Diversity of Fungi

The main taxa of fungi isolated from the high Arctic glaciers include melanized fungi that were mainly represented by the oligotrophic genus Cladosporium, taxonomically and phylogenetically closely related to the black yeast-like halotolerant genus Aureobasidium. Among melanized fungi the genera Alternaria and Phoma occured with low frequencies. Filamentous fungi, isolated with high frequency were represented by the cosmopolitan anamorphic genera Aspergillus and Penicillium, together with the teleomorphic form Eurotium, with the prevalence of g. Penicillium over Aspergillus and Eurotium [Gunde-Cimerman et al., 2003; Gunde-Cimerman et al., 2005b; Sonjak et al., 2006]. Other filamentous genera that appeared with low frequency were Mucor and Trichoderma [Gunde-Cimerman et al., 2005b].

Non-melanized yeasts that were detected in the glaciers include Bulleromyces, Candida, Cryptococcus, Cystofilobasidium, Debaryomyces, Filobasidium, Leucosporidiella, Pichia, Protomyces, Rhodosporidium, Rhodotorula and Trichosporon [Butinar et al., 2007; sub.).

Species diversity in glacier ice depended considerably on the occurrence of mineral inclusions. In samples with mineral sediments penicillia prevailed, and their species diversity was high, while in samples of clear ice, overlying the ice reach in sediment inclusions, the species diversity was low, although individual species of non-melanized yeasts occurred with high frequency [Gunde-Cimerman et al., 2003; Gunde-Cimerman et al., 2005b; Sonjak et al., 2006].

1.1 Aureobasidium

Aureobasidium is a genus of xerotolerant fungi, primarily inhabiting oligotrophic environments. The most ubiquitous species is the halotolerant A. pullulans, which is regularly detected in salterns, where it represents one of the core species of saltern mycobiota at lower salinities (up to 10% NaCl). Besides salterns, sea and fresh water its ecological niches are oligotrophic inert surfaces such as glass or stone or it can be frequently isolated from xeric phylloplane of plants. It is also known for its ability to grow at high levels of radioactive gamma contamination, since some strains have been isolated even from the walls of the Chernobyl reactor. In polar environments A. pullulans has been previously isolated from continental Antarctic soil samples [Onofri et al., 2004].

The dominant genus of black yeast-like fungi isolated from subglacial samples containing abundant crystal gypsum inclusions was Aureobasidum. Based on multi-loci sequencing, isolates were identified as diverse genotypes of A. pullulans [data not published]. A. pullulans strains were primarily isolated on media with 5% NaCl after one week of incubation at 22°C, or two weeks at 10°C [Gunde-Cimerman et al., 2003; Gunde-Cimerman et al., 2005b].

1.2 Cladosporium

The genus Cladosporium is taxonomically and phylogenetically closely related to black yeasts belonging to the order Capnodiales [Crous et al., 2007]. It comprises about 500 plant-pathogenic and saprophytic species, many of which are among the most airborne species in the fungal kingdom.

The species C. cladosporioides, C. herbarum, C. sphaerospermum, C. tenuissimum, and C. oxysporum are often isolated from salty/ sugary food [Samson et al., 2004] and other environments with low water activity such as saline (coastal) soils and salt marshes, the phylloplane of Mediterranean plants, and the rhizosphere of halophytic plants [Abdel-Hafez et al., 1978]. During the study on the occurrence of fungi in hypersaline environmnts, strains from this genus were isolated from hypersaline waters worldwide. They were in general among the most abundant and most consistently detected fungi in all natural hypersaline waters sampled, at environmental salinities between 15-25% [Zalar et al., 2001; Butinar et al., 2005a; Gunde-Cimerman et al., 2005a).

The majority of cladosporia isolated from Kongsfjorden comprise the same species as most frequently isolated from Antarctic soil and hypersaline waters in the salterns: C. cladosporioides complex of species and C. herbarum complex of species, although several yet unidentified strains were isolated as well [Abyzov, 1993; Ma et al., 1999, 2000; Gunde-Cimerman et al., 2005b]. These Cladosporium species were primarily isolated from glacial ice samples and considerably less frequently from sea water and sea ice [Gunde-Cimerman et al., 2003; Gunde-Cimerman et al., 2005b]. The highest frequency of occurrence was obtained on media with 50% glucose added at 10 °C, although all tested strains grew well on 17% NaCl media [Gunde-Cimerman et al., 2003; Gunde-Cimerman et al., 2005b].

The genus Cladosporium is known for its psychrotolerance. The reported temperature range for growth of C. cladosporoides is from 0-32 °C, with slight growth possible at -3 °C and even at -10 °C. Some isolates of C. herbarum were shown to grow even at -6 °C. All three species can grow at high levels of radioactive gamma irradiation [Onofri et al., 2004].

1.3. Non-Melanized Yeasts 1.3.1 Yeast Diversity

Around 500 yeast isolates were obtained from the four glaciers sampled. Most of these were of basidiomycetous origin, while ascomycetous yeasts represented ~15% of all of the isolates. The ratio of basidiomycetous towards ascomycetous yeasts changed with the aw and the solute used to increase the osmotic pressure of the media. On media with added NaCl, the ratio between ascomycetous and basidiomycetous yeasts increased above 50% (Fig. 2). However, on media with aw between ~1 and 0.95, basidiomycetous yeasts prevailed (Fig. 2), they constituted ~85% of all identified strains from subglacial ice samples [Butinar et al., 2007; Butinar et al., sub.]. Although basidiomycetous yeasts are generally recognized as being more nutritionally versatile and tolerant to low temperatures than ascomycetous yeasts [Sampaio, 2004], these results also reflect the biased approach of most isolation procedures.

Overall, 22 different basidiomycetous species of the classes Hymenomycetes and Urediniomycetes were found [Butinar et al., 2007] (Table 3). With respect to their abundance and species diversity, the dominant group corresponded to the hymenomycetous yeasts and amongst them the non-pigmented Filobasidium/ Cryptococcus albidus taxa of the Tremellales predominated [Butinar et al., 2007]. Our isolates fell into the Albidus clade [Fonseca et al., 2000], where Cr. adeliensis, Cr. albidosimilis, Cr. albidus, Cr. liquefaciens and Cr. saitoi were identified, and into the Magnus clade [Fonseca et al., 2000], where Cr. gilvescens and Cr. magnus, Cr. oeirensis and F. uniguttulatum were recognised. Differentiation between Cr. magnus and F. floriforme required additional ITS analysis. No representatives of the Aerius clade were found [Butinar et al., 2007].

The remaining species of the Tremellales belong to the Bulleromyces (Bulleromyces albus, species from Cr. laurentii complex) and the Dimennae/Victoriae clades (Cr. carnescens, Cr. victoriae) [Scorzetti et al., 2002; Takashima et al., 2003]. Cryptococcus sp. MZKI K-282 from Cr. laurentii complex differed by 10 bp when compared with Cr. laurentii CBS 139 (Butinar et al., 2007). The remaining strains from this complex were grouped into the phylogenetic group II [Takashima et al., 2003].

Two species assigned to the Cystofilobasidium clade of the Cystofilobasidiales [Scorzetti et al., 2002], Cr. macerans and Cystofilobasidium sp., were also found [Butinar et al., 2007]. Sequence of the strain EX-F 1547 differed significantly (2%) from the type strain of Cystofilobasidium bisporidii [Butinar et al., 2007].

In the clade Cutaneum of the Trichosporonales [Scorzetti et al., 2002] Trichosporon mucoides, which is characterized by the presence of septate hyphae and the production of arthroconidia, was identified [Butinar et al., 2007].

The urediniomycetous yeasts were represented by four carotenoid-pigmented and one non-pigmented species and were distributed in three lineages: the Sporidiobolus lineage, with Rhodosporidium diobovatum from the clade Glutinis and Rhodotorula mucilaginosa belonging to the clade Sphaerocarpum (Scorzetti et al., 2002); the Erythrobasidium lineage, with Rh. laryngis and Rh. lysiniphila from the clade Occultifur, and the Microbotryum lineage, with Leucosporidiella fragaria from the clade Leucosporidium [Scorzetti et al., 2002; Sampaio et al., 2003; Butinar et al., 2007].

Table 3. Occurrence of asco- and basidiomycetous yeast species in the glaciers studied.

Conwaybreen

Kongsvegen

Austre Lovénbreen

Austre Braggerbreen

Subglacial

Supraglacial

Ice from

Subglacial

Glacier

Supraglacial

Subglacial

Supraglacial

Subglacial

samples

samples

cryokarst

samples

melt-

samples

samples

samples

samples

formations

water

Bulleromyces albus

+

Candida parapsilosis

+

C. pseudorugosa-like

+

Cryptococcus adeliensis

+

Cr. albidosimilis

+

Cr. albidus

+

+

+

+

Cr. carnescens

+

+

Cr. gilvescens

+

Cr. laurentii complex

+

+

Cr. liquefaciens

+

+

+

+

+

+

+

Cr. macerans

+

Cr. magnus

+

+

+

+

Cr. oeirensis

+

Cr. saitoi

+

Cr. victoriae

+

+

+

+

Cystofilobasidium sp.

+

Debaryomyces hansenii

+

+

+

Filobasidium uniguttulatum

+

+

Leucosporidiella fragaria

+

+

Pichia guilliermondii

+

+

Protomyces inouyei

+

Rhodotorula laryngis

+

Rh. lysiniphila

+

Rh. mucilaginosa

+

+

+

+

+

+

+

+

+

Rhodosporidium diobovatum

+

+

Trichosporon mucoides

+

The majority of ascomycetous yeasts identified to the species or genus levels were assigned to the Hemiascomycetes lineage, Order Saccharomycetales (Table 3) [Kurtzman and Robnett, 1998]. Within the Debaryomyces/ Lodderomyces clade [Kurtzman and Robnett, 1998], Candida parapsilosis, Pichia guilliermondii and Debaryomyces hansenii were identified [Butinar et al., sub.].

Sequence analysis of the D1/D2 domains of the 26S rDNA revealed that the sequences of two strains (MZKI K-259 and K-269) differed (3 and 4 nucleotides, respectively) from the type strain of C. pseudorugosa. Only one representative, Protomyces inouyei, was found to belong to the "Archiascomycetes" lineage (Protomycetales) [Butinar et al., sub.].

1.3.2 Yeast Distribution in the Glaciers

Basidiomycetous yeasts were always recovered in low numbers (up to 25 x 103 CFU L-1) from supraglacial samples, whereas their counts increased approximately 15-fold (up to 4 x 105 CFU L-1) in samples from diverse cryokarst formations (Fig. 1) [Butinar et al., 2007]. In comparison, the ascomycetous yeasts were absent in supraglacial samples, however they were detected in subsurface samples (up to 103 CFU L-1) (Fig. 1) [Butinar et al., sub.].

The counts of basidomycetous yeasts from the subglacial samples were two orders of magnitude greater when compared with those recovered from supraglacial samples (Fig. 1) [Butinar et al., 2007]. A similar trend was observed for ascomycetous yeasts [Butinar et al., sub.]. The ratio shifted in favour of ascomycetous yeasts in particular when the aw of the media was lowered with NaCl (Fig. 2) [Butinar et al., sub.]. In subglacial ice, the highest mean values recorded for ascomycetous yeasts (with a maximum around 104 CFU L-1; Fig. 2) were obtained on media with 5% NaCl and 20% glucose (>300 CFU L-1) [Butinar et al., sub.]. On the remaining media, with few exceptions, the mean counts remained below 102 CFU L-1 (Fig. 1) [Butinar et al., sub.].

In this study, yeasts were isolated primarily from the subglacial ice at the bottom of the glaciers. Their frequency of occurrence was surprisingly high, reaching 4 x 103 CFU ml-1 [Butinar et al., 2007; sub.], while total bacterial counts in thawed subglacial ice was within the range 104-107 cells ml-1 [Foght et al., 2004]. It is worth noting that yeast counts in freshwater rivers and lakes of glacial origin are normally in the range of 0-0.25 cells ml-1 [Botha and Wolfaardt, 2000; Libkind et al., 2003). In contrast to subglacial bacteria, primarily associated with debris-rich subglacial ice [Skidmore et al., 2000], the highest yeast counts were obtained for the clear glacier ice, just above the ice with sediments [Butinar et al., 2007, sub.].

Taxonomic characterisation of the isolates showed that the dominant yeasts are of basidiomycetous affinity [Butinar et al., 2007, sub.] and belong to the same genera that occur frequently on Antarctic soils, Siberian permafrost and polar offshore sea-waters, where they represent the most important eukaryotic microorganisms [Jones, 1976; Vishniac and Klinger, 1986; Abyzov, 1993; Dmitriev et al., 1997; Vishniac, 2002; Onofri et al., 2004]. Cr. liquefaciens was the predominant yeast species and it was isolated primarily from sedimentfree subglacial ice, reaching counts as high as 4 x 106 CFU L-1 [Butinar et al., 2007].

Although strains in the Albidus clade are widely distributed in nature, both in natural and man-made environments, they have been isolated only sporadically from soil and plants in the polar regions [Middelhoven, 1997; Vishniac and Onofri, 2002; Onofri et al., 2004]. Since this species was reinstated not long ago [Fonseca et al., 2000], the ecology of Cr. liquefaciens (Fig. 3), apart its dominant presence in subglacial environments, is not well known. The second most frequent species, Rh. mucilaginosa, was always isolated together with Cr. liquefaciens, although it was the prevailing yeast in subglacial ice with sediment inclusions (up to 105 CFU L-1) [Butinar et al., 2007]. Interestingly, both Cryptococcus sp. and Rh. mucilaginosa were the only microorganisms that has been isolated from Antarctic Vostok ice core [Christner, pers. comm.] and at various depths of the Greenland ice sheet [Starmer et al., 2005]. Rh. mucilaginosa has been associated to human infections, besides it is rather common in diverse aquatic environments, including polar waters of glacial origin [Libkind et al., 2004]. Interestingly, it has been isolated from hypersaline waste water ponds in Israel as one of the two dominant microorganisms [Lahav et al., 2002] as well as from deep sea [Nagahama et al., 2001]. Cr. liquefaciens was detected with the highest numbers on MEA medium, while the highest counts for Rh. mucilaginosa were obtained on MEA with the addition of 5% NaCl [Butinar et al., 2007].

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