepposus isolates under these conditions. Moliné Schinner, F. & Margesin, R. 2005. et al., (2011) have stated that MGG efficiently Biodegradation of phenol and phenol-related absorbs UVB radiation, is highly stable, and compounds by psychrophilic and cold- could act as a natural sunscreen in these tolerant alpine yeasts. Chemosphere, 59: 909- microorganisms. 918. Brown, M.E.; Ouyang, H.; Habib, S.; Shrestha, The phylogenetic distribution of MGG is B.; Shrestha, M.; Panday, P.; Tszortziou, M.; more widespread compared to carotenoids, Policelli, F.; Artan, G.; Giriraj, A.; although less chemically complex since only Bajracharya, S.R. & Racoviteanu, A. 2010. one micosporine molecule has so far been HIMALA: Climate Impacts on Glaciers, detected for yeasts. Production of MGG in Snow, and Hydrology in the Himalayan yeasts is favored under high UV irradiances as Region. Mountain Research and denoted by the large numbers of cold-adapted Development, 30(4): 401-404. species that are able to synthesis such metabolite. Given that UVR is an important Branda, E.; Turchetti, B.; Diolaiuti, G.; Pecci, force in structuring yeast community M.; Smiraglia, C. & Buzzini, P. 2010. Yeast composition, the ability of yeasts to synthesize and yeast-like diversity in the southernmost carotenoids and mycosporines becomes at least glacier of Europe (Calderone Glacier, part of the adaptive suite of mechanisms Apennines, Italy). FEMS Microbiology needed for exploiting highly UV-exposed cold Ecology, 72: 354-369. habitats (Moliné et al. 2013). Brandao, R.L.; Libkind, D.; Vaz, B.M.A.; Espirito Santo, L.C.; Moliné, M.; de Garcia, Final Remarks V.; van Broock, M. & Rosa, C.A. 2011. has a great potential in Yeasts from an oligotrophic lake in Patagonia terms of macro- and micro-biodiversity (Argentina): diversity, distribution and (Caravajal et al. 2011) much of which may still synthesis of photoprotective compounds and be undiscovered. In this sense the Patagonian extracelular enzymes. FEMS Microbiology Andes possess glaciers with great potential for Ecology, 76: 1-13. glaciological and paleoclimatic studies, and also Brizzio, S.; Turchetti, B.; de Garcia, V.; Libkind, for microbiological surveys (de Garcia et al. D.; Buzzini, P.; Gasparetti, C. & van Broock, 2012a). Biodiversity studies of cold-adapted M. 2007. Extracellular enzymatic activities yeasts from glacial environments of Patagonia (EEA) in basidiomycetous yeasts isolated have contributed not only to the conservation from glacial and subglacial waters of of genetic resources in these extreme northwest Patagonia (Argentina). Canadian environments in Patagonia, but also to fungal Journal of Microbiology, 53: 519-525.

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Butinar, L.; Spencer-Martins, I. & Gunde- de Garcia, V.; Brizzio, S.; Libkind, D.; Rosa, Cimerman, N. 2007. Yeasts in high Arctic C.A. & van Broock, M. 2010b. glaciers: the discovery of a new habitat for Wickerhamomyces patagonicus, a novel yeasts eukaryotic microorganisms. Antonie van isolated from Patagonia, Argentina. Journal Leeuwenhoek, 91(3): 277-289. of Systematic and Evolutionary Buzzini, P.; Turchetti, B.; Diolaiuti, G.; D’Agata, Microbiology, 60(7): 1693-1696. C.; Martini, A. & Smiraglia, C. 2005. Cultivable de Garcia, V.; Brizzio, S. & van Broock, M. yeasts in melt waters draining from two glaciers 2012a. Yeasts from Glacial Ice of Patagonian in the Italian Alps. Annals of Glaciology, 40(1): Andes, Argentina. FEMS Microbiology 119-122. Ecology, 82(2): 540-50. Buzzini, P.; Branda, E.; Goretti, M. & Turchetti, de Garcia, V.; Zalar, P.; Brizzio, S.; Gunde- B. 2012. Psychrophilic yeasts from worldwide Cimerman, N. & van Broock, M. 2012b. glacial habitats: diversity, adaptation strategies Cryptococcus species (Tremellales) from glacial and biotechnological potential. FEMS biomes in the southern (Patagonia) and Microbiology Ecology, 82: 217-241. northern (Svalbard) hemispheres. FEMS Carvajal, J.; Libkind, D.; Briones, A.; Úbeda, J.; Microbiology Ecology, 82(2): 523-39. Roberts, I.; James, S.; Portero, P.; Alejo, J. & de Garcia, V.; Libkind, D.; Moliné, M.; Rosa, Rosa, C. 2011. Yeasts biodiversity significance: C.A. & van Broock, M. 2013. Cold-Adapted case studies in natural and human-related Yeasts in Patagonian Habitats. In: Buzzini, P. environments, ex situ preservation, applications & Margesin, R (Eds.) Cold-Adapted Yeasts: and challenges. In: Grillo, O. & Venora, G. Biodiversity, Adaptation Strategies and (Eds.) Changing Diversity in Changing Biotechnological Significance. Springer- Environment. InTech, Croatia, pp. 55-86. Verlag, Berlín, pp. 123-148. Connell, L.B.; Redman, R.; Craig, S.; Scorzetti, G.; de Garcia, V.; Coelho M.; Maia T.; Rosa L.; Iszaard, M. & Rodriguez, R. 2008. Diversity of Martins Vaz A.; Rosa C.A.; Sampaio J.; soil yeasts isolated from South Victoria Land, Goncalves P.; van Broock M.; Libkind D. Antarctica. Microbial Ecology, 56: 448-459. 2015. Sex in the cold: taxonomic Correa, M.N. 1998. Flora Patagónica (República reorganization of psychrotolerant yeasts in Argentina) Tomo VIII. In: Correa, M. N. (Ed.) the order Leucosporidiales. FEMS Yeasts Flora Patagónica (República Argentina), Research, 15(4): pii: fov019. doi: Colección científica del INTA, Buenos Aires, 10.1093/femsyr/fov019. pp 31-38. Fell, J.W. 2011. Mrakia Y. Yamada & Komagata Delgado Granados, H.; Miranda, P. J.; Huggel, C.; (1987). In: Kurtzman, C.P.; Fell, J.W. & Ortega del Valle, S. & Alatorre Ibarguengoitia, Boekhout, T. (Eds.) The Yeasts: a M.A. 2007. Chronicle of a death foretold: Taxonomic Study, 5th edn, Elsevier, Extinction of the small-size tropical glaciers of Amsterdam, pp. 1503-1510. Popocatépetl volcano (Mexico). Global Planet Frisvad, J.C. 2007. Fungi in cold ecosystems. In: Change, 56:13-22. Margesin, R.; Schinner, F.; Marx, J.C. & de Garcia, V.; Brizzio, S.; Libkind, D.; Buzzini, P. Gerday, C. (Eds.) Psychrophiles: From & van Broock, M. 2007. Biodiversity of cold- Biodiversity to Biotechnology. Springer- adapted yeasts from glacial meltwater rivers in Verlag, Berlin, pp. 137-156. Patagonia, Argentina. FEMS Microbiology Gerday, C. 2013. Fundamentals of Cold- Ecology, 59(2): 331-341. Active Enzymes. In: Buzzini, P. & de Garcia, V.; Brizzio, S.; Russo, G.; Rosa, C.A.; Margesin, R. (Eds.). Cold-Adapted Yeasts: Boekhout, T.; Theelen, B.; Libkind, D. & van Biodiversity, Adaptation Strategies and Broock, M. 2010a. Cryptococcus spencermartinsiae Biotechnological Significance, Springer- sp. nov., a basidiomycetous yeast isolated from Verlag, Berlin, pp. 325-352. glacial waters and apple fruits. Journal of Systematic and Evolutionary Microbiology, 60(3): 707-711. 8 Lundiana

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Gostincar, C.; Grube, M.; de Hoog, S.; Zalar, P. & Margesin, R.; Neuner, G. & Storey, K. B. 2007. Gunde-Cimerman, N. 2011. Extremotolerance Cold-loving microbes, plants, and animals- in fungi: evolution on the edge. FEMS fundamental and applied aspects. Microbiology Ecology, 71: 2-11. Naturwissenschaften, 94: 77-99. Huston, A.L. 2007. Biotechnological aspects of Margesin, R. & Miteva, V. 2011. Diversity and cold-adapted enzymes. In: Margesin, R.; ecology of psychrophilic microorganisms. Schinner, F.; Marx, J.C. & Gerday, C. (Eds.) Research in Microbiology, 162: 346-361. Psychrophiles: From Biodiversity to Medeiros, A.O.; Rosa, C.A.; Brandao, R.L.; Biotechnology. Springer-Verlag, Berlin, pp. Giani, A.; Gomes, A.N.L. & Libkind, D. 347-363. 2011. Microbial Quality of Freshwater Kuhn, M. 2007. The climate of snow and ice as Ecosystems of South America. In: Ertuo, K. boundary condition for microbial life. In: & Mirza, I. (Eds.) Water Quality: Physical, Margesin, R.; Schinner, F.; Marx, J.C. & Chemical and Biological characteristics. Nova Gerday, C. (Eds.) Psychrophiles: From Science Publishers, Inc. New York, pp. 43- Biodiversity to Biotechnology. Springer-Verlag, 69. Berlin, pp. 3-16. Mestre, M.C.; Rosa, C.; Safar, S.C.; Libkind, D. Kurtzman, C.P.; Fell, J.W.; Boekhout, T. & & Fontenla, S. 2011. Yeast communities Robert, V. 2011. Methods for the isolation, associated with bulk-soil, rhizosphere and phenotypic characterization and maintenance of ectomycorrhizosphere of a Nothofagus pumilio yeasts. In: Kurtzman, C.P.; Fell, J.W. & forest in Northwestern Patagonia, Argentina. Boekhout, T. (Eds.), The Yeasts, a Taxonomic FEMS Microbiology Ecology, 78: 531–541. Study, 5th edn, pp. 87-110. Amsterdam, Miteva, V. 2008. Bacteria in snow and Glacier Elsevier. Ice. In: Margesin, R.; Schinner, F.; Marx, J.C. Lee, J.K.; Park, K.S.; Park, S.; Park, H.; Song, & Gerday, C. (Eds.) Psychrophiles: From Y.H.; Kang, S.H. & Kim, H.J. 2010. An Biodiversity to Biotechnology. Springer- extracellular ice-binding glycoprotein from an Verlag, Berlin, pp. 31- 50. Arctic psychrophilic yeasts. Cryobiology, 60: Moliné, M.; Libkind, D.; del Carmen Diéguez, 222-228. M. & van Broock, M. 2009. Photoprotective Libkind, D.; Brizzio, S.; Ruffini, A.; Gadanho, M.; role of carotenoids in yeasts: response to van Broock, M.R. & Sampaio, J.P. 2003. UV-B of pigmented and naturally-occurring Molecular characterization of carotenogenic albino strains. Journal of Photochemistry and yeasts from aquatic environments in Patagonia, Photobiology B: Biology, 95(3): 156-161. Argentina. Antonie van Leeuwenhoek, 84: 313- Moliné, M., Flores, M.R.; Libkind, D.; del 322. Carmen Diéguez, M.; Farías, M.E. & van Libkind, D.; Sommaruga, R.; Zagarese, H. & van Broock, M. 2010. Photoprotection by Broock, M. 2005. Mycosporines in carotenoid pigments in the yeast Rhodotorula carotenogenic yeasts. Systematic and Applied mucilaginosa: the role of torularhodin. Microbiology, 28: 749-754. Photochemistry & Photobiological Sciences, Libkind, D.; Gadanho, M.; van Broock, M.R. & 9(8): 1145-1151. Sampaio, J.P. 2008. Studies on the Moliné, M.; Arbeloa, E.M.; Flores, M.R.; heterogeneity of the carotenogenic yeast Libkind, D.; Farías, M.E.; Bertolotti, S.G.; Rhodotorula mucilaginosa from Patagonia, Churio, M.S. & van Broock, M.R. 2011. UVB Argentina. Journal of Basic Microbiology, 48: photoprotective role of mycosporines in 93-98. yeast: photostability and antioxidant activity Libkind, D.; Moliné, M.; Sampaio, J. & van of mycosporine-glutaminol-glucoside. Broock, M. 2009. Yeasts from high altitude Radiation Research, 175(1): 44-50. lakes: influence of UV radiation. FEMS Microbiology Ecology, 69: 353–362.

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Moliné, M.; Libkind, D.; de Garcia, V. & van Turchetti, B.; Buzzini, P.; Goretti, M.; Branda, Broock, M. 2013. Production of Pigments and E.; Diolaiuti, G.; D´Agata, C.; Smiraglia, C. & Photo-Protective Compounds (Carotenoids and Vaughan-Martini, A. 2008. Psychrophilic Mycosporines). In: Buzzini, P. & Margesin, R. yeasts in glacial environments of Alpine (Eds.) Cold-Adapted Yeasts: Biodiversity, glaciers. FEMS Microbiology Ecology, Adaptation Strategies and Biotechnological 63(1):73-83. Significance, Springer-Verlag, Berlin, pp. 193- Turchetti, B.; Thomas-Hall, S.R.; Connell, L.B.; 224. Branda, E.; Buzzini, P.; Theelen, B.; Müller, Price, P.B. 2007. Microbial life in glacial ice and W.H. & Boekhout, T. 2011. Psychrophilic implications for a cold origin of life. FEMS yeasts from Antarctica and European Microbiology Ecology, 59(2): 217-231. glaciers: description of Glaciozyma gen. nov., Rabassa, J.; Rubulis, S. & Suárez, J. 1978. Los Glaciozyma martinii sp. nov. and Glaciozyma Glaciares del Monte Tronador Parque Nacional watsonii sp. nov. Extremophiles, 15:573–586. Nahuel Huapi (Río Negro, Argentina). Anales Turchetti, B.; Goretti, M.; Buzzini, P. & Parques Nacionales, 14:259–318. Margesin, R. 2013. Cold-Adapted Yeasts in Ruiz, L.; Masiokas, M.H. & Villalba, R. 2012. Alpine and Apennine Glaciers. In: Buzzini, P. Fluctuacions of Glaciar Esperanza Norte in the & Margesin, R. (Eds.) Cold-Adapted Yeasts. north Patagonian Andes of Argentina during Biodiversity, Adaptations Strategies and the past 400 yr. Climate of the Past, 8: 1079- Biotechnological Significance. Springer, 1090. Berlin, pp. 99-122. Sampaio, J.P. 2004. Diversity, phylogeny and Uetake, J.; Kohshima, S.; Takeuchi, N.; Fujita, classification of basidiomycetous yeasts. In: K.; Miyake, T.; Narita, T.; Aizen, V. & Agerer, R.; Blanz, P. & Piepenbring, M. (Eds.) Nakawo, M. 2011. Evidence for propagation Frontiers in Basidiomycote Mycology. of cold-adapted yeasts in an ice core from a IHWVerlag, Eching, pp. 49–80. siberian Altai glacier. Journal of Geophysical Skvarca, L.; Raup, B. & De Angelis, H. 2003. Research, 116: G01019. Recent behaviour of Glaciar Upsala, a fast- Vaz, A.B. M.; Rosa, L.H.; Vieira, M.; de Garcia, flowing calving glacier in Lago Argentino, V.; Brandão, R.L.; Teixeira, L.; Moliné, M.; Southern Patagonia. Annals of Glaciology, Libkind, D.; van Broock, M. & Rosa, C.A. 36:184-188. 2011. The diversity, extracellular enzymatic activities and photoprotective compounds of Starmer, W.T. & Lachance, M. 2011. Yeast Ecology. In: Kurtzman, C.P.; Fell, J.W. & yeasts isolated in Antarctica. Brazilian Journal of Microbiology, 42: 937-947. Boekhout, T. (Eds.) The yeasts, A Taxonomy study. Elsevier, London, pp. 65-83. Villarosa, G.; Outes, V.; Masiokas, M.; Villalba, R. & Rivas, S. 2008. El Monte Tronador: Stuefer, M.; Rott, H. & Skvarca, P. 2007. Glaciar Perito Moreno, Patagonia: climate sensitivities Historias de hielo y fuego. In: Rastelli, D. (Ed.) Sitios de Interés Geológico de la and characteristics preceding 2003/04 and 2005/06 damming events. Journal of República Argentina. SEGEMAR (Servicio de Geología y Minería de la República Glaciology, 53(180):3-16 Argentina), Buenos Aires, pp. 627-641. Thomas-Hall, S.; Turchetti, B.; Buzzini, P.; Zalar, P. & Gunde-Cimerman, N. 2013. Cold- Branda, E.; Boekhout, T.; Theelen, B. & Watson, K. 2010. Cold adapted yeasts from Adapted Yeasts in Arctic Habitats. In: Buzzini, P. & Margesin, R. (Eds.) Cold- Antarctica and the Italian Alps. Description of three novel species: Mrakia robertii sp. nov., Adapted Yeasts. Biodiversity, Adaptations Strategies and Biotechnological Significance. Mrakia blollopis sp. nov. and Mrakiella niccombsii sp. nov. Extremophiles, 14: 47-59. Springer, Berlin, pp. 49-74.

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