Yeasts from an oligotrophic lake in Patagonia (Argentina) diversity

7/28/2019 Yeasts from an oligotrophic lake in Patagonia (Argentina) diversity

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R E S E A R C H A R T I C L E

Yeasts from an oligotrophic lake in Patagonia (Argentina): diversity,

distribution and synthesis of photoprotective compounds and

extracellular enzymes

Luciana R. Brandao1, Diego Libkind2, Aline B.M. Vaz1, Llia C. Esprito Santo1, Martn Moline2,Virginia de Garca2, Maria van Broock2 & Carlos A. Rosa1

1Departamento de Microbiologia, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil; and 2Laboratorio de Microbiologa

Aplicada y Biotecnologa, Instituto de Investigaciones en Biodiversidad y Medioambiente, UNComahue-CONICET, Bariloche, Rio Negro, Argentina

Correspondence: Diego Libkind,

Laboratorio de Microbiologa Aplicada y

Biotecnologa, Instituto de Investigaciones en

Biodiversidad y Medioambiente,

UNComahue-CONICET, Quintral 1250,

Bariloche 8400, Rio Negro, Argentina. Tel.:

1154 2944 428505; fax: 1154 2944

423111; e-mail: [email protected]

Received 7 September 2010; revised 12

November 2010; accepted 12 November 2010.

Final version published online 11 January 2011.

DOI:10.1111/j.1574-6941.2010.01030.x

Editor: Gary King

Keywords

yeasts diversity; oligotrophic lake; carotenoids;mycosporines; extracellular enzymes.

Abstract

Nahuel Huapi (NH) Lake is an oligotrophic temperate lake of glacial origin with

high transparency, surrounded by well-developed forests and located at San Carlos

de Bariloche, Nahuel Huapi National Park, in Patagonia, Argentina. In this lake, we

characterized yeast distribution and diversity along a south-to-north transect and

established a relationship between the ability to produce photoprotective com-

pounds (PPCs) (carotenoid pigments and mycosporines) and the occurrence of

yeast at different collection points. Subsurface water samples were filtered for yeast

isolation. Total yeast counts ranged between 22 and 141 CFU L1, and the highest

values corresponded to the most impacted sites. Littoral sites had a low proportion

of yeast-producing PPCs and this group prevailed in pelagic sites. This is probably

a result of the high transparency of the water and the increased UV exposure. The

yeast community from NH Lake showed a high species richness and a uniform

distribution of taxa between pelagic and border collection points. Yeasts were

identified as belonging to 14 genera and 34 species. Rhodotorula mucilaginosa and

Cryptococcus victoriae were the most frequently found species, representing 14.4%

and 13.6% of the total yeast isolates, respectively. Most of the yeast isolatesdemonstrated at least one extracellular enzymatic activity (mainly cellulase and

lipase activities), which suggested that these microorganisms are metabolically

active in the lake.

Introduction

Yeasts occur in a variety of freshwater systems, including

eutrophic to ultra-oligotrophic lakes, lagoons, rivers,

groundwater, glaciers and glacial meltwaters (Hagler &

Ahearn, 1987; Libkind et al., 2003; Nagahama, 2006; de

Garca et al., 2007; Medeiros et al., 2008; Brandao et al.,

2010). The yeast diversity in these ecosystems is highly

affected by a variety of abiotic and biotic factors, such as

temperature, pressure, UV radiation (UVR), salinity, fauna,

flora, soil run-off and anthropogenic effluents. The condi-

tions prevailing in these natural habitats determine the

metabolic activity, growth and survival of the yeast popula-

tions (Deak, 2006). Despite the fact that yeasts are common

in different aquatic systems, our knowledge of the ecology of

freshwater yeasts remains incipient. Notable gaps in our

knowledge are mainly related to the factors that drive the

distributional patterns, diversity and functional significance

of yeasts in aquatic systems (Spencer & Spencer, 1997).

The majority of the studies on yeasts from freshwater

ecosystems have focused on the association of yeasts with

contaminated waters (de Almeida, 2005; Hagler, 2006;

Nagahama, 2006; Medeiros et al., 2008; Brandao et al.,

2010). These studies identified the presence of opportunistic

yeast pathogens belonging to species such as Candida

albicans, Candida parapsilosis, Candida krusei, Candida

guilliermondii, Candida glabrata and Candida tropicalis in

polluted water ecosystems. These yeasts are part of the faecal

microbiota of many animals, including humans (Medeiros

et al., 2008). Less research has been performed on yeast

FEMS Microbiol Ecol 76 (2011) 113 c 2011 Federation of European Microbiological Societies

Published by Blackwell Publishing Ltd. All rights reserved

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occurrence in pristine aquatic environments. The oligo-

trophic to ultra-oligotrophic lakes of glacial origin in

Patagonia, Argentina, are examples of this type of minimally

polluted water ecosystem (Daz et al., 2000). Lakes near the

city of San Carlos de Bariloche, which is located within

Nahuel Huapi National Park (NHNP), can be considered to

be pristine and have minimal anthropogenic influence.These Patagonian natural lakes are typically exposed to

elevated UVR due to their transparency and ultra-oligo-

trophic conditions (Villafane et al., 2001). Recent reports

have demonstrated that UVR is a selective factor that

favours the occurrence of more UV-resistant yeast species

in these lakes (Libkind et al., 2009). To minimize UV-

induced damage, several yeast species synthesize antioxi-

dants and UV sunscreen compounds such as carotenoids

and mycosporines (Libkind et al., 2009; Moline et al., 2009,

2010b).

The biodiversity of basidiomycetous yeasts in certain

lakes in Andean Patagonia (Argentina) was investigated by

Libkind et al. (2003, 2005b, 2009). These studies exclusively

focused on carotenoids and mycosporine-producing basi-

diomycetous yeasts and detected several novel species,

including Rhodotorula meli, Sporidiobolus longiusculus, Sporo-

bolomyces patagonicus and Cystofilobasidium lacus-mascardii

(Libkind et al., 2005a, 2009, 2010). Studies of the entire

cultivable yeast community of these pristine lakes have not

been conducted previously. In this work, we studied the

diversity and frequencies of yeast species along a south-to-

north transect of Nahuel Huapi (NH) Lake in Andean

Patagonia. In addition, we investigated extracellular enzyme

production and photoprotective compound (PPC) synthesis

by yeast species and correlated our results with theirdistribution in the lake.

Materials and methods

Collection area and yeast sampling

NH Lake is the largest water body in NHNP (411S711W),

Patagonia, Argentina, with an area of 557 km2 and a max-

imum depth of 465 m (Calcagno et al., 1995). It is consid-

ered an oligotrophic temperate lake of glacial origin

(Pedrozo et al., 1993; Daz et al., 2000) and has an elevated

transparency (20.9 m) (Zunino & Diaz, 2000). Its waters

have a very low dissolved organic carbon (o 0.5gm3),

which results in a low vertical attenuation of UVR in the

water column (Morris et al., 1995). Most energetic wave-

lengths of UVB radiation can occur down to 10-m deep,

which means that organisms living or migrating to the

upper 10 m would be affected by this deleterious band or

the indirect effect of UVR (i.e. reactive oxygen species

generation) (Balseiro et al., 2008).

The city of San Carlos de Bariloche (c. 100000 inhabi-

tants) is located on the southern coast of the lake, and this

coast has a significant periphyton growth (Baffico, 2001).

Although the southern coast of the lake is strongly influ-

enced by urban discharges, no relevant industrial or exten-

sive agricultural activities exist in the area (Guevara et al.,

2002), tourism being the main commercial activity. Thenorthern coast of the lake is surrounded by a dense native

forest ofNothofagus dombeyi and Austrocedrus chilensis and

the anthropogenic impact is negligible.

On 18 December 2007, five independent 300400-mL

water samples were collected in sterile bottles from seven

sites (NH0, 1, 2, 3, 4, 5 and 6) that were located along a

south-to-north transect of the lake (Fig. 1, Table 1). Samples

were collected from the subsurface at a depth of c. 30cm.

The first sampling site (NH0) was located in the city coast, at

4m from the edge of the lake close to the Nireco river

discharge that collects runoff and urban discharges from the

city. Point 1 (NH1) was located 30 m from the city border, at

the western Nireco river inflow, and point 6 was located

approximately 5 m from the northern edge of the lake, near

a forest ofN. dombeyi and A. chilensis (Fig. 1). The distances

between sampled points (NH1NH6) were approximately

1.3 km. The samples were stored at 5 1C and processed until

a maximum of 10 h after collection. Sampling was per-

formed at midday (12:0013:00 hours) of a cloudless and

windless day. Water temperature was recorded in situ;

pH and conductivity were measured in the laboratory with

a 3310 JENWAY metre and an Orion 135 apparatus,

respectively.

UV irradiance data acquisition

The irradiance at different wavelengths in the UV range

(305, 320, 340 and 380 nm) was acquired for the period

1620 November 2007; the total-day doses for each wave-

length was calculated and then averaged. The maximum

irradiance detected for each day was registered and then

averaged. Ground level irradiance data were obtained by

means of a radiometer GUV 511 (Biospherical Instruments)

and provided by Laboratorio de Fotobiologa (INIBIOMA-

CONICET, UNComa, Bariloche). The radiometer was

placed 12 km from the city of S.C. Bariloche and 5 km from

the NH Lake.

Yeast isolation and quantitative analysis

Variable volumes of water (300400 mL) were filtered

through Millipore nitrocellulose membranes (0.45-mm pore

size, 47-mm diameter) with a sterilized Nalgene device. The

filters were placed on the surface of yeast extractmalt

extract agar plates (YMA, yeast extract 0.3%, malt extract

0.3%, peptone 0.5%, dextrose 1%, agar 2%, pH 4.0)

containing 200mg L1 chloramphenicol and incubated at

FEMS Microbiol Ecol 76 (2011) 113c 2011 Federation of European Microbiological Societies


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15 1C for up to 1 week. The yeasts were chosen for isolation

based on colony morphology. When possible, at least three

randomly selected colonies of each different morphotype

were purified by repeated streaking on YMA plates and

preserved at 80 1C or with liquid nitrogen for later

identification. Yeast CFU were registered for quantitative

analysis of yeast occurrence using a stereoscopic microscope

(Olympus SZX9) on the seventh day of incubation. The

percentages of pigmented yeast colonies from each sample

were also calculated. The averages and SDs of the total viable

yeast cells (CFU) per litre of water of each sampling point of

the lake were calculated. The same was carried out for the

percentage of pigmented colonies.

Yeast identification

All yeasts were preliminarily grouped based on their

cultural morphology, urease production and physiological

characteristics based on assimilation tests of carbon and

nitrogen sources and the production of amyloid compounds

(Yarrow, 1998). The yeasts were also characterized by PCR

Fig. 1. Locationof NH Lake andsampling points.

Table 1. Physicochemical characteristics, yeast counts and percentages of pigmented basidiomycetous yeasts, mycosporine (MYC)-positive yeasts and

yeasts producing photoprotective compounds (pigmented and/or mycosporine-positive yeasts)

Sites Temperature ( 1C) pH

Conductivity

(mS cm1)

Yeast counts

(CFUL1)

% Pigmented

yeasts

% MYC

positive

% Pigmented

and/or MYC1yeasts

NH0 16 6.90 34.5 141 94 24.0 24.0 14.2 14.1 40.0 28.4

NH1 10 6.49 35.0 97 78 46.9 31.8 52.1 27.6 72.9 19.8

NH2 11 6.58 36.0 49 43 71.0 23.9 69.8 15.5 81.3 22.5

NH3 11 6.40 35.0 43 43 67.9 19.0 77.3 17.7 81.6 19.1

NH4 11 6.58 35.5 73 47 73.6 19.6 73.5 16.7 75.5 14.6

NH5 11 6.42 36.0 25 13 69.7 27.5 68.7 36.2 72.3 20.9

NH6 14 6.52 35.0 22 3 8.3 12.0 83.3 23.6 83.0 24.0



3Yeast diversity of Nahuel Huapi Lake


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fingerprinting, using the mini/microsatellite-primed PCR

technique (MSP-PCR) (Libkind et al., 2003).

DNA extraction

For DNA extraction, yeast colonies were grown on modified

Sabouraud agar (glucose 2%, peptone 1%, yeast extract

0.5% and agar 2%) at 15 1C overnight, transferred to 2-mL

sterile tubes (Eppendorf) containing 100mL extraction

buffer solution (50 mmolTris L1, 250mmol NaClL1,

50mmol EDTA L1, 0.3% w/v SDS, pH 8) and incubated at

65 1C for 30 min. After incubation, 100mL phenol/chloro-

form/isoamilic alcohol (25 : 24 : 1) was added. The mixture

was vortexed vigorously, incubated for 3 min and centri-

fuged for 3 min at 7558g. DNA was dried overnight at room

temperature, suspended in 100 mL TE buffer (10 mM Tris,

10 mM Na-EDTA, pH 8.0) and stored in a refrigerator.

PCR fingerprinting

The synthetic oligonucleotide (GTG)5 and the core sequence

of the phage M13 (GAGGGTGGCGGTTCT) were used in

MSP-PCR experiments, respectively. The PCR reactions

were performed according to Libkind et al. (2003). Yeast

strains with identical DNA banding patterns were grouped

and putatively considered to belong to the same species

(Gadanho & Sampaio, 2002). At least one representative

strain of each MSP-PCR group was subjected to sequence

analysis of the D1/D2 domains of the large subunit of the

rRNA gene as described below. Physiologically distinct

strains with unique MSP-PCR banding patterns were also

selected for direct identification by sequencing of the D1/D2

region of the rRNA gene. When necessary, the internal

transcribed spacer (ITS) domains of the rRNA gene were

also sequenced.

Sequencing analysis

Total DNA was extracted using the methods described

above. The D1/D2 variable domains of the large subunit of

the rRNA gene were amplified as described previously by

Lachance et al. (1999) using the primers NL-1 (50-GCATAT

CAATAAGCGGAGGAAAAG-30) and NL-4 (5 0-GGTCCGT

GTTTCAAGACGG-3 0). The ITS regions of rRNA genes

were amplified with the universal primers ITS1 (5 0-TCCG

TAGGTGAACCTGCGG-30) and ITS4 (5 0-TCCTCCGCTT

ATTGATATGC-3 0) as described by White et al. (1989).

Sequencing of the D1/D2 region and ITS domains was

performed directly from purified PCR products using a

MegaBaceTM 1000 automated sequencing system (Amer-

sham Biosciences). The sequences obtained were compared

with those included in the GenBank database using the BASIC

LOCAL ALIGNMENT SEARCH TOOL (BLAST at http://www.ncbi.nlm.

nih.gov) (Altschul et al., 1997).

Mycosporine production and extracellular

enzymatic activities

The ability to synthesize mycosporine was tested according

to the method described previously by Libkind et al.

(2005b). The yeast isolates were tested for their ability to

degrade starch, protein (casein), pectin, carboxymethyl-

cellulose and lipids (Tween-80) according to the proceduresdescribed by Brizzio et al. (2007). Calibrated suspensions of

106 cells mL1, which were grown for 2448 h, were inocu-

lated on the surface of agar plates using a multipoint

inoculation device (de Garca et al., 2007). Plates containing

each substrate were incubated at 4 or 20 1C. Enzymatic

activity was analysed after 5 days in the samples incubated

at 20 1C and after 21 days in those incubated at 4 1C. The

enzymatic activities for specific substrates were evaluated as

described by Brizzio et al. (2007).

Statistical analyses

A one-way ANOVA was used to test the differences in the

percentages of pigmented basidiomycetous yeasts and/or

mycosporine-producing yeasts and the nonproducing PPCs

among the sampling sites. The variable total yeast count

(CFU) was analysed with a MannWhitney rank sum test.

When possible, a post hoc multiple comparison was per-

formed by applying the Tukey test (a= 0.05). Otherwise,

sampling sites were grouped into coastal (NH0 and 6) and

pelagic (NH1, 2, 3, 4 and 5) categories and compared using

the Student t-test. The same was done to compare the yeast

community composition at impacted sites (NH0 and 1)

with that at pristine sites (NH2, 3, 4, 5 and 6).

Species diversity at coastal and pelagic sites was measuredin terms of the richness, evenness and dominance given by

three indexes: (1) Shannon H= Sni/n ln (ni/n), (2)

Simpsons index =S(ni/n)2 and (3) Dominance

D = sum((ni/n)2), where ni is the number of individuals of

the taxon i and n is the total number of individuals. All

results were obtained with 95% confidence, and bootstrap

values were calculated from 1000 iterations. Species richness

refers to the number of species in a community, and species

dominance refers to the contribution of individuals. The

index calculations were performed using the computer

program PAST, version 1.90 (Ryan et al., 1995).

Results and discussion

Physical and chemical characteristics

The physical and chemical characterization of the water

samples are summarized in Table 1. The pH was nearly

constant along the transect, returning values between 6.4

and 6.9. The conductivity also did not change significantly

among the sampled sites. The water temperature was higher



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at sites near the coast than at pelagic sites due the greater

depth of the lake at points NH2 through NH5.

Table 2 shows an estimate of UV doses and maximum

irradiances to which the NH Lake surface was exposed

during sampling. Due to the low attenuation coefficients

(Kd) of the NH water (Morris et al., 1995) and the low

sampling depth (30 cm), it can be assumed that the micro-

organisms under study were exposed to almost 100% of the

UV irradiance reaching the water surface.

Yeast counts and quantitative analyses

Average yeast counts for each sampling site are shown in

Table 1. The counts ranged from 22 to 141CFU L1, with the

highest values corresponding to the most anthropogenically

influenced sites (NH0 and NH1). In general, the yeast

counts observed in the NH Lake were typical of clean lakes,

which usually contain o 100CFUL1 and rarely exceed

200CFUL1 (Hagler & Ahearn, 1987). The highest yeast

counts, which were found at sites NH0 and NH1, could be

related to the proximity of these sites to the city border (4and 30 m, respectively), where the effect of the Nireco river

inflow and human activities is the largest. Significant

differences were found (P= 0.001) when comparing sites

affected by anthropogenic activities (NH0 and NH1) with

sites that experienced lesser human influence (NH2, 3, 4, 5

and 6). Yeast occurrence decreased with increasing distance

to the south-edge of the lake (Table 1). To find higher yeast

counts in the south-coast waters than in pelagic zones is

reasonable due to the organic matter (and probably yeast)

input of the Nireco river into the NH waters, which are then

subjected to a dilution effect. Baffico (2001) found that the

area of influence of the Nireco river had relatively high levels

of readily assimilable forms of phosphorus and nitrogen

than other nonanthropogenically impacted coasts of NH.

The north coast, even though it has several small inflows,

showed the lowest yeast counts. This is probably because

such streams are not as anthropogenically impacted as the

Nireco river and do not carry much organic matter (and

yeasts).

Additional factors limiting yeast propagation in the water

body are the lack of nutrients and the lower water tempera-

tures to which yeasts are subjected once they enter NH

waters. These, together with the effect of UVR, which in the

case of NH is highly significant in the upper layer due to the

high transparency of its waters (Morris et al., 1995), are

important factors conditioning yeast survival in the water

column and thus determining its distribution in the lake. It

was then hypothesized that a significant fraction of the

yeasts entering the littoral areas of the lake is rapidly

eliminated, and thus in pelagic areas mostly yeasts able to

cope with such extreme conditions are found.

Pigmented basidiomycetous yeasts are common in most

aquatic yeast communities and often comprise 4 50% of

the yeast population, especially in oligotrophic marine or

fresh waters (Hagler & Ahearn, 1987; Libkind et al., 2003).

In the present study, these yeasts were present in all water

samples, and relatively high numbers were found in the

pelagic sampling points. Carotenoid pigments (antioxi-

dants), which are synthesized by several pigmented basidio-

mycetous yeast species from oligotrophic aquatic

environments, have been reported to have a photoprotective

function in yeasts (Moline et al., 2009, 2010b). For example,the yeast Rhodotorula mucilaginosa, an abundant species in

patagonian lakes (Libkind et al., 2003), produces large

quantities of torularhodin, a carotenoid that affords UVB

photoprotection (Moline et al., 2010b). Significant differences

were found when comparing the percentage of pigmented

yeasts found at pelagic and border lake sites (Po 0.005).

These differences could be related to the general lower

susceptibility of carotenogenic yeasts to UV than nonpigmen-

ted ones (Moline, 2004). The latter possibly survive shorter

periods in the water column than the former. In a previous

study, focused mainly on pigmented yeasts of high-altitude

lakes (mountain lakes) from Patagonia, Libkind et al. (2009)

found that pigmented yeasts prevailed only in highly trans-

parent lakes. Due to the high transparency of NH water,

allowing an extraordinary penetration of solar radiation

(Morris et al., 1995; Balseiro et al., 2008), UV appears to be a

strong selective factor in favour of more UV-resistant yeast

species. This has also been demonstrated for planktonic

organisms (Villafane et al., 2001; Marinone et al., 2006).

Another type of PPC that can be synthesized by yeasts is

mycosporines, which are UVB screening compounds that

Table 2. Daily UV dose and maximum irradiance detected at the time of sampling in the NH lake at different wavelengths

Wavelength (nm) Daily UV dose (kJ m2)

Maximum irradiance

(mW cm2nm1 s1 )]

NH UV attenuation

coefficient: Kd (m1)

305 1.857 0.034 8.864 0.262 0.543

320 8.416 0.279 31.775 0.944 0.407

340 20.045 0.720 71.476 0.303 0.303

380 25.370 1.014 89.640 0.177 0.177

Media and SD are provided.Obtained from Morris et al. (1995); average values of data from three sampling sites.





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also have antioxidant properties (Moline et al., 2010a). The

percentage of mycosporine-positive yeasts found at the

south coast (NH0) was significantly (Po 0.005) lower

(14%) than the rest of the sampling sites (452%). Con-

trary to what was observed for pigmented yeasts, the highest

percentage of mycosporine-positive yeasts was observed in

the north coast (83%; NH6), while pelagic sites had valuesranging 5277%. When the distribution of yeasts producing

at least one of the two PPCs was analysed, a trend similar to

that found for mycosporine-positive yeasts was observed

(Table 1). A situation similar to that postulated for pigmen-

ted yeasts is also observed for mycosporine-positive yeasts,

in which the extreme conditions (particularly UV) may

select for mycosporine-synthesizing yeasts, explaining their

prevalence in pelagic sites. Even though, the high values

observed for the north coast (NH6 site is 5 m from the forest

border) are not in agreement with such hypotheses, this

could be due to the influx of yeasts from phylloplane run-

off. The phyllosphere is a notable and common niche for

yeasts (Fonseca & Inacio, 2006), which is highly exposed to

solar radiation. We have recently found that the surface of

Nothofagus spp. leaves harbour a peculiarly large proportion

of mycosporine-positive yeasts (Munoz, 2010) similar to

that observed in NH6. Interestingly, the proportion of

pigmented yeasts in such leaves rarely exceeded 10%.

Libkind et al. (2009) found that mycosporine-synthesizing

species were poorly represented in high-altitude lakes, an

environment exposed to high UVR in which such PPC could

be a useful adaptation for survival. A plausible explanation

arises from the fact that due to the high altitude, the

vegetation surrounding those mountain lakes is limited to a

fewNothofagus shrubs, and therefore a much lower run-offfrom the phylloplane (and thus of mycosporine-positive

yeasts) is expected.

It can be hypothesized that the north coast receives

already UV-adapted (mycosporine-positive) yeasts from the

nearby Nothofagus phyllosphere, while the south coast

receives mostly yeasts without PPC (less adapted) or ubiqui-

tous pigmented yeasts normally related to human activity

(e.g. R. mucilaginosa and Aureobasidium pullulans) from

urban discharge through the Nireco river. Additionally, our

data support the idea that UVR is an important factor that

determines yeast community structures in Andean oligo-

trophic lakes and that yeasts producing carotenoids and/or

mycosporine possess an adaptative advantage in highly UV-

exposed habitats than those incapable of producing them.

Comparative analyses among pelagic and coastal zones

using the Shannon H, Simpsons, and Dominance indexes

are shown in Table 3. The pelagic zone of the lake presented

the highest Shannon Hand Simpsons indexes. However, the

Dominance index was higher at the coastal points of the lake

than at the pelagic points. The values of these indexes

showed that the yeast community from NH Lake has a

relatively higher richness index (H=2.5 0.2) and a uni-

form distribution of taxa among pelagic and border lake

sites (Simpsons index = 0.85 0.6). Consequently, a very

low species dominance (D = 0.09 0.06) was observed.

Table 4 shows the distribution among the NH sampling

sites of the various yeast species in terms of their production

of PPC (mycosporines and carotenoids). Interesting cases

include the yeast-like fungus A. pullulans, which has higher

counts in the anthropogenically impacted coast and gradu-

ally reduces its numbers in sites away from that coast. This is

in agreement with the fact that A. pullulans is a ubiquitous

organism and that it is closely related to human activities

(Zalar et al., 2008). A similar case is that of R. mucilaginosa,

a pigmented but mycosporine-negative yeast, also consid-

ered ubiquitous (Libkind & Sampaio, 2009) and that

appears to be introduced into the lake from the city coast.This is in agreement with the fact that R. mucilaginosa was a

minor component of the yeast community of Nothofagus

phylloplane (Munoz, 2010). Contrary to A. pullulans,

R. mucilaginosa is apparently well-adapted to survive in

NH waters given that it occurred in all sampling sites. This

might be partially related to the high tolerance of R.

mucilaginosa to UVR (Moline et al., 2010b; Libkind et al.,

2011). The category including yeasts producing only mycos-

porines comprised mostly Cryptococcus species, and these

were mainly found in pelagic sites. Yeasts not producing any

of the two PPCs included most ascomycetous yeasts and also

several Cryptococcus species.

Yeast identification and ecology

We obtained 94 pigmented yeasts (including the yeast-like

fungi A. pullulans and Delphinella strobiligena) and 55

nonpigmented yeast isolates. The isolates with white to

cream colonies were grouped as nonpigmented yeasts. All

yeasts were preliminarily grouped based on their cultural

and physiological characteristics, and the groups with

Table 3. Diversity indexes of yeasts from coastal and pelagic sites in NH

Lake

Diversity indexes

Sampling sites Nahuel Huapi

Coast sites Pelagic sites Mean SE

Shannon H 2.2 2.8 2.5 0.2

(2.1/2.8) (2.6/3.0)

Simpsons index 0.8 0.9 0.85 0.6

(0.8/0.9) (0.8/0.9)

Dominance D 0.1 0.08 0.09 0.06

(0.06/0.1) (0.06/0.1)

Coastal sites: NH0 and NH6; Pelagic Sites: NH1, NH2, NH3, NH4, NH5.

The numbers in parentheses represent the lower and upper diversity

values, respectively, with 95% confidence and bootstrap values calcu-

lated from 1000 iterations.





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similar or identical morphological and physiological char-

acteristics were subsequently subjected to MSP-PCR finger-

printing. Characterization by MSP-PCR fingerprinting

allowed the formation of seven groups of identical DNA

banding patterns among the pigmented yeasts and five

patterns among nonpigmented yeasts (data not shown).

One representative strain from each group was selected

for sequence analysis of the D1/D2 domains of the 26S

rRNA gene. The pigmented species of these MSP-PCR

fingerprinting groups were represented by A. pullulans,

Cystofilobasidum infirmominiatum, Cystofilobasidum capitatum,

R. mucilaginosa, Rhodotorula laryngis, Rhodotorula slooffiae

and two isolates of the genus Dioszegia, identified as

Dioszegia sp. 1. The nonpigmented yeast groups were

identified as Candida railenensis, Cryptococcus adeliensis,

Cryptococcus magnus, Cryptococcus saitoi and Cryptococcus

victoriae. Among the pigmented yeasts, five isolates showed

unique MSP-PCR fingerprinting patterns and were identi-

fied by sequencing as Dioszegia hungarica, Rhodosporidium

diobovatum, Rhodosporidium colostri, Rhodosporidium

Table 4. Identification, distribution and occurrence of yeasts isolated from NH Lake

Yeast species

Sampled points

NH0 NH1 NH2 NH3 NH4 NH5 NH6

Pigmented and MYC-positive species

Aureobasidium p ullulansw 4.4 3.7 3.3 2.3 2.6 2.5 1.8 2.8

Delphinella strobiligenaw 0.6 1.4

Dioszegia hungarica 0.6 1.4

Dioszegia sp.1 1.3 1.8

R. laryngis 9.7 13.7 17.5 39.1 16.8 29.9 4 8.9 5.9 11.6

R. pinicola 13.5 30

R. slooffiae 43.5 31 8.5 12.4

Pigmented and MYC-negative species

C. victoriae 6.6 13 11 10.3 6.6 11.7 17.5 21.9 3.1 4

Cystofilobasidium capitatum 1.8 2.6 2 4.4 9 12.4 4 8.9

C. Infirmominiatum 1.3 2.9 0.6 1.4

R. colostri 2.6 5.9

Rhodosporidium diobovatum 0.5 1.1

Rhodotorula mucilaginosa 22.9 25.6 6.6 6.6 1 2.2 0.5 1.1 0.6 1.4 1 1.3 2 2.8

Rhodotorula sp.1 0.6 1.4

Nonpigmented and MYC-positive species

Bullera dendrophila 1.3 1.8Cryptococcus adeliensis 3.3 7.4 7.8 8.4 2.6 5.9 0.5 1.1

C. diffluens 0.6 1.4 0.5 1.1

C. festucosus 1.3 2.9

C. heveanensis 4 5.6

C. magnus 1.3 1.8 0.6 1.4 0.5 1.1 0.6 1.4

C. saitoi 10.5 22.7 1.5 2.2 1 2.2

C. stepposus 0.6 1.4

C. wieringae 3.3 7.4

Guehomyces pullulans 0.5 1.1 0.8 1.7

Species without PPC

Candida parapsilosis 1.5 3.3

C. railenensis 12.6 28.3 5.9 11.6 0.5 1.1

C. sake 0.6 1.4 5.3 7.6 0.6 1.4

Candida sp. 1 4 5.6C. carnescens 1.3 2.9

Cryptococcus sp.1 2 4.4

C. tephrensis 2.5 5.5 4 5.6

Debaryomyces hansenii 6.6 14.8

G. pullulans 0.6 1.4

Hanseniaspora uvarum 1.3 1.8 0.6 1.4 0.6 1.4

Pichia fermentans 2 4.4

Media and SD of CFU L1 of yeast in the water samples.wYeast-like fungus.

MYC, Mycosporines; PPC, Photoprotective compounds.





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pinicola and Rhodotorula sp. 1. Ten yeast isolates showed

unique physiological characteristics and carbon assimilation

patterns and were identified as C. parapsilosis, Candida sp. 1,

Cryptococcus sp. 1, Cryptococcus carnescens, Cryptococcus

festucosus, Cryptococcus haveanensis, Cryptococcus stepposus,

Cryptococcus weringae, Debaryomyces hansenii and the yeast-

like fungus D. strobiligena. The remaining isolates weregrouped based on morphological similarity and identical

results for physiological tests. These yeasts were identified as

Bullera dendrophila, Candida sake, Cryptococcus diffluens,

Cryptococcus tephrensis, Guehomyces pullulans, Hansenia-

spora uvarum and Pichia fermentans (Table 4).

Some yeast isolates showed five or more nucleotide

differences in D1/D2 domains of the rRNA gene when

compared with the most closely related known species;

therefore, they could represent novel yeast species. Accord-

ing to Kurtzman & Robenett (1998), isolates of the same

species usually have only zero to two nucleotide differences

in the D1/D2 region of the large subunit of the rRNA gene.

One isolate ofCryptococcus (strain identified as Cryptococcus

sp. 1, GenBank accession number HM990974) differed by

nine nucleotide substitutions in the D1/D2 region of the

rRNA gene from the closest known species, Cryptococcus

spencermartinsiae, a yeast isolated from glacial melting

waters in Patagonia by de Garcia et al. (2010). Our isolate

probably represents a new Cryptococcus species that is

related to C. spencermartinsiae. Another isolate, identified

as Rhodotorula sp. 1, showed five nucleotide substitutions in

the D1/D2 region of the rRNA gene compared with the

Rhodotorula sp. NBRC 105035 (Sporidiobolales, GenBank

accession number AB462346) and 24 substitutions from the

closest known relative species Sporobolomyces inositophilus(GenBank accession number AF189987). This isolate prob-

ably also represents a new yeast species of the genus

Rhodotorula because it does not produce ballistoconidia.

The isolate identified as Candida sp.1 presented 100%

sequence similarity to Candida sp. SDY 211 (GenBank

accession number AY731817) and Candida sp. AS 2.3084

(GenBank accession number DQ451013), which were iso-

lated from acidic aquatic environments in Portugal and

from an unidentified flower in Tibet, respectively. These

three isolates represent new yeast species that are most

closely related to several Candida species but have 470

indels of difference in their D1/D2 domains. The isolate

identified as Dioszegia sp. 1 presented one substitution in the

D1/D2 domain sequence compared with the strain Dioszegia

sp. CRUB 1147 (GenBank accession number EF595753),

which was isolated from altitudinal lakes in Argentina.

These isolates probably represent a new species that is

closely related to D. hungarica.

The yeast isolates from NH Lake were identified as

belonging to 13 genera and 34 species (Table 4). Basidiomy-

cetous yeasts were represented by 73.8% of the isolates. In

general, these yeasts are more nutritionally versatile and

more tolerant of extreme environmental conditions than

ascomycetous yeasts (Sampaio, 2004). In addition, basidio-

mycetous yeasts are often found in association with the

phyllosphere of terrestrial plants (Fonseca & Inacio, 2006);

their occurrence in aquatic environments could be consid-

ered the result of a run-off from this substrate (Hagler &Ahearn, 1987; Lachance & Starmer, 1998).

Species of Cryptococcus were common to all of the sites

sampled in NH Lake. These yeasts, which represented 34.8%

of the total isolates, were the most frequent and diverse

group, followed byRhodotorula, which represented 26.7% of

the total isolates. These genera have been reported in other

studies from Patagonian aquatic environments (Libkind

et al., 2003, 2009; de Garca et al., 2007; Russo et al., 2008),

suggesting that these yeasts occur frequently in such envir-

onments. The wide nutritional plasticity and the adaptabil-

ity to harsh environmental conditions of many yeasts species

of these genera explain their high frequencies of isolation in

NH Lake.

Rhodotorula. mucilaginosa was the most frequently isolated

yeast (21 isolates; 28.7% of total pigmented strains) and was

present at all of the sampled points. This species is ubiquitous

and has been isolated in all kinds of natural substrates

(Gadanho et al., 2006). Libkind et al. (2009) reported that

87.5% of the pigmented yeasts occurring in Negra Lake, an

ultra-oligotrophic freshwater from Patagonia, Argentina,

were R. mucilaginosa. According to these authors, an increase

in the population of this species may be related to a

temporary increase in the organic matter in the lakes. Moline

et al. (2010b) suggest that R. mucilaginosa enhances UVB

survival by producing the carotenoid pigment torularhodin;however, it does not produce mycosporines. This yeast

species appeared in the NH Lake at a lower frequency

than in the highly transparent Patagonian mountain lakes

(4 1400 m.a.s.l.), where it was prevalent (Libkind et al.,

2009). Other pigmented species such as D. hungarica, Diosze-

gia sp. 1, R. diobovatum, R. colostri, R. pinicola, R. slooffiae and

Rhodotorula sp. 1 were less frequently isolated. Species of the

pigmented mycosporine-negative yeasts C. infirmominiatum,

C. capitatum, R. diobovatum and Rhodotorula sp. 1 were

poorly represented, suggesting that they have a low resistance

to the UVR conditions found in NH Lake. In a previous

work, we reported the low tolerance of Cystofilobasidium

species to UVB (Libkind et al., 2009). Dioszegia strains have

been found recently in glacial meltwaters and mountain lakes

in Patagonia (de Garca et al., 2007; Libkind et al., 2009). In

this work, two Dioszegia species were observed at low

frequencies in NH Lake. These yeasts are pigmented and able

to produce mycosporine and show high tolerance to UVB

(Libkind et al., 2009). However, our results suggest that

Dioszegia species are minor components of the yeast commu-

nities in Andean aquatic environments. This yeast genus is



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frequently found in association with plants and terrestrial

substrates (Fonseca & Inacio, 2006).

The pigmented species C. victoriae occurred frequently in

NH Lake, and this is the first report on its occurrence in

Patagonian lakes. This yeast was originally isolated from soil,

moss, lichen, soil, Granite Harbour soil, Lichen Valley and

Vestfold Hills in Antarctica (Thomas-Hall et al., 2002).However, the habitat of this yeast is wider than previously

thought because it was isolated from glaciers in Italy and

from indoor dust in central Finland (Pitkaranta et al., 2008;

Branda et al., 2010). Other Cryptococcus species occurred in

minor frequencies, including C. adeliensis, C. carnescens, C.

diffluens, C. magnus and C. saitoi, which are frequently

reported in cold habitats (Vishniac, 2006; de Garca et al.,

2007; Russo et al., 2008; Libkind et al., 2009) and therefore

appear to be autochthonous in cold ecosystems.

Among the ascomycetous yeasts isolated in our study, the

yeast-like fungi A. pullulans and D. strobiligena were able to

produce mycosporines. This is the first report of the

presence of mycosporine-producing ascomycetous yeasts in

the lakes of Patagonian Argentina. Aureobasidium pullulanswas the most frequent ascomycetous species. This yeast-like

species is often isolated from many different types of water

(Slavikova & Vadkertiova, 1997a). Debaryomyces hansenii, a

ubiquitous yeast species found in aquatic environments

(Nagahama, 2006), was found at site NH1, which was

relatively highly impacted by human activities. The Candida

species C. parapsilosis, C. sake, C. railenensis and Candida

Table 5. Comparison of yeast species isolated from NH Lake and from other cold and tropical aquatic environments

Species from Nahuel

Huapi Lake

Tropical aquatic

environmentsAlpine

glacierswArctic

environmentszAntarctic

environments

Oligo to ultra-oligotrophic aquatic

environments from Patagonia,

Argentinaz

Aureobasidium pullulans 1 1 1

Bullera dendrophila

Candida sake 1 1

C.railenensis

C. parapsilosis 1 1 1

Cryptococcus adeliensis 1 1 1

C. carnescens

C. diffluens 1

C. festucosus 1

C. heveanensis

C. magnus 1

C.saitoi 1 1 1 1

C. stepposus 1

C. tephrensis

C. victoriae 1 1 1

C. wieringae

Cystofilobasidium capitatum 1

C. infirmominiatum 1 1

Debaryomyces hansenii 1 1 1

Dioszegia hungarica 1 1

Delphinella strobiligena

Guehomyces pullulans 1

Hanseniaspora uvarum

Pichia fermentans

Rhodotorula mucilaginosa 1 1 1 1

R. colostri 1

R. laryngis 1 1 1 1

R. pinicola 1

R. sloofiae 1

Rhodosporidium diobovatum 1 1

Hagler & Mendonca-Hagler (1981), Rosa et al. (1995), Morais et al. (1996), Medeiros et al. (2008), Valdez-Collazo et al. (1987).wTurchetti et al. (2008).zGostincar et al. (2006), Connell et al. (2006), Butinar et al. (2007).Vishniac (2006).zBrizzio et al. (2007), de Garca et al. (2007), Russo et al. (2008), Libkind et al. (2009).

, Absent; 1, present.





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sp.1 represented 8% of the total yeast isolates. A single

isolate of C. parapsilosis was observed at site NH3 in the

middle of the lake. This species is often reported in aquatic

environments that have high levels of organic matter from

industrial and domestic wastes (Hagler, 2006; Nagahama,

2006; Medeiros et al., 2008). However, our results indicated

that the NH Lake has low levels of anthropogenic organicpollution because only one opportunistic yeast species of

faecal origin was obtained. Most isolates of C. sake were

obtained at point NH1. Candida sake occurs in different

aquatic environments, including lagoons (Boguslawska-Was

& Dabrowski, 2001), algae, shore soil, lakes and penguin

dung from Antarctic environments (Goto et al., 1969). This

species is able to grow in habitats ranging from c. 5 to 30 1C

(Vishniac, 1996). C. railenensis occurred in higher amounts

at points NH0 and NH1, which are near the city coast. The

species was described based on isolates from a rotten trunk

ofN. dombeyi and Nothofagus obliqua (Ramrez & Gonzalez,

1984) and is probably associated with the forest vegetation

found in Patagonia.

When the yeast diversity of NH Lake was comparedwith that of other cold and tropical aquatic environments

(Table 5), a remarkable resemblance to Antarctic habitats

and other oligotrophic aquatic environments in Patagonia

was observed. Thirty-five per cent of the species isolated in

our study are present in Antarctic habitats (Vishniac, 2006),

and 54% of the species are present in other water bodies in

Patagonia (Libkind et al., 2003, 2009; de Garca et al., 2007;

Table 6. Extracellular enzymatic activities of yeasts from NH Lake

Species Number strains

AmA PrA PecA CelA LpA

5 1C 20 1C 5 1C 20 1C 5 1C 20 1C 5 1C 20 1C 5 1C 20 1C

Aureobasidium pu llulans 20 16 17 17 19 18 18 20 20 19 19

Bullera dendrophila 2 2 2 1 1

Candida sake 4 1 1 1 2 2 2 2

C. railenensis 6 1 2 2 1 1 2 1

C. parapsilosis 1 1 1 1 1 1 1

Cryptococcus adeliensis 10 3 1 1 1 5 5 3 3 6 6

C. carnescens 1 1 1 1 1

C. diffluens 2 1 2 2 2

C. festucosus 1 1 1 1 1

C. heveanensis 1

C. magnus 6 4 3 3 3 2 2 6 6 6 6

C. Saitoi 6 3 1 1 2 5 5 4 4 5 6

Cryptococcus sp. 1 1 1 1 1 1

C. stepposus 1 1 1 1 1

C. tephrensis 2 2 2 1 2

C. victoriae 20 4 3 2 2 3 3 16 17 18 18

C. wieringae 1 1 1 1 1 1 1 1

Cystofilobasidium capitatum 7 1 1 3 2 4 4

C. infirmominiatum 2 2 2

Debaryomyces hansenii 1 1 1 1 1

Dioszegia sp. 1 2 1 1 2 1 2 2

Dioszegia hungarica 1 1 1 1

Delphinella strobiligena 1 1

Guehomyces pullulans 3 2 1 2 2 3 3 2 2

Hanseniaspora uvarum 4 1 1 1 4 4 4 4

Pichia fermentans 2

Rhodotorula colostri 1

Rhodotorula mucilaginosa 21 1 1 5 11 1 3 7 7

R. laryngis 9 8 8R. pinicola 1 1 1 1

R. sloofiae 6 1 1 1 3 3

Rhodotorula sp. 1 1

Rhodosporidium diobovatum 1 1 1 1 1 1 1

Total number of strains 148

Total number of positive strains for

the tested enzymatic activities

36 28 27 32 53 62 74 77 102 101

AmA, amylolytic activity; PrA, proteolytic activity; PcA, pectinolytic activity; CelA, cellulolytic (degradation of carboxymethyl-celullose); LpA, lipidic

(esterasic) activity.





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Russo et al., 2008). In this work, only five ubiquitous yeast

species (A. pullulans, C. parapsilosis, C. infirmominiatum,

D. hansenii and R. mucilaginosa) were also found in tropical

aquatic habitats (Table 5).

Screening for extracellular enzymatic activity

The extracellular enzymatic activity profiles of the yeast and

yeast-like isolates are shown in Table 6. Of the 148 tested

strains, 82% showed at least one extracellular enzymatic

activity at 5 and/or 20 1C. These yeasts were represented by

72.4% of basidiomycetous isolates and 24.8% of ascomyce-

tous isolates. The percentage of yeasts producing extracel-

lular enzymes was slightly higher in the pelagic sites of the

lake (56.5%). Esterasic activity (hydrolysis of Tween-80) was

the most widely expressed extracellular enzyme activity

(positive for 71.8% of the total isolates), followed by the

degradation of carboxymethyl-cellulose (cellulase activity,

53.0%), pectinase (42.9%), amylase (26.8%) and protease

(22.1%) activities. Among the ascomycetous yeasts, 19isolates of A. pullulans, four isolates each of D. strobiligena

and H. uvarum, three isolates each ofC. parapsilosis and C.

railenensis, two isolates of C. sake and one isolate of D.

hansenii showed esterasic activity. Cellulase was the second

most prevalent enzymatic activity observed among the

ascomycetous yeast isolates; 29 isolates were positive for this

trait. Almost all of the strains of A. pullulans showed the

ability to produce all of the tested enzymes. Among the

basidiomycetous species of the genus Cryptococcus, 50 iso-

lates showed at least one extracellular enzymatic activity at 5

and/or 20 1C. Cryptococcus adeliensis, C. magnus, C. saitoi

and C. victoriae exhibited activity for all enzymes tested.

Lipolytic and cellulolytic activities were expressed mainly by

species of Cryptococcus and Rhodotorula. Candida sp. 1,

Cryptococcus heveanensis, P. fermentans, R. colostri and

Rhodotorula sp. 1 were not able to hydrolyse any of

compounds tested. de Garca et al. (2007) also showed that

a significant proportion of yeast isolates from glacial melt-

water rivers in Patagonia, Argentina, were capable of de-

grading natural compounds. The fact that a significant

proportion of yeasts are able to hydrolyse natural com-

pounds such as lipids, starch, protein and pectin suggests

that these strains are metabolically adapted to cold environ-

ments and have a significant ecological role in organic

matter decomposition and nutrient cycling.

Conclusion

The occurrence and distribution of yeasts along a transect of

NH Lake showed peculiar distributional patterns probably

influenced by inputs of allochthonous organic matter from

the borders of the lake and by abiotic factors such as UVR.

PPC-producing yeasts were mainly found in pelagic points

of the lake, suggesting that both mycosporine and carote-

noid production capacities are important for yeast survival

under high UVR conditions expected in the upper layer of

NH Lake. The significant relationship between the ability of

the yeasts to produce PPC and their distance from the lakes

shore also indicates that yeasts entering NH Lake are

subjected to extreme conditions that imply a significant

force towards the selection of UV-tolerant yeasts. Most ofthe species isolated in our work were typical of cold aquatic

environments. In addition, most of the yeast isolates pre-

sented at least one active extracellular enzyme, which

suggests that these microorganisms are metabolically active

in the lake and could contribute to organic matter recycling

in this cold freshwater environment. This work represents

the first comprehensive survey of the cultivable ascomyce-

tous and basidiomycetous yeast community of an oligo-

trophic lake in the Patagonia region.

Acknowledgements

This work was accomplished with financial aid from the

Universidad Nacional del Comahue (Project B143) and

ANPCYT (Project PICT-1176). This work was also sup-

ported by the Fundacao de Amparo a Pesquisa do Estado de

Minas Gerais (FAPEMIG) and Conselho Nacional de De-

senvolvimento Cientfico e Tecnologico (CNPq) of Brazil.

We would like to thank the authorities of Parques Nacio-

nales (Argentina) for providing permission to collect water

samples within the NHNP. Special thanks are given to V.d.G.

for assistance in enzymes assays and A. Denegri for map

design.

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Yeasts from an oligotrophic lake in Patagonia (Argentina) diversity

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