Microbial Ecology

Microbial Ecology Penicillium Mycobiota in Arctic Subglacial Ice Silva Sonjak1, Jens C. Frisvad2 and Nina Gunde-Cimerman1 (1) Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecˇna pot 111, SI-1000 Ljubljana, Slovenia (2) Center for Microbial Biotechnology, Biocentrum-DTU, Building 221, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark Received: 12 April 2006 / Accepted: 18 April 2006 / Online publication: 8 August 2006

Abstract

Fungi have been only rarely isolated from glacial ice in extremely cold polar regions and were in these cases considered as random, long-term preserved Aeolian deposits. Fungal presence has so far not been investigated in polar subglacial ice, a recently discovered extreme habitat reported to be inhabited exclusively by heterotrophic bacteria. In this study we report on the very high occurrence (up to 9000 CFU L–1) and diversity of filamentous Penicillium spp. in the sediment-rich subglacial ice of three different polythermal Arctic glaciers (Svalbard, Norway). The dominant species was P. crustosum, representing on the average half of all isolated strains from all three glaciers. The other most frequently isolated species were P. bialowiezense, P. chrysogenum, P. thomii, P. solitum, P. palitans, P. echinulatum, P. polonicum, P. commune, P. discolor, P. expansum, and new Penicillium species (sp. 1). Twelve more Penicillium species were occasionally isolated. The fungi isolated produced consistent profiles of secondary metabolites, not different from the same Penicillium species from other habitats. This is the first report on the presence of large populations of Penicillium spp. in subglacial sediment-rich ice.

Introduction

Certain species of fungi, one of the ecologically most successful eukaryotic lineages, can be considered as rare examples of eukaryotic extremophiles. Although they display different strategies from those encountered in prokaryotic extremophiles [39], they have been isolated from extreme environments such as hypersaline waters of the salterns [25], dry and hot deserts [1], deep sea basins [33], arid rocks [51], very low pH waters [28, 32] as well Correspondence to: Silva Sonjak; E-mail: [email protected] DOI: 10.1007/s00248-006-9086-0

& Volume 52, 207–216 (2006) & *

as in extremely cold polar environments. Cold-tolerant species have been primarily reported in connection with sub-Arctic vegetation [3, 12, 52], in snow and below snow-covered tundra [3, 38, 45, 53], and in permafrost [3, 8, 23, 49, 52] and offshore polar waters [4]. Very few studies describe their presence in Arctic glaciers. Viable fungi have, however, been isolated from Arctic and Antarctic ice, ranging in age from 10,000 and up to 140,000 years [2, 6, 7, 34, 35]. In these cases the few isolated filamentous fungi were considered as randomly entrapped fungal Aeolian propagules originating from close and distant locations. In our previous study we isolated fungi from different types of ice in the Arctic coastal environment in Svalbard, Spitzbergen, Norway. The isolations were performed by using media with low water activity (aw) to prevent osmotic imbalances due to sudden melting of ice. Surprisingly, considerably higher fungal counts than previously reported were detected in randomly sampled marginal glacial ice. The mycobiota was dominated by nonmelanized yeasts, whereas filamentous fungi, primarily of the genus Penicillium, comprised in certain cases up to 25% of all isolates [24]. Therefore, in the present study we focused on the systematic isolation of Penicillium spp. primarily from glacier basal ice, exposed at the glacier margins, but originating from subglacial environments of three different high Arctic polythermal glaciers. In addition, penicillia were isolated from the glaciers’ surface ice and diverse cryokarst formations (caves, moulins) within the three glaciers. This is the first report on the presence of large populations of Penicillium spp. in subglacial environments. We present their diversity, frequency of occurrence, selected physiological characteristics, and extrolite (secondary metabolite) profiles. Methods Site and Sampling. Kongsfjorden is one of the large Arctic fjords found on the western coast of Spitsbergen,

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Svalbard, located at 79-N, 12-E. It is 26 km long and 8 km wide and stretches from ESE to WNW at the Greenland Sea. Most of the drainage basin is covered by glaciers. Many pieces of glacial ice are floating in the fjord during most of the year. Annual mean temperature is around –5-C [27]. Sampling was performed in June and August 2001 [24] and in September 2003. Glacial ice was sampled in Conwaybreen, Kongsvegen, and Austre Loveńbreen glaciers. These glaciers have polythermal characteristics: cold (subfreezing) ice at the surface, margins, and terminus of the glacier. They are drained by a stable, open-channel system with ice temperature below zero in the ablation areas (80–100 m). Liquid water at the glacier bed derives from pressure melting, friction, surface flow, cryokarst formations, and groundwater infiltration. When unfrozen sediments and water freeze onto the basal glacial ice, this subglacial ice is transported to the glacier margins where it can be easily accessed and aseptically sampled [30]. Ice from the glaciers was sampled at the surface, in diverse caves and moulins, but primarily on glacier snout and margins where subglacial ice was exposed. Sediment-rich and overlying clear basal ice was sampled by chopping with surface-sterilized tools from the glacier margins. Ice samples were transported to the laboratory, where they were melted and processed as previously described [24]. In summer, glacial meltwater was directly sampled as well. Determination of Physicochemical Parameters of Ice In all samples of melted ice and and Water Samples.

glacial meltwater pH (ISO 10523:1994E, electrochemical method using a pH meter), Na+, Mg2+, and K+ cation concentrations [inductively coupled plasma atomic emission spectrometry (ICP–AES), Thermo Jarell Ash, USA], total phosphorous content (ISO 6878/1:1986E, spectrometrically after mineralization with persulfate), Kjeldahl nitrogen [trivalent negative nitrogen; DIN EN 25663 (ISO 5663:1984), after mineralization with selenium], ammonium (ISO 5664:1984, distillation and titration method), and water activity (aw; DECAGON CX-1 Water Activity System, Campbell Scientific Ltd.) were determined [25]. Isolation and Preservation of Strains. The surface layer of glacial ice was aseptically melted at room temperature and discarded. The ice sample was than transferred to another sterile container, melted, and immediately filtered in aliquots of 100 mL using Millipore membrane filters (0.45 mm). Membrane filters were placed on solid media, a drop of the original sample water was applied on the membrane, and particles trapped on the filter surface were dispersed over the whole agar plate with a Drigalski spatula [25]. Mineral ice inclusions were collected aseptically and 1 g of this sediment was spread

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directly over the agar plate with a Drigalski spatula. At least four and up to 10 replicates of each general or selective agar medium and incubation temperature were used in each method. All plates were incubated up to 14 weeks at 10-C and 24-C. After incubation, colonyforming units (CFU) were counted on all the replicates, average CFUs calculated and expressed as CFU per liter for meltwater and CFU per gram for the direct spreading of the sediment. The total CFU per liter or CFU per gram for each Penicillium sp. in a particular sample was obtained by summing the CFU per liter or CFU per gram for all the media and incubation temperatures used. All isolated strains are maintained in the EXF Culture Collection of the Department of Biology, Biotechnical Faculty, University of Ljubljana, Slovenia, and in IBT Culture Collection of Center for Microbial Biotechnology, BioCentrum-DTU, Technical University of Denmark. Media. The general-purpose isolation and enumeration media used were dichloran rose bengal chloramphenicol agar (DRBC, aw = 1.0) [29], malt extract agar (MEA, aw = 1.0) [40], and a medium for detection of moderate xerophiles, dichloran 18% glycerol agar (DG18, aw = 0.946) [26]. Because ice formation results in little biologically available water, additionally selective media with high concentrations of salt or sugar and thus low water activity were used [24]: malt yeast 10% glucose and 12% NaCl agar (MY10–12, aw = 0.88) [40], malt yeast X% glucose agar, X = 20, 35, 50 (MY20G, aw = 0.941; MY35G, aw = 0.915 [24]; MY50G, aw = 0.89 [40]), and malt extract agar with X% NaCl, X = 5, 10, 15, 17, 24, 30 (MEAXNaCl, aw = 0.951 – 0.782) [24]. For prevention of bacterial growth, chloramphenicol (50 mg L–1) was added to all media. Water activities of the media were determined using the DECAGON CX-1 Water Activity System (Campbell Scientific Ltd, Shepshed, UK). Taxonomic Identification. Isolates of the genus Penicillium were identified to the species level by comparison with ex-type cultures using morphology, physiology, and extrolite profiles [16, 17, 20, 43, 44]. Macroscopic parameters were observed and colony diameters measured on media used for identification of Penicillium spp.: Czapek yeast autolysate agar (CYA), Czapek yeast autolysate agar with 5% NaCl (CYAS), malt extract agar (MEA), yeast extract sucrose agar (YES), creatine sucrose agar (CREA), and nitrite sucrose agar (NO2). Strains were inoculated in three-point cultures and incubated for 7 days in the dark at 25-C. A duplicate set of CYA plates was incubated in the dark at 15-C. Degree of sporulation was determined on YES and described as none or very thin and poor sporulation (0), sporulation in the center of the colony (1), strong

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sporulation on more than 90% of the colony (2) [16]. Microscopic parameters were determined on MEA and CYA incubated at 25-C. For selected Penicillium isolates (P. bialowiezense: EXF-1091, EXF-1102, EXF-1129; P. species 1: EXF-1307, EXF-1319; P. species 2: EXF-1010; P. species 3: EXF-1190, EXF-1481) DNA was extracted with mechanical lysis [22] from ca. 200 mg of 4-day-old cultures. Amplification of partial b-tubulin gene was carried out using primers T1 and T22, as described by O’Donnell and Cigelnik [37]. Automated DNA sequencing was provided by Macrogen Company (Korea). Sequences were deposited in GenBank under accession numbers DQ486640–DQ486647. High-Performance Liquid Chromatography For high-performance liquid chromatogAnalysis.

raphy (HPLC) extraction plugs (6 mm in diameter) were cut from the Penicillium sp. colonies growing on CYA (three plugs) and YES (three plugs) media. Plugs from each medium were transferred to a 1.5-mL disposable autosampler screw-cap vial with 500 mL of the solvent mixture, methanol–dichloromethane–ethyl acetate (3:2:1), containing 0.5% (v/v) formic acid. Extracts were obtained ultrasonically for 60 min and then transferred to a clean vial, the organic phase was evaporated to dryness in a fume hood and with centrifugation in vacuum. The residues were redissolved ultrasonically for 10 min in 500 mL methanol. All samples were filtered through 0.45-mm Minisart RC4 filters (Sartorius, Germany) into clean vials before analyses [48]. The HPLC analyses were based on the methods of Frisvad and Thrane [18, 19], as modified by Smedsgaard [48]. The analyses were performed on an A1100 HPLC (Agilent, Germany) using 5 mL injections. The metabolites were detected at 210 nm, but each peak detected was characterized by its UV spectrum from 200 to 600 nm (diode array detection) with a peak width of 0.2 min. Separations were done on a 2 100-mm Luna2 OOD4251-BO-C18 column (Phenomenex, Germany) with a C18 precolumn, both packed with 3-mm particles. The column was maintained at 40-C. A linear gradient starting from 85% water (A) and 15% acetonitrile (B) going to 100% acetonitrile in 20 min, then maintaining 100% acetonitrile for 5 min, was used at a flow rate of 0.4 mL min–1. Both eluents contained 0.005% (v/v) trifluoroacetic acid (TFA). Chemicals used were Merck or Riedel-de Hoen (acetonitrile) analytical grade. An alkylphenone retention index was calculated for each peak detected. The metabolites were identified by comparison with standards and by their characteristic UV spectra. Multivariate Statistical Analyses. Data were analyzed by means of correspondence analysis, an explorative computational method for study of associations among objects and variables, using the program Statistica

(StatSoft\). A low-dimension (plane) projection of the data was displayed to reveal correlation between objects (habitats—six different glacial ice and water samples) and variables (the 22 respective Penicillium spp. with their total counts per liter; Table 2).

Results Physicochemical Conditions at the Isolation Sites. pH of the melted glacial ice within individual glaciers varied between 7.1 and 7.4. The cation concentrations in glacial ice ranged from 5 to 340 mg kg–1 for sodium, from 20 to 310 mg kg–1 for potassium, and from 70 to 550 mg kg–1 for magnesium. The highest phosphorus content was determined in Kongsvegen glacier ice sample, 9 mg kg–1; otherwise the content was low, G1–3 mg kg–1. The Kjeldahl nitrogen content ranged from 3.45 to 7.1 mg L–1, whereas ammonium nitrogen content ranged from 0.8 to 1.57 mg L–1. The water activity of melted ice ranged between 0.95 and 0.98, indicating a salt concentration of less than 5%. Frequency of Occurrence of Penicillum spp. in the The highest penicillia CFU counts (52% of Glaciers.

the total) were detected on the DG18 enumeration medium with lowered water activity (aw = 0.946) at 10-C and 24-C, whereas on selective media the CFU counts were considerably lower (up to 10% of total); however, the highest CFUs were obtained on media containing 20% glucose (aw = 0.941), 35% glucose (aw = 0.915), and 5% NaCl (aw = 0.951), respectively (Table 1). Statistical analysis of the data performed with Student’s t test showed that means of the two temperatures were not significantly different for most of the media, except for DG-18 and media with 10–17% NaCl. Regardless of the medium used, the highest counts of penicillia were detected in sediment-rich basal ice with mineral inclusions deriving from the glacier bed (Table 2).

Table 1. Penicillia obtained on different media at 10--C or 24--C, expressed as percentage of total fungal colony-forming units

Medium MEA DRBC DG18 MY20G MY35G MY50G MY10-12 MEA5NaCl MEA10NaCl MEA15NaCl MEA17NaCl MEA24NaCl MEA30NaCl

aw

% (10-C)

% (24-C)

1 1 0.946 0.941 0.915 0.890 0.916 0.951 0.924 0.881 0.861 0.828 0.782

3.6 0.2 30.2 5.5 3.0 2.0 1.6 3.2 0.1 0.1 0.01 0 0

5.8 3.5 21.6 4.4 5.8 2.1 2.3 3.7 0.5 0.8 0.04 0 0

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Table 2. Frequencies of occurrence of isolated Penicillium species in Conwaybreen (C), Kongsvegen (K), and Austre Loveńbreen (A)

glaciers Filtration method (CFU L–1) Ca

Penicillium spp.

Cb

d

P. bialowiezense P. brasilianum P. brevicompactumd P. chrysogenumd P. communed P. corylophilumd P. crustosumd P. discolord P. echinulatumd P. expansumd P. glabrumd P. lanosumd P. nordicumd P. olsoniid P. palitansd P. polonicumd P. roquefortid P. solitumd P. thomiid P. tulipae P. species 1d P. species 2 P. species 3d Total

Direct spreading of the sediment (CFU g–1)

Ka

Kb

Kc

Aa

88

161

11,280

410

C

K

A

160 2

2 2 783

9000

3 48 7 7 552 10

14 65 1180

828 548 1720

1600 248

100

25 169

36

1 2

3 42

105 10

20

26 10 106 35

2 26

102

5 42 2 53 28

1 1 1

40 10 40 200 550

600 1

5 43

200

5 935

2480

1

20 60

1

4429

203

1 58 1 15

10 1069

9112

13,000

301

3

a

Clear glacial ice samples. Sediment rich glacial ice samples. Outflow water samples d Penicillia isolated also on media with aw from 0.9 to 0.78. b c

The samples with sediment from Kongsvegen glacier contained, on the average, 2480 CFU L–1 of penicillia. Surprisingly high values were obtained in sediment-rich basal ice samples of Conwaybreen glacier, where counts in individual samples increased up to 9112 CFU L–1. Samples of clear ice, overlying the sediment-rich subglacial ice, contained, on the average, 1000 CFU L–1 of penicillia, although counts in individual samples increased up to 4429 CFU L–1. However, the highest counts of penicillia, up to 13,000 CFU L–1, were detected in glacier outflow water, which contained a very fine mineral sediment. The counts of penicillia in the surface layers of glacial ice of all three glaciers were considerably lower, only up to 50 CFU L–1. Identification of Penicillium spp. Based on Extrolite Production, Morphology, and Selected Physiological Selected isolates of each Penicillium Characteristics.

species were tested for their extrolite production. Extrolite profiles for each isolate were consistent within each species and similar to those reported by Samson et al. [43], Frisvad and Samson [16], and Frisvad et al. [17] and not different from the extrolite profiles of isolates of the same Penicillium species from other habitats. Table 3 shows profiles of extrolites produced by isolated Penicillium

species along with some relevant morphological and physiological characteristics. Diversity of Penicillium spp. and Distribution in the Identification of Penicillium spp. revealed Glaciers.

that basal, sediment-rich ice, with the highest penicillia counts, is colonized by only few species. The dominant species, isolated from samples from all three glaciers, was P. crustosum. In certain cases it represented up to 95% of all isolated Penicillium strains with counts up to 9000 CFU L–1. In others, the proportion was less, but never below 30%. P. crustosum was found as well by direct spreading of the mineral inclusions of all three glaciers. Besides P. crustosum, P. bialowiezense was the only species detected in high counts in the glacier outflow water. Although it was present also in samples of clear ice, overlying the sediment-rich basal ice, the average counts were considerably lower (250 CFU L–1). The second species that appeared in sediment-rich ice with high counts (550 CFU L–1) was P. thomii, although it was detected only in samples of one glacier and not in the clear, overlying ice (Table 2). Besides the two above-mentioned dominant species, four more Penicillium species (P. chrysogenum, P. solitum, P. bialowiezense, and P. species 1), were relatively fre-

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Table 3. Selected morphological and physiological characteristics of isolated Penicillium species including profiles of extrolites produced

Species (Series)

S

CREA

NO2

Cs

Ct

Extrolites

Subgenus: Penicillium, Section: Chrysogena P. chrysogenum (Chrysogena) 2 Weak

Poor

1.0

0.7

Roquefortine C, meleagrin, chrysogine, xanthocillins, secalonic acid D

Subgenus: Penicillium, Section: Coronata P. bialowiezense (Olsonii) 2

Poor

Poor

1.1

1.4

Two Raistrick phenols, quinolactacin, mycophenolic acid, mycochromenic acid, asperphenamate, xanthoepocin Four Raistrick phenols, mycophenolic acid, mycochromenic acid, brevianamide A and B, asperphenamate Verruculone, asperphenamate, 2(4-hydroxyphenyl)-2-oxo acetaldehyde oxime, two breviones

P. brevicompactum (Olsonii)

2

Poor

Good

0.9

0.8

P. olsonii (Olsonii)

2

Weak

Good

0.8

0.7

Good

0.9

0.8

Patulin, chaetoglobosin A, B, and C, communesin A and B, roquefortine C, expansolide A and B, citrinin

Subgenus: Penicillium, Section: Penicillium P. expansum (Expansa) 2 Very good or poor Subgenus: Penicillium, Section: Roqueforti P. roqueforti (Roqueforti) 2

Very good

Good

1.2

0.7

Andrastin A, citreoisocoumarin, mycophenolic acid, PR toxin, roquefortine C and D, isofumigaclavine A and B

Subgenus: Penicillium, Section: Viridicata P. commune (Camemberti) 2/1

Very good

Good

1.0

0.9

Cyclopaldic acid, chromanols, palitantin, rugulovasine A and B, cyclopiazonic acid. Penitrem A–G, terrestric acid, viridicatol, andrastin A, viridicatic acid, cyclopeptin, dehydrocyclopeptin, cyclopenin, cyclopenol, viridicatin, roquefortine C–E Palitantin, cyclopiazonic acid, fumigaclavine A and B, cyclopeptin, dehydrocyclopeptin, cyclopenin, cyclopenol, viridicatin, viridicatol Terrestric acid, chrysogine, roquefortine C, meleagrin, neoxaline, penitrem A Palitantin, cyclopeptin, dehydrocyclopeptin, cyclopenol, cyclopenin, viridicatol, viridicatin, chaetoglobosin A, B, and C, daldinin D Palitantin, territrem B and C, arisugacins, cyclopeptin, dehydrocyclopeptin, cyclopenin, cyclopenol, viridicatin, viridicatol Compactin, cyclopeptin, cyclopenin, cyclopenol, solistatin, viridicatol, viridicatin, dehydrocyclopeptin Verruculone, ochratoxin A and B, sclerotigenin, viridic acid, anacine Penicillic acid, verrucosidin, puberuline, pseurotins, verrucofortine, cyclopeptin, viridicatol, cyclopenin, dehydrocyclopeptin, anacine, cyclopenol, rugulosuvine, 3-methoxyviridicatin, leucyltryptophanyldiketopiperazine

P. crustosum (Camemberti)

2

Very good or weak

Poor

1.2

0.7

P. palitans (Camemberti)

2/1

Very good

Good

0.9

1.0

P. tulipae (Corymbifera)

2

Poor

Weak

1.2

0.8

P. discolor (Solita)

2

Good

Poor

1.1

0.9

P. echinulatum (Solita)

2

Very good

Weak

0.9

0.9

P. solitum (Solita)

2/1

Good

Weak

0.8

0.9

P. nordicum (Verrucosa)

2/1

Weak

Good

0.8

1.2

P. polonicum (Viridicata)

2

Good

Weak

0.9

0.9

1–2 1–2

Weak Weak

Good Good

1.6 1.3

0.4 0.9

1–2

Weak

Very good

1.4

0.4

Subgenus: Furcatum P. corylophilum (Citrina) P. lanosum (Lanosa) P. brasilianum (Simplicissima)

Citreoisocoumarin Kojic acid, griseofulvin, sclerotigenin, cycloaspeptide A and D Penicillic acid, verrucologen, fumitremorgin A and B, viridicatum toxin

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Table 3. Continued Species (Series)

S

Subgenus: Aspergilloides P. glabrum (Glabra)

CREA

NO2

Cs

Ct

Extrolites

Citromycetin, sulochrin, bisdechlorogeodin, asterric acid, PI-4

2

Weak

Good

1.2

0.6

P. thomii (Glabra) Penicillium sp. Species 1 Species 2

2

No

Very good

1.4

0.7

2 2

Good Very good

Weak Very good

5.3 1.1

0.6 0.9

Species 3

0–1

Weak

Weak

1.5

1.1

Xanthoepocin Viridicatol, viridicatin, cyclopenol, cyclopeptin, cyclopenin, dehydrocyclopeptin

S, spore production level; CREA, growth on CREA medium; NO2, growth on NO2 medium; Cs, CYA/CYAS growth ratio; Ct, CYA 15-C/CYA 25-C growth ratio.

The Penicillium species diversity in clear ice overlying the ice with mineral inclusions was higher in comparison. The dominant Penicillium spp. in the clear ice was again P. crustosum, followed by P. bialowiezense, which prevailed in the glacier outflow water. Four more penicillia were detected with high counts (up to 830 CFU L–1): P. chrysogenum, P. polonicum, P. commune, and P.

quently isolated from the sediment-rich basal ice and the sediments of at least two sampled glaciers, although with considerably lower counts (up to 200 CFU L–1) (Table 2). In ice with mineral inclusions, additional six species were detected with counts up to 40 CFU L–1 : P. brevicompactum, P. glabrum, P. nordicum, P. olsonii, P. polonicum, and P. roqueforti (Table 2).

Dimension 2; Eigenvalue: ,43387 (25.56% of Inertia)

1.0

ech, lan, pal, sp.2

sp.2 ech lan pal C

0.5 0.0 -0.5

sol Cs nor cru tho Ks roq bre ols cor sp.1 K sp.3 gla

sol, nor, tho, roq, bre, cru

Kwbia

ols, cor, sp.1 gla, sp.3

Group 3

Group 1

-1.0 -1.5 tul A

-2.0 -2.5

chr pol

-3.0

dis com exp

Group 2

-3.5 -1.0

-0.5

0.0

0.5

Dimension 1; Eigenvalue: ,72640 (42,80% of Inertia)

1.0

Samples (glaciers) Penicillium sp.

Figure 1. Correspondence analysis of frequencies of Penicillium spp. isolated from different samples originating from three glaciers.

Samples of clear glacial ice: C, Conwaybreen; K, Kongsvegen; A, Austre Loveńbreen glacier. Samples of glacial ice with sediment: Cs and Ks. Kw, Kongsvegen outflow water; bia, P. bialowiezense; bre, P. brevicompactum; chr, P. chrysogenum; com, P. commune; cor, P. corylophilum; cru, P. crustosum; dis, P. discolor; ech, P. echinulatum; exp, P. expansum; gla, P. glabrum; lan, P. lanosum; nor, P. nordicum; ols, P. olsonii; pal, P. palitans; pol, P. polonicum; roq, P. roqueforti; sol, P. solitum; tho, P. thomii; tul, P. tulipae; sp.1, sp.2, sp.3, Penicillium species 1, 2, and 3, respectively.

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discolor. Except for P. discolor all the listed species were also detected in the sediment-rich ice, although their occurrence was considerably lower. Four more species appeared in counts up to 110 CFU L–1: P. palitans, P. echinulatum, P. expansum, and P. species 1. The diversity of Penicillium further increased by 10 different species: P. glabrum, P. olsonii, P. solitum, P. corylophilum, P. nordicum, P. brevicompactum, P. lanosum, P. tulipae, P. species 2, and P. species 3. None of these exceeded 40 CFU L–1 in any sample (Table 2). Correspondence Analysis of Penicillium spp. The frequencies of Penicillium spp. isolated from ice samples of three glaciers were analyzed by correspondence analysis to statistically determine the correlation between the species and samples. The first two dimensions described approximately 68% of the data variation presenting grouping of all the isolates into three distinct groups (Fig. 1). The first and the largest group showed correlation among P. glabrum, P. crustosum, P. corylophilum, P. nordicum, P. olsonii, P. solitum, P. brevicompactum, P. thomii, P. roqueforti, P. palitans, P. lanosum, P. echinulatum, P. species 1, P. species 2, and P. species 3 with Kongsvegen and Conwaybreen glacier ice samples. Some of these species grouped more closely with ice samples with sediment. The second group was composed of Penicillium species isolated in high counts only from Austre Loveńbreen glacier—P. tulipae, P. chrysogenum, P. polonicum, P. discolor, P. commune, and P. expansum— thus showing correlation between these species and the glacier. Due to the very high number of P. bialowiezense isolates in the glacier outflow water, this species was located separately. The third group thus revealed correlation between P. bialowiezense and glacier outflow water (Fig. 1).

Discussion

The cosmopolitan genus Penicillium comprises more than 225, mainly food-, soil-, or airborne species [41]. The genus shows tolerance for cold environments as demonstrated by the fact that many species grow on food preserved in refrigerators [40] or are isolated from alpine, tundra [9], and even polar soil [14, 36]. Being often psychrotolerant and sturdy as well as having prolific production of conidia, it is not surprising that penicillia are among the few viable fungi isolated from glacial ice cores even up to 38,600 years old [2]. Spores and fragments of mycelia trapped within ice are exposed to several stressful factors such as freezing, desiccation, and starvation. However, ice also protects microorganisms from UV irradiation, oxidation, and chemical damage [42].

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Recent investigations have shown that life in glacial ice is more dynamic on the geomorphological level than previously assumed. Particularly polythermal glaciers, known for massive surface ablations, are capable of rapid movements [10, 11]. These sudden shifts, which may happen even daily, can relocate cryokarst formations and generate frictional and geothermal melting of ice at the glacier base. Thus, these subglacial waters originate as groundwater, basal meltwater, and additionally as supraglacial waters that have reached the glacier bed through cryokarst formations. These subglacial waters flow and interact with rocks and sediments that underlie the ice. When they freeze onto the basal glacial ice they contain high solute and suspended mineral sediment concentrations deriving from the glacier bed [13]. These processes create subglacial environments, until recently considered abiotic. However, recent studies have revealed prokaryotic microbial communities dominated by aerobic heterotrophic Betaproteobacteria [13]. They have been mainly associated with sediment particles [13, 21, 31, 46, 47]. Thin films of liquid water around embedded mineral grains have been reported as potential microniches (J. R. Reeve, pers. comm.). So far there were no reports on the presence of fungi or any other eukaryotic microorganisms in subglacial environments. Our study revealed that surface ice contains only up to 50 CFU L–1 of penicillia, whereas subglacial ice harbors a surprisingly rich diversity and high occurrence of penicillia, equivalent to subglacial bacteria isolated primarily from subglacial debris-rich ice [47]. It seems that penicillia were present in the soils and sediments that glaciers overrode and became selectively enriched through the processes of melting and freezing that occurred at the glacier bed. The dominant species, both in sediment-rich and in overlying clear glacial ice, is the cosmopolitan P. crustosum, which is typically reported as being foodborne. This species was isolated from all three sampled glaciers and represents one of the two dominant species in the glacier outflow water. P. crustosum strains, isolated from the Conwaybreen glacier, differed from all other Arctic strains and also from known strains isolated worldwide. Although growth on CREA medium is one of the basic characteristics of this species [16], these isolates showed surprisingly weak growth. However, they did not differ in other tested chemical, physiological, and morphological characteristics (Table 3) [50]. In the clear glacial ice only few Penicillium species, P. commune, P. discolor, and P. polonicum, were detected with significantly higher counts than in the sedimentrich ice. These species might have originated in the sediment but they may also have been washed into the subglacial environment from the glacial surface. The only species besides P. crustosum that was detected with high counts in the glacial outflow water was P. bialowiezense.

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Although the total CFU number of penicillia obtained from Conwaybreen and Kongsvegen glaciers was much higher in subglacial ice, the diversity of Penicillium species was significantly lower [Simpson’s index of diversity (1 – D) = 0.225] in comparison to overlying clear glacial ice (1 – D = 0.545). The three Penicillium taxa P. species 1, P. species 2, and P. species 3 that were discovered in the glacial ice of the three glaciers appear to be new to science and will be described in a separate article. Of all the species of Penicillium recovered, most species found in subglacial ice are also among the very common foodborne penicillia, including P. crustosum, P. polonicum, P. discolor, P. commune, P. palitans, P. nordicum, P. solitum, P. echinulatum, P. expansum, P. brevicompactum, P. chrysogenum, and P. tulipae. In contrast, these species are very rare in soil [16]. The only soilborne forms of Penicillium found in the subglacial ice were P. brasilianum from series Simplisissima, P. lanosum from series Lanosa, P. corylophilum from series Citrina, (all from Penicillium subgenus Furcatum) and P. glabrum and P. thomii from series Glabra, subgenus Aspergilloides. Thus, penicillia that are primarily foodborne are clearly much more prevalent in subglacial ice than soilborne penicillia. A similar observation was made for penicillia found in the Antarctic, which were isolated from soil or bird nest material on Antarctica. McRae et al. [36] reported on P. aurantiogriseum, P. brevicompactum, P. chrysogenum, P. commune, P. echinulatum, P. expansum, P. palitans, and P. solitum from subgenus Penicillium, whereas they found P. antarcticum, P. corylophilum, P. fellutanum, P. glabrum, P. janthinellum, P. jensenii, and P. waksmanii of the more soilborne type of penicillia from the subgenera Aspergilloides and Furcatum. This mycobiota is actually rather similar to that found by us in the Arctic subglacial ice. The foodborne penicillia from subgenus Penicillium are extremely rarely found in isolations from soil regardless of climatic zone [5, 15], but apparently they are very common in the polar regions both in soil (Antarctica) and in ice samples (Arctic). Several of the species from subgenus Aspergilloides and Furcatum, including P. glabrum, P. corylophilum, and P. antarcticum, have also been found regularly on foods, whereas some of the fungi reported from Antarctica, such as P. janthinellum, P. jensenii, and P. waksmanii, are common fungi in soil worldwide. These common soilborne types were not found in the subglacial samples. In addition to these species, some new species from soil in Greenland or alpine habitats were also found in the subglacial samples. Penicillia strains that populate glacial ice must be physiologically adaptable and able to retain their viability throughout the dynamic processes of ice melting and freezing and extremes in pressure. Such enrichment would select for Penicillium populations best adapted to

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dark, cold, oligotrophic environments with shifting osmotic pressures. These predictions were confirmed by our isolation of the highest Penicillium counts on the enumeration medium used for the isolation of moderate xerophiles at 10-C (Table 1), whereas almost all penicillia were isolated additionally also on media with even lower water activity (Table 2). The dominant species, P. crustosum, and the other frequently occurring species apparently can propagate in this extreme habitat due to their sturdy nature, ability to withstand osmotic imbalances, and to adjust to nutritionally poor habitats. Besides, they are characterized by a high production of small-sized conidia, able to spread and colonize pockets and microchannels of the ice. In cold and oligotrophic conditions, small size could be advantageous for more efficient nutrient uptake and for the occupation of microenvironments. Our results indicate that subglacial environments may represent a significant, previously unrecognized reservoir not only of prokaryotic, but also of eukaryotic diversity. Penicillia enclosed in ice will be released during periods of glacial melts and thus contribute to the biogeochemical processes and biodiversity in polar regions.

Acknowledgments

The work in Ny-A˚lesund was funded by EU Large-Scale Facility Fund. Laboratory experiments were partially supported by a FEMS Fellowship and partly by EU under the Major Research Infrastructure (MRI) Centre for Advanced Food Studies (LMC). We also thank the Danish Technical Research Council for support of the Program for Predictive Biotechnology and Center for Microbial Biotechnology.

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