Yeast occurrence and quantitative analyses
Average yeast counts for each sampling site are shown in Table 1. Yeast counts between sampling sites were not significantly different (P > 0.117), nor were counts for different selective media within each sampling site (P > 0.117). No yeasts colonies were observed in selective media with casein and Tween 80 substrates (Table 1). Yeast counts were similar to those obtained for meltwater rivers of Mount Tronador (de Garcia et al., 2007) and in other aquatic environments of Patagonia Argentina (1–2 × 103 ± 1–4 × 102 CFU L−1; Libkind et al., 2003; Brandao et al., 2011). Studies in similar environments of the Italian Alps also registered similar values of yeast counts, 1 × 101 and 4 × 103 CFU L−1 (Buzzini et al., 2005; Turchetti et al., 2008; Branda et al., 2010). These values are relatively low when compared with coastal or polluted aquatic systems (Nagahama, 2006), because of the oligotrophic nature of the ice samples (Hagler & Ahearn, 1987; Foght et al., 2004).
Table 1. Location of sampling sites and viable yeast counts
|Sampling sites||Frías Glacier (Mount Tronador)||Perito Moreno Glacier|
|Location||41°08′43″S, 71°49′01″W||49°S, 73°W|
|pH of the sample||6.5||6.4|
|Yeast viable counts in general and selective media * Media ± SD (CFU L−1)|
| MYP||3.3 × 103 ± 9.6 × 102||3.3 × 103 ± 2.1 × 103|
| YNB + Pectin pH 5||2.5 × 103 ± 8.0 × 102||5.3 × 103 ± 3.7 × 103|
| YNB + Pectin pH 7||2.1 × 103 ± 6.8 × 102||3.6 × 103 ± 1.0 × 103|
| YNB + CMC||0||4.0 × 103 ± 2.8 × 103|
A total of 153 isolates were classified. Of these, 115 corresponded to the ice samples of the Frias and Perito Moreno glaciers. These were assigned to 16 genera and 29 species (Table 2). The remaining 38 corresponded to the unidentified isolates from a previous study on yeasts in meltwaters of Mount Tronador (de Garcia et al., 2007). These were classified into five genera and 16 different species (Table 2). Results in this section will refer to all 153 isolates. Ninety percent of the total yeast strains studied (153 isolates) belonged to the phylum Basidiomycetes.
Table 2. Yeast species isolated from glacial meltwater and ice from Patagonia Argentina, number of isolates and origin
|Species||Total number of isolates||Origin of strains|
|Mount Tronador||Frias Glacier||Perito Moreno Glacier|
|Dioszegia crocea ||24 (15.6)||21||3||–|
|Psychrophilic strain sp. 1a||19 (12.5)||–||–||19|
|Sporobolomyces ruberrimus ||15 (9.8)||–||15||–|
|Dioszegia fristingensis ||12||1||11||–|
|Udeniomyces pannonicus ||8||–||8||–|
|Cryptococcus victoriae ||7||6||–||1|
|Udeniomyces pyricola ||5||–||5||–|
|Mrakiella aquatica ||4||–||4||–|
|Rhodotorula sp. 1a||3||3||–||–|
|Rhodotorula mucilaginosa ||3||–||–||3|
|Dioszegia butyracea ||3||–||3||–|
|Cr. spencermartinsiae b ||3||3||–||–|
|Udeniomyces megalosporus ||3||–||–||3|
|Mrakia robertii ||3||–||3||–|
|Aureobasidium pullulans ||3||–||3||–|
|Candida sp. 1a||3||3||–||–|
|Cryptococcus sp. 1a||2||2||–||–|
|Cryptocccus sp. 2a||2||2||–||–|
|Holtermanniella festucosa ||2||–||–||2|
|Candida mesenterica ||2||1||–||1|
|Mastigobasidium intermedium ||1||–||1||–|
|Rhodotorula sp. 2 CRUB 1756a||1||–||–||1|
|Cryptococcus sp. 3 CRUB 1267a||1||1||–||–|
|Holtermanniella sp. 1 CRUB 1256a||1||–||–||1|
|Udeniomyces sp. 1 CRUB 1695a||1||–||1||–|
|Mrakia sp. 1 CRUB 1707a||1||–||1||–|
|Mrakia sp. 2 CRUB 1706a||1||–||1||–|
|Guehomyces pullulans ||1||–||–||1|
|Bensingtonia yamatoana ||1||–||1||–|
|Cryptococcus sp. 4 CRUB 1245a||1||1||–||–|
|Cryptococcus terrícola ||1||–||1||–|
|Cryptococcus wieringae ||1||1||–||–|
|Ascomycota sp. 1 CRUB 1755a||1||–||–||1|
|Phaeococcomyces sp. 1 CRUB 1760a||1||–||–||1|
|Wickerhamomyces patagonicus b ||1||1||–||–|
|Debaryomyces hansenii ||1||–||1||–|
|Candida sp. 2 CRUB 1719a||1||–||–||1|
|Candida sp. 3 CRUB 1220a||1||1||–||–|
|Candida sp. 4 CRUB 1295a||1||1||–||–|
|Candida maritima ||1||1||–||–|
All isolates were adapted to living at cold temperatures, 75% were psychrotolerant (growth at 5–25 °C), while the remaining 25% were psychrophilic (growth at 5–15 °C). The occurrence of psychrophilic yeasts in cold environments reported here was similar to that found in Alpine glaciers (17%) by Turchetti et al. (2008) and Branda et al. (2010), and in Arctic glaciers (30%) by Pathan et al. (2010).
Species diversity values, measured with the Shannon–Weaver index, were higher in the ice from Frias glacier and meltwaters from Mount Tronador (H = 2.23 and H = 2.52, respectively) than in ice from Perito Moreno (H = 1.72). There were no significant differences for Shannon–Weaver diversity indices among all compared communities (P > 0.05). Jaccard index for community similarity analysis showed that Frias and meltwater yeast communities and Perito Moreno and meltwater yeast communities were the most similar (J = 0.15 and J = 0.13, respectively); however, the index values were low for all the compared communities, and no similarity was found when yeasts communities of Perito Moreno and Frias were compared.
A relatively higher richness index of taxa among ice and meltwater samples was observed, compared to the values reported for soil samples in Patagonian forest by Mestre et al. (2011). In addition, Brandao et al. (2011) mentioned similar values in water samples from Nahuel Huapi Lake (Patagonia, Argentina; coast sites H = 2.2 and pelagic sites H = 2.8).
Yeast identification, diversity, and ecology
Basidiomycetous yeasts were the predominant group in ice from Patagonian glaciers, belonging mostly to subphylum Agaromycotina, particularly to Tremellales (55 isolates) and Cystofilobasidialles orders (27 isolates). Similar results from different cold environments of the world (Antarctica, Alpine glaciers, Alaska, and Arctic) have been reported (Thomas-Hall et al., 2002, 2010; Bergauer et al., 2005; Margesin et al., 2005, 2007b; Butinar et al., 2007; Connell et al., 2008, 2010; Turchetti et al., 2008; Shivaji & Prasad, 2009; Branda et al., 2010; Uetake et al., 2011; Vaz et al., 2011) and also from other aquatic environments of Patagonia (Libkind et al., 2003; de Garcia et al., 2007; Brandao et al., 2011).
Connell et al. (2008) mentioned that in Antarctic soils (South Victoria Land), 90% of yeast isolates were basidiomycetous, 43% of these corresponded possible new species. In this study, 40% (11 species) of basidiomycetous isolates were also possible new species. Several authors have suggested that the predominance of basidiomycetous yeasts in these extreme environments is because they are more nutritionally versatile and more tolerant to extreme environmental conditions than ascomycetous yeasts (Sampaio, 2004; Brandao et al., 2011).
The species most frequently recovered from Perito Moreno glacier ice was a yeast identified as psychrophilic yeasts sp. 1 (19 isolates), followed by and Sporobolomyces ruberrimus (15 isolates) identified by MSP-PCR fingerprinting (data not shown) isolated from ice from Frias glacier. While Dioszegia species were most frequently recovered (D. crocea 24 and D. fristingensis 12 isolates) from Mount Tronador Glaciers (ice and meltwaters). Species belonging to the Dioszegia genus are frequently found associated with plants and terrestrial substrates (Inácio et al., 2005), while D. crocea species more commonly related to cold environments.
Some cosmopolitan species, such as Rhodotorula mucilaginosa (three strains from Perito Moreno glacier), Cryptococcus victoriae (six strains from meltwaters of Mount Tronador and one strain from Perito Moreno Glacier), and Aureobasidium pullulans (three strains from Frias glacier) were isolated.
Psychrophilic yeasts sp. 1 was shown to be identical in D1/D2 sequence to yeasts isolated in Alaska (Basidiomycota sp. GU 74), and considering ITS region, the closest related strain was an Antarctic yeast (CBS 8941), with 94% similarity in blast result. This species is closely related to Camptobasidium hydrophilum and together with the unidentified species from Alaska and Antarctica (GU 54 and CBS 8941) formed a new clade within the class (Fig. 1). The presence of teliospores without mating was observed in some isolates of this species.
Figure 1. Phylogenetic placement of psychrophilic basidiomycetous Patagonian yeasts species obtained by neighbor-joining (distance K2P method) of the LSU rRNA gene D1/D2 domains. Names in bold type are strains described in this work. Bar, substitutions accumulated every 100 nucleotides. Bootstrap values higher than 50% are shown (1000 replicates). TType strain.
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Strains related to the genus Rhodotorula were found (Rhodotorula sp. 1 and Rhodotorula sp. 2 CRUB 1756). Species Rhodotorula sp. 1 was related to Rh. glacialis and had four nucleotide differences in the D1/D2 region. Further analyses are needed to determine whether these strains represent new species. Rhodotorula sp. 2 CRUB 1756 had 15 nucleotide differences in D1/D2 region and 11 in ITS region, with the closest species Rhodotorula laringysT, having a basal position in Rh. laringys clade.
Three potential new Cryptococcus species, related to Tremellales order, were identified, Cryptococcus sp. 1 (related to Cr. foliicola), Cryptococcus sp. 2 (related to Cr. laurentii clade), and Cryptococcus sp. 3 (related to Kwoniella clade formal Cr. heaveanensis clade). Formal description of these species is in progress. Species Cr. spencermartinsiae sp. nov. has been recently described (de Garcia et al., 2010a).
Species of genera Udeniomyces and Guehomyces were identified (U. pannonicus, U. pyricola, U. megalosporus, Udeniomyces sp. 1 CRUB 1697 and G. pullulans); Udeniomyces sp. 1 CRUB 1697 had 11 nucleotide differences in the D1/D2 region and 16 in the ITS region with the closest species being U. pseudopyricolaT.
Hortemanniella festucosa (Cryptococcus festucosus) of the recently described order Holtermanniales (Wuczkowski et al., 2010) was also isolated.
Udeniomyces pannonicus, G. pullulans and H. festucosa, species are psychrophilic or psychrotolerant and have been isolated from different cold terrestrial regions of the world (Fell & Guého-Kellermann, 2011; Takashima & Nakase, 2011). Wuczkowski et al. (2010) concluded that Holtermanniella species possess significant amounts of polyunsaturated fatty acids, a fatty acid composition typical of yeasts adapted to cold environments.
Regarding the Filobasidialles order, Cryptococcus terricola, Cr. wieringae and a possible new species Cryptococcus sp. 4 CRUB 1245 (13 nucleotide difference in D1/D2 region and 9 in ITS region with the closest species Cryptococcus arrabidensis CBS 8678T) were identified.
Representatives of the psychrophilic genus Mrakia and the anamorphic-related genus Mrakiella were isolated in this survey: three strains of Mrakia robertii, two possible new species (Mrakia sp. 1 CRUB 1706 and Mrakia sp. 2 CRUB 1707), and four strains of Mrakiella aquatica.
Yeasts belonging to phylum Ascomycota were less abundant, being approximately 10% of total isolates. Species Candida maritima, Debaryomyces hansenii, Candida mesenterica, and A. pullulans were identified, and the new species Wickerhamomyces patagonicus was recently described (de Garcia et al., 2010b).
From 11 identified species, seven (63%) failed to match the already-described species, and most probably represent new ones. Four species were related to Candida in Saccharomycetales order, one strain was related to the species Hyalodendriella betulae (Helotiales), and one to the melanin-producing species Phaecoccomyces nigricans (Chetothyriales; Fig. 2).
Figure 2. Phylogenetic placement of Ascomycetous Patagonian yeasts species obtained by neighbor-joining (distance K2P method) of the LSU rRNA gene D1/D2 domains. Names in bold type are strains described in this work. Bar, substitutions accumulated every 100 nucleotides. Bootstrap values higher than 50% are shown (1000 replicates). TType strain.
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As was mentioned, isolates of species Rhodotorula sp. 1 related to Rh. glacialis were identified. Gadanho & Sampaio (2009) proposed the ‘ecoclade’ concept, which refers to species that are phylogenetically related and show metabolic adaptations associated with physicochemical conditions present in the environment from which they were recovered. These authors suggest the existence of two ‘ecoclades’, acidic and psychrophilic. The psychrophilic ecoclade is defined by species from glacial environments from the Alps and mountainous areas from Himalaya. Rhodotorula sp. 1, isolated from glacier meltwater from Mount Tronador belongs to this clade, supporting this ecoclade proposal.
All Mrakiella strains isolated here produced mycelium and teliospores in culture medium without nitrogen (Yeasts Carbon Base). Teliospores were placed in distilled water and incubated at 5 °C for up to 1 year; after this period, agar blocks containing teliospores were placed in agar-water and, after 2 months, germinating teliospores were observed in two strains (Mrakiella sp. CRUB 1272 and M. aquatica CRUB 1209). Hyphae and teliospores developed directly from a single cell, without mating, and clamp connections were not observed. Germinating teliospores of strain CRUB 1272 produced three to five single-celled structures (Fig. 3), a septate structure was observed (Fig. 3c).
Figure 3. Phase-contrast micrograph of teliospores of Mrakiella strains isolated from Patagonian glaciers. Mrakiella sp. CRUB 1272 (a–c), after 7 weeks in YCB agar media at 10 °C followed by 1 year in distilled water, (a, b) Teliospores germinated, (c) Teliospores germinated, arrow is showing septated structure. (d) Mrakiella aquatica CRUB 1709, germinated teliospores. (e, f) Teliospores of M. aquatica CRUB 1708 and CRUB 1721, respectively. On 2% agar after 2 months. Bar = 10 μm.
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Is not clear whether teliospores found here in Mrakiella species were from asexual or sexual origin. Generally, germination of sexual teliospores in Mrakia species is not common and in some species has rarely been observed (Mrakia frigida and M. gelida; Fell, 2011). Further studies will be necessary to determine the sexual state of Mrakiella strains.
The presence of teliospores has been observed in almost all psychrophilic species described (Mrakia and Glaciozyma; Thomas-Hall et al., 2010; Fell et al., 2011; Turchetti et al., 2011). Production of this structure can enhance survival in a diverse array of harsh environmental conditions, including cold habitats.
The presence of these extremophilic microorganisms in geographically distant regions could be the result of ecological fitting and genetic adaptation that allowed them to increase and improve their survival in these specific environments (Margesin et al., 2007b; Rossi et al., 2009).
In summary, and given the hypothesis that microorganisms in extreme environments could have differential evolutionary ratios compared with those in temperate environments (Skidmore et al., 2000; Rosenberg & Hastings, 2003, 2004), studying and understanding the evolution of extremophiles will increase the basic knowledge of evolutionary processes, allowing a better evaluation of potential ecological consequences of environmental changes and possible effects on human health (Gostincar et al., 2011).
Several selective media containing different substrates were included in the isolation step, attempting to improve the recovery of yeast strains with the ability to metabolize these different substrates. Results were not significantly different to those obtained with MYP agar, indicating that yeast diversity in the samples was adequately reflected by culturing in this generic medium.
Extracellular activity of selected yeast strains was previously reported (de Garcia et al., 2007). However, in that study, the evaluation was not complete, as it included only some strains for the enzymatic analyses. Those strains were therefore included in this work, for a complete analysis of enzymatic production (from ice and meltwater samples) at 5 and 18 °C. Thus, a total of 212 strains were assessed (115 strains from ice and 97 from meltwaters), and the results are shown in Fig. 4.
Figure 4. Extracellular enzymatic activity of yeast strains from ice and meltwater rivers from Perito Moreno glacier and Mount Tronador glaciers. Bars indicate the SD of halo/colony mean values. N indicates the number of positive strains (of 212 strains) for the corresponding enzymatic activity. a, Significantly different activity at 5 and 18 °C (P = 0.001).
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Eighty-five percent (85%) of the 212 yeasts strains were able to produce at least one enzymatic activity, and 18% produced five different enzymatic activities. Differences in qualitative (number of positive strains) and semiquantitative (intensity of degrading activity) expression of extracellular enzymatic activities at 5 and 18 °C were investigated. Higher activity at 5 °C was observed for all extracellular activities analyzed (both in number of positive strains and in intensity), and statistically significant differences were found for proteolysis (P ≤ 0.001), hydrolysis of carboxymethyl-cellulose (cellulose activity; P = 0.025), and hydrolysis of Tween-80 (P = <0.001; Fig. 4).
Turchetti et al. (2008) reported similar results for yeasts isolated from Alpine glaciers, indicating that 73 selected isolates had enzymatic activity at low temperatures (4 °C). However, Pathan et al. (2010) observed higher degradation at 22 than at 8 °C in psychrotolerant yeasts from Arctic glacier meltwaters, but in this case, the authors used an incubation period of only 10 days at 8 °C, when, in general, 20 days are needed at this temperature for psychrophilic and psychrotolerant strains to achieve stationary growth phase, in which extracellular enzymes are produced.
Hydrolysis of Tween-80 (esterase activity) was the most common activity, being present in 146 isolates (69%). These results are in agreement with those reported by Brandao et al. (2011) for enzymatic activity of yeasts from an oligotrophic lake of Patagonia (71.8% of the total isolates) and by Margesin et al. (2005), Buzzini et al. (2005) and Turchetti et al. (2008) for yeasts isolates from Alpine glacial environments (89%, 46%, and 86% respectively). Hydrolysis of carboxymethyl-cellulose (96 isolates) was the second most frequent activity, followed by proteolysis (60 isolates) and pectinolysis (62 isolates), amylase activity was the least frequent of the activities (27 isolates).
An association between yeast genera and the ability to produce extracellular enzymes was found through multiple correspondence and hierarchical classification analysis. Six different classes were proposed:
- Class 1: Sporobolomyces isolates produce amylase at 5 and 18 °C.
- Class 2: Leucosporidiella and Udeniomyces isolates produce protease, CMC-cellulose, pectinase, and esterase at 5 and 18 °C.
- Class 3: Cryptococcus isolates produce CMC-cellulase at 5 and 18 °C.
- Class 4: Dioszegia isolates produce esterase at 5 and 18 °C.
- Class 5: Mrakia and Mrakiella isolates produce protease, esterase, and pectinase at 5 °C.
- Class 6: Ascomycota and Rhodotorula isolates were not associated with enzymatic activity tested in the essay conditions. It must be noted that even though, according to multiple correspondence and hierarchical classification analysis, ascomycetous yeasts were not associated with any enzymatic activity; A. pullulans isolate had more than two extracellular activities, which is in agreement with other reports for this species (Turk et al., 2007; Zalar et al., 2008). Buzzini et al. (2005) found similar results for ascomycetous yeast isolated from Alpine glacier.
In conclusion, results of the laboratory cultures carried out in this study support that the yeasts in these extreme (cold) habitats possess metabolic adaptation to low temperatures. Cold-adapted Cryptococcus isolates with the ability to produce more than one hydrolytic cold-active enzyme were obtained from Patagonian glaciers. Also Mrakia and Mrakiella isolates able to produce up to five different extracellular cold-active enzymes, and resistance structures (teliospores) were recovered and characterized. These microorganisms are heterotrophic, and their ability to degrade organic macromolecules through the secretion of extracellular hydrolytic cold-adapted enzymes suggest that, as proposed by Turchetti et al. (2008), they may have a significant ecological role in organic matter decomposition and nutrients in glacial environments. This role is also supported by the presence of organic carbon and organic and inorganic nitrogen in glacial meltwater and ice (Skidmore et al., 2000; Foght et al., 2004; Margesin et al., 2007a).
The biotechnological (and industrial) relevance of cold enzymes from psychrophilic yeasts has been emphasized (Thomas-Hall et al., 2010). Association observed here between certain taxa of basidiomycetous yeasts and extracellular enzymatic activities facilitates a directed search of genera of interest, both in culture collections and in the environment, to find strains with possible biotechnological applications.
Basidiomycetous yeasts are a diverse group of fungi with considerable industrial and medical importance and have undeniable potential for economic exploitation (Abadias et al., 2003; Qin et al., 2004; Schisler et al., 2011). This study has contributed to the understanding of their biodiversity and ecological roles.