Yeasts from high-altitude lakes: influence of UV radiation

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

Yeasts fromhigh-altitude lakes: in£uenceofUVradiationDiego Libkind1, Martın Moline1, Jose Paulo Sampaio2 & Maria van Broock1

1Laboratorio de Microbiologıa Aplicada y Biotecnologıa, Universidad Nacional del Comahue, Centro Regional Universitario Bariloche (CRUB) – CONICET

(Consejo Nacional de Investigaciones Cientıficas y Tecnologicas), Rıo Negro, Argentina; and 2Centro de Recursos Microbiologicos, Seccao Autonoma de

Biotecnologia, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal

Correspondence: Diego Libkind,

Laboratorio de Microbiologıa Aplicada y

Biotecnologıa, Universidad Nacional del

Comahue, Centro Regional Universitario

Bariloche (CRUB) – CONICET (Consejo

Nacional de Investigaciones Cientıficas y

Tecnologicas), Quintral 1250, Bariloche, Rıo

Negro, Argentina. Tel.: 154 2944 428505;

fax: 154 2944 423111; e-mail:

[email protected]

Received 19 December 2008; revised 24 April

2009; accepted 26 May 2009.

Final version published online 14 July 2009.

DOI:10.1111/j.1574-6941.2009.00728.x

Editor: Riks Laanbroek

Keywords

carotenoids; MSP-PCR fingerprinting; mountain

lakes; photoprotection; Patagonia; UVB

resistance.

Abstract

Mountain lakes located at a high elevation are typically exposed to high UV

radiation (UVR). Little is known about the ecology and diversity of yeasts

inhabiting these extreme environments. We studied yeast occurrence (with special

emphasis on those producing carotenoid pigments) at five high-altitude

(4 1400 m a.s.l.) water bodies located in the Nahuel Huapi National Park

(Bariloche, Argentina). Isolates were identified using a polyphasic approach.

Production of photoprotective compounds (carotenoids and mycosporines) by

yeast isolates, and UVB resistance of selected species were studied. All water

samples contained viable yeast cells in variable numbers, generally ranging from

49 to 209 cells L�1. A total of 24 yeast species was found; at least four represented

novel species. Carotenogenic yeasts prevailed in lakes with low water conductivity

and higher transparency and chlorophyll a levels. Apparently, the ability to

produce photoprotective compounds in yeasts was related to the transparency of

mountain lake waters, and strains from more transparent waters developed

increased UVB resistance. Our results indicate that UVR is an important environ-

mental factor affecting the yeast community structure in aquatic habitats.

Introduction

The northern Andean Patagonia (Argentina) has a great

variety of glacially formed water bodies with different

trophic status, mean precipitation and altitude, among other

characteristics. Mountain lakes located at 4 1400 m a.s.l.

are typically exposed to increased UV radiation (UVR) not

only due to the natural increase of the UVR flux with

elevation but also due to the transparency level and shallow-

ness of their water (Zagarese et al., 1998, 1999; Sommaruga,

2001). UVR is now recognized as a strong selective force in

aquatic ecosystems, and accumulated evidence demonstrates

that UV levels that are considered normal have significant

impacts on natural communities (Williamson, 1995).

Although considerable knowledge has accumulated con-

cerning the effect of UVR on aquatic microbial communities

in general (Sommaruga, 2001), very little is known about the

importance of UVR in determining the structure of yeast

populations in these extreme environments.

Among the known strategies for the minimization of UV-

induced damage in organisms are the synthesis of anti-

oxidants and UV sunscreen compounds such as carotenoids

and mycosporines, respectively (Roy, 2000). Several yeast

species have long been known to accumulate carotenoid

pigments (mainly b-carotene, torulene and torularhodine);

as a result, red-pink-colored colonies are formed (Barnett

et al., 2000). These secondary metabolites are believed to

have a photoprotective function in yeasts due to their

antioxidant properties (Moore et al., 1989; Gunasekera

et al., 1997; Tsimako et al., 2002; Moline et al., 2009). The

presence of an additional putative photoprotective com-

pound with UV-screening properties (Bandaranayake, 1998;

Volkmann et al., 2003) has been reported for some yeast

species grown under strong light or UVR (Libkind et al.,

2004a, b, 2005a, b). This UV-absorbing metabolite belongs

to the family of the so-called mycosporines, and was

identified as mycosporine–glutaminol–glucoside (MGG)

(Sommaruga et al., 2004).

FEMS Microbiol Ecol 69 (2009) 353–362 c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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Yeast ecological studies on freshwater aquatic environ-

ments are scarce and identifications have been mostly based

on physiological tests (Slavikova et al., 1992; Rosa et al.,

1995; Slavikova & Vadkertiova, 1997; Bogusławska-Wasa &

Dabrowskia, 2001), an approach that frequently leads to

wrong identifications. For a rapid and accurate identifica-

tion of yeast isolates in ecological studies, we have used

the mini/microsatellite-primed PCR method (MSP-PCR)

(Gadanho & Sampaio, 2002; Gadanho et al., 2003) in

combination with other conventional and molecular tech-

niques (Libkind et al., 2003).

Studies on molecular characterization (Libkind et al.,

2003), killer activity (Brizzio & van Broock, 1998), carote-

noid pigment production (Libkind et al., 2004a, b; Libkind

& van Broock, 2006) and photoprotective compounds

synthesis (Libkind et al., 2004a, b, 2005a, b) of yeast strains

isolated from Patagonian aquatic environments, including a

few from high-altitude lakes, have been carried out. How-

ever, as far as we know, yeast biodiversity and ecology in

these extreme Patagonian environments, or from any other

part of the world, has not yet been assessed.

Five Patagonian mountain lakes in the Nahuel Huapi

National Park (Bariloche, Argentina) were selected for a

yeast biodiversity survey. Special attention was paid to the

carotenoid-producing yeast species. A quantitative estima-

tion of the yeast community, as well as the ability of these

yeasts to produce photoprotective compounds is included.

A relationship between these abilities and the resistance to

UVB radiation is established. Given that a previous report

dealt with yeasts from most piedmont lakes (around

750 m a.s.l.) in the same region (Libkind et al., 2003), yeast

diversity in high- and low-altitude lakes was compared.

Materials and methods

Area description and sampling

Patagonian Andean lakes cover an ultra to mesotrophic

range of small and large lakes including small high-elevation

water bodies, sometimes surrounded by Nothofagus pumilio

forest or shrubs. A complete description of Patagonian high-

altitude lakes may be found in Zagarese et al. (1999). The

five selected aquatic environments surveyed (Lakes Verde,

Negra, Azul, Ilon and Toncek) are located in the Nahuel

Huapi National Park (c. 411050S, 711300W) and occur at

altitudes ranging from 1400 to 1750 m a.s.l. A total of 15

water samples were obtained during a 2003 summer cam-

paign (Fig. 1) as described by Libkind et al. (2003) with

modifications. Subsurface water samples were collected with

sterile bottles and filtered in situ (pressure o 15 in.

of mercury) using a sterile Nalgene filtering apparatus.

Bottles were submerged as far off the lake coast as possible

(4 5 m), filled, sealed and used to vacuum filter known

volumes (150–500 mL depending on the trophic status of

the lake) of water under aseptic conditions. Three individual

samples were taken per lake at a single point on each water

body.

Physicochemical characterization of the lakes

Temperature, pH and conductivity were measured using an

Orions 115 pH meter. For Ilon and Azul lakes, chlorophyll

a concentration was determined as described by Wetzel &

Likens (2000). Water transparency was measured using the

Secchi disk.

Yeast isolation and quantitative analysis

Filter membranes (Millipore, +0.47mm) were maintained

on sterile Petri dishes and kept refrigerated (not longer than

36 h). Filters were placed on MYP agar plates (g L�1; malt

extract 7, yeast extract 0.5, soytone 2.5, agar 15, chloram-

phenicol 0.2; pH 5), and incubated at 15–17 1C until colony

emergence was observed. Yeast CFU were registered using a

stereoscopic microscope (Olympus SZX9) at the seventh day

of incubation. The percentage of carotenoid-producing

(carotenogenic) colonies was calculated over the total yeast

CFU for each sample. Controls, performed in the same way,

but filtering air instead of water, showed negative results in

all cases. The average and SDs of the total viable yeast cells

(CFU) of each lake were calculated. The same was done for

the percentage of carotenogenic colonies.

All red colonies from a representative plate as well as at

least one colony of each macromorphological type of non-

pigmented colonies were picked for purification on MYP

agar plates (pH 5.5, no antibiotic added) and preserved on

MYP and PDA slants at 4 1C by periodical subculturing. All

strains were included in the Centro Regional Universitario

Bariloche Culture collection under the CRUB acronym.

Physiological characterization and sexualcompatibility studies

A selection of five physiological tests (assimilation of

inositol, erythritol and D-glucuronate as the sole carbon

sources, assimilation of nitrate as the sole nitrogen source

and production of amyloid compounds) was performed

according to Yarrow (1998). Urease test and fermentation

assays were also performed for the nonpigmented yeast

isolates. For sexual compatibility studies, pairs of 2–3-day

old cultures were crossed on SG agar (g L�1; soytone 2,

glucose 2, agar 15). After a 1-week incubation at room

temperature, the plates were examined upside down under

the optical microscope at a low magnification to check for

the production of mycelium and teliospores.

FEMS Microbiol Ecol 69 (2009) 353–362c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

354 D. Libkind et al.

MSP-PCR fingerprinting

DNA extraction and PCR procedures were performed

according to Libkind et al. (2003). The core sequence of the

phage M13 (50-GAGGGTGGCGGTTCT-30) was used in

MSP-PCR experiments as described in Libkind et al.

(2003). In some cases, the synthetic oligonucleotides

(GTG)5 and (GAC)5 were used for additional MSP-PCR

assays. DNA banding patterns were visualized under UV

transilluminator and images were acquired using a Kodak

Digital Science EDA 120 System and Kodak Digital Science

1D image analysis software. DNA banding patterns were

analyzed using the GELCOMPAR software package, version 4.1

(Applied Maths, Kortrijk).

rRNA sequence analysis

DNA was extracted using the methods described for PCR

fingerprinting and amplified using primers ITS5 (50-GGA

AGT AAA AGT CGT AAC AAG G-30) and LR6 (50-CGC

CAG TTC TGC TTA CC-30). Cycle sequencing of the

600–650 bp region at the end of the 26S rRNA gene D1/D2

domain used the forward primer NL1 (50-GCA TAT CAA

TAA GCG GAG GAA AAG-30) and the reverse primer NL4

(50-GGT CCG TGT TTC AAG ACG G-30). Sequences were

obtained with an Amersham Pharmacia ALF express II

automated sequencer using standard protocols. Alignments

were made with MEGALIGN (DNAStar) and visually corrected.

GenBank accession numbers of the sequences determined in

this study are EF595745–EF595769.

Mycosporine production studies

Synthesis of mycosporines was induced by transferring young

cultures (24 h) to YPD agar medium (g L�1; yeast extract 10,

peptone 20, glucose 20, agar 15) and the plates were incubated

for 4 days at 18 1C in an environmental test chamber (SANYO

MLR 350) with a 12 : 12 light : dark photoperiod. The chamber

was illuminated with 10 white light fluorescent tubes (SANYO,

40 W) and five Q-Panel 340 fluorescent tubes, resulting in

photosynthetically active radiation (PAR), UVA and UVB

irradiances of 66, 15 and 0.7 Wm�2, respectively. For the

screening analysis, Petri dishes containing isolated colonies

were shielded with a Ultraphan-395 film (UV Opak, Digefra,

Munich, Germany, cutoff: 395 nm) and exposed to PAR only.

After exposure, colonies were transferred to distilled water,

centrifuged and conserved at � 20 1C until mycosporine

extraction was carried out. Mycosporine extraction and spec-

trophotometric detection were performed following the pro-

tocols described by Libkind et al. (2005a, b).

UVB survival experiments

In a first experiment, a set of five (one per lake) randomly

selected strains of Rhodotorula mucilaginosa were used for

UVB survival assays. The type strain of this species (CBS

316T) was included for comparison purposes. The second

experiment consisted in comparing the UVB resistance of

three Dioszegia strains (two species) with that of three

Cystofilobasidium strains (three species). The UVB resistance

of a strain of Sporobolomyces ruberrimus was also tested.

Rhodotorula mucilaginosa strains were propagated in Erlen-

meyer flasks with 100 mL of minimal medium salt (MMS)

without CaCl2 in an incubator shaker INNOVA4000 set at

180 r.p.m. and 24 1C. Cells were harvested by centrifuging

culture broth (5 min, 1000 g), rinsed twice with sterile-

distilled water and transferred to quartz test tubes containing

20 mL of sterile-distilled water to reach a final concentration

of 2� 105 cells mL�1. The tubes were exposed to a Spectroline

XX15-B UVB lamp covered with a cellulose acetate (UVC

Fig. 1. Map showing the location of the five

mountain lakes surveyed.


355Yeasts from high-altitude lakes

filter), resulting in PAR, UVA and UVB irradiances of

6.35� 10�5, 1.6� 10�4 and 1.27� 10�5 W s�1 cm2, respec-

tively. Aliquots of 1 mL were taken after 60 and/or 120 min

of exposure, conveniently diluted and plated (four replicates)

on solid media in order to obtain 30–300 colonies per plate.

Dioszegia and Cystofilobasidium yeasts were propagated in

MMS agar medium under the same conditions and sus-

pended in distilled water at the desired cell concentration

(2� 105 cells mL�1) before UVB exposure. The experiments

were performed at least twice for each strain. After 4–7 days

of incubation at 20–22 1C, the number of colonies was

recorded under a microscope (Olympus SZX9). SDs of the

mean values obtained in controls (considered as 100% of

survival) were considered by error propagation as described

by Harris (1991).

Statistical analysis

The results of quantitative yeast studies and UVB experi-

ments were analyzed by means of one-way ANOVA and all

pairwise multiple comparison procedures (Tukey’s test).

Normality and homoscedasticity were tested.

Statistical comparisons of yeast quantitative data between

high- and low-altitude lakes were performed by Student’s t-

tests. The relationship between the percentage of pigmented

yeasts and water transparency, conductivity and chlorophyll

a level was determined using standard linear correlations.

Results and discussion

Yeast occurrence and quantitative studies

All water samples collected from five mountain lakes in

northwestern Patagonia had viable yeast cells. In general, the

total cell counts ranged from 59� 10 cells L�1 (Azul lake) to

208� 23 cells L�1 (Ilon lake) (Table 1). These values were

not significantly different (P4 0.01) from those reported

for Patagonian piedmont lakes (Libkind et al., 2003), except

for Negra lake, which had the highest yeast count

(890� 108 CFU L�1, 93% of which were pigmented yeasts).

This value cannot be attributed to a high anthropic influ-

ence because of the secluded location of this lake. Phyllo-

plane run-off is also unlikely because it is surrounded by

scattered shrubs. The fact that 87.5% of the pigmented

yeasts occurring in Negra lake were R. mucilaginosa suggests

that possibly an occasional surge of organic matter caused a

temporary increase of this yeast population. All other lakes

surveyed showed yeast values typical of open waters of

nonpolluted lakes (Hagler & Ahearn, 1987; Nagahama, 2006).

The percentages of pigmented yeasts over the total yeast

counts were variable, as was the case for other Patagonian

aquatic environments (Libkind et al., 2003) (Table 1). No

significant differences were found between the percentage of

pigmented yeasts of high-altitude lakes and that of Patago-

nian piedmont lakes (t =� 0.4424, P = 0.6604). However, in

terms of only mountain lake data, a significant positive

correlation was found between lake transparency and the

percentage of pigmented yeasts in the water samples

(R2 = 0.67, P4 0.001). Moreover, transparent lakes typically

show low conductivity and chlorophyll a, and in agreement

with this, we observed that both variables were negatively

correlated to the percentage of pigmented yeasts, R2 = 0.75

and 0.92, respectively (Verde lake was considered an outlier

and was thus excluded from this analysis). Apparently,

extremely transparent mountain lakes are more likely to

have elevated proportions of pigmented yeasts compared

with less transparent ones. Future studies including lakes

Table 1. Geographic location, some optical and chemical characteristics of the lakes studied and results on yeast quantification and percentage of

pigmented yeasts

Lake Lat. Long.

Alt.

(m)

Max. depth

(m)

Area

(km2)�Transp.

(m)

Cond.

(mS cm�1)

Chl-a

(mg m�3)

Yeast

counts

(CFU L�1)w

% of

pigmented

yeastsw

Ilon 411110 711560 1370z 4 12z 0.45 4 10z 17.5z 0.47z 208� 23a 16�4a

Negra 411080 711350 1450z 4 15z 0.11 4 15z 5.8‰ 0.12z 890� 108b 93�3b

Verde 411150 711170 1545k 5k 0.005 2.1�� 29.0‰ 9.72ww 132� 68a 21�5a

Azul 411120 711590 1520z 4 15�,z 0.67 4 15z 5.6z 0.15z 59� 10a 70�4b

Toncek 411120 711290 1700z 12z 0.05 6�� 12.3‰ 0.55z 87� 20a 20�2a

�Estimated.wMean values and SD are shown.zThis study.‰Tartarotti et al. (2004).zZagarese et al. (1999).kZunino & Dıaz (2000).��Romero (1986).

Values with different letters are significantly different (Po 0.001).

Lat., latitude; Long., longitude; Alt., altitude; Max., maximum; Transp., transparency; Cond., conductivity; Chl-a, chlorophyll a.



over a broader altitude range will be carried out to verify this

trend. The relationship between pigment production and

water transparency is in agreement with recent results that

indicate a higher tolerance to UVR of yeasts that contain

carotenoids over those that do not (Moline, 2004; Moline

et al., 2009).

Polyphasic identification of the isolates

Seventy-five pigmented yeast strains and 17 nonpigmented

ones were isolated. These yeasts were preliminarily grouped

based on their cultural and physiological characteristics. It

was found that inositol and erythritol tests did not discri-

minate the pigmented isolates (mostly negative). A more

detailed grouping of the isolates was achieved with MSP-

PCR experiments. The direct comparison of MSP-PCR

banding patterns with those obtained for selected type

strains (based on the previous cultural and physiological

results) led to the rapid identification of 71% of the

pigmented strains under study. The species detected by this

method were R. mucilaginosa, Rhodotorula minuta, Rhodo-

torula laryngis, S. ruberrimus and Sporidiobolus longiusculus

(data not shown). The latter species was found to mate with

a strain isolated from a piedmont lake (Libkind et al., 2003)

and was described as a novel species (Libkind et al., 2005a).

A group of 15 pigmented non-ballistoconidia-forming

strains, which were inositol negative, erythritol negative,

D-glucuronate negative and nitrate positive, had similar M13

DNA banding patterns, and resembled profiles (character-

ized by a predominant band of 575 bp) observed previously

in species belonging to the Rhodosporidium babjevae/Rhodo-

torula glutinis clade of the Sporidiobolales order (Sampaio

et al., 2001; Gadanho et al., 2003; Libkind et al., 2003).

Further studies using the (GAC)5 primer allowed the

formation of five groups (data not shown). Representative

strains of these groups were subsequently subjected to 26S

rRNA gene sequence analysis of the D1/D2 domains for final

identification. Three of these MSP-PCR classes were

R. babjevae, although certain heterogeneities in nucleotide

sequences were found (Table 2). The rest belonged to

Rhodosporidium diobovatum (one isolate), and to a new

species of Rhodotorula (three isolates). Neither of the two

teleomorphic Rhodosporidium species showed sexual

activity when crossed together or with their respective

mating types.

The isolates with white to cream colonies, as well as those

resembling yeast-like fungi or black yeasts, were grouped as

nonpigmented yeasts. The 17 isolates studied were classified

into 12 different species: two ascomycetous yeasts, eight

basidiomycetous yeasts, one yeast-like (Aureobasidium pull-

ulans) and one black yeast (Venturia hanliniana) (Table 2).

Representative strains of the remaining MSP-PCR classes

of pigmented and nonpigmented yeasts were selected for

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

gene. The BLAST-based (Altschul et al., 1997) identification

results indicated the existence of 12 pigmented and 12

nonpigmented yeast species (Table 2). Identification down

to the species level through sequence analysis for all of the

isolated yeasts was not possible, because significant differ-

ences were found when comparing with GenBank reference

sequences. In those cases, the name of the closest species

found in the BLAST search was retained if less than three base

substitutions were found for the D1/D2 region. Two pig-

mented species showed nine or more nucleotide differences

in comparison with the closest known species, and were

hence considered novel taxa.

Only five out of 12 nonpigmented species found pre-

sented a 100% identical D1/D2 sequence when compared

with the closest GenBank BLAST match (Table 2). Noteworthy

are the strains Cryptococcus sp. 1 CRUB 1165 and Crypto-

coccus sp. 2 CRUB 1154, showing 14 and seven nucleotide

differences when compared with Filobasidium globisporum

CBS 7642T and Cryptococcus festucosus VKM Y-2930T,

respectively.

Yeast diversity

Pigmented isolates were classified into seven genera and 12

species. Rhodotorula mucilaginosa was the most frequently

isolated species, followed by R. babjevae, S. ruberrimus,

Sporobolomyces roseus, R. diobovatum, R. minuta, R. laryngis,

Dioszegia hungarica, Dioszegia fristingensis and S. longiuscu-

lus. Basidiomycetous species were the predominant group

between nonpigmented species, and there were no common

species among the lakes.

Comparative analyses of the pigmented yeast biodiversity

found both in high-altitude water bodies and in piedmont

lakes could be performed. In the latter aquatic environ-

ments, it was observed that in most water samples

R. mucilaginosa was present, representing c. 50% of the total

number of the isolates (Libkind et al., 2003). From previous

results it was apparent that in water bodies with a low

anthropic influence, Sporobolomyces spp. species prevailed

and R. mucilaginosa was absent. In this case, in all five

mountain lakes sampled, the latter species constituted the

most frequent taxon (40–92% of the total pigmented

isolates), whereas Sporobolomyces spp. were not abundant.

The low occurrence of Sporobolomyces, in particular

S. ruberrimus, in these extreme environments may be

explained by the relatively low UVB resistance of this species

(data not shown).

The initial presumed specialization of R. babjevae to

terrestrial substrates (Golubev, 1993) has been recently

questioned, because strains of this species have been fre-

quently isolated from freshwater (Libkind et al., 2003; and

the present study) or even marine environments (Gadanho



et al., 2003). In this work, R. babjevae was the second most

frequently isolated species, and was present in three of the

five lakes studied, and has been cited in several piedmont

lakes (Libkind et al., 2003). Other less frequent species such

as S. ruberrimus, S. roseus, R. laryngis and R. minuta have

also been found in piedmont lakes. This was not the case for

R. diobovatum, a species frequently, if not exclusively,

isolated from seawater (Nagahama et al., 2001; Gadanho

et al., 2003; Almeida, 2005), which was isolated here,

although at a very low frequency and incidence, in freshwater

from of Patagonian high-altitudes lakes. A similar case is that

of D. hungarica (formerly Cryptococcus hungaricus), of which

several strains have been isolated from marine environments

together with other terrestrial substrates (Fell & Statzell-

Tallman, 1998). The isolation of two strains of D. fristingensis

is noteworthy since only one strain of this recently described

species was known (Inacio et al., 2005). Several Dioszegia

strains have been found recently in glacial melt waters in

Patagonia (de Garcıa et al., 2007). These results suggest that

Dioszegia species are frequent in Andean aquatic environ-

ments, probably as consequence of phylloplane run-off.

Differences in pigmented yeast species distribution were

observed between high- and low-altitude aquatic environ-

ments, particularly within taxa of the Hymenomycotina sub-

phyla. The presence of species of the Tremellales (Dioszegia

spp.) was detected in high-altitude lakes, whereas such yeasts

had not been previously isolated in lowland Patagonian lakes

surveyed to date. The inverse situation was observed for

Cystofilobasidiales species such as Cystofilobasidium capita-

tum, Cystofilobasidium infirmominiatum, Cystofilobasidium

macerans and Cystofilobasidium lacus-mascardii (Libkind

et al., 2003, 2009), which were frequently found in piedmont

lakes, but were absent in mountain lakes. A possible explana-

tion for these findings may be provided by the study of

photoprotective compounds’ production and UVB survival

experiments. Both lowland and elevated lakes shared yeast

Table 2. Identification, taxonomic classification, distribution, occurrence and mycosporine production of yeasts isolated from mountain lakes

Species

Fungal

group� n

Lakesw

MSP-PCRC MYC

BLAST match % similarity

(no. nt differences)V A T I N

Pigmented species

Rhodotorula mucilaginosa B/P/Sp 50 16 38 13 13 753 1T � 100

Rhodosporidium babjevae B/P/Sp 10 16 – 3 – 13 3 � 100–99 (0–1)

Sporobolomyces ruberrimus B/P/Sp 2 – 3 – 4 – 1T � –

Sporidiobolus longiusculus B/P/Sp 2 – – – 2 – 1T � 100

Rhodotorula sp.z B/P/Sp 3 – – – – 10 1 � 97 (10)

Sporobolomyces marcillae B/P/Sp 1 – – – 2 – 1 � –

Rhodosporidium diobovatum B/P/Sp 1 – – – – 3 1 � 100

Rhodotorula laryngis B/P/C 1 – – – 2 – 1T 1 100

Rhodotorula minuta B/P/C 1 – – – – 3 1T 1 100

Dioszegia hungarica B/H/T 1 – – – 2 – 1 1 100

Dioszegia sp.z B/H/T 1 – – 1 – – 1 1 98 (9)

Dioszegia fristingensis B/H/T 2 – – – 2 3 2 1 99 (1)

Nonpigmented species

Candida coipomoensis A/S/Sa 1 4 1 – – – – 1 � 99 (1)

Leucosporidiella creatinivora B/P/L 1 4 1 – – – – 1 � 99 (2)

Leucosporidiella muscorum B/P/L 1 – – – – 4 1 1 � 100

Cryptococcus sp. 1z B/H/F 1 – – – – 4 1 1 1 98 (14)

Cryptococcus sp. 2z B/H/F 1 – – – – 4 1 1 1 98 (7)

Cryptococcus albidus B/H/F 1 – – – – 4 1 1 1 99 (1)

Venturia hanliniana A/Pe/Pl 1 – 4 1 – – – 1 ND 100

Cryptococcus antarticus B/H/F 2 – 4 1 – – – 1 1 99 (3)

Cryptococcus gastricus B/H/F 1 – – 4 1 – – 1 � 99 (3)

Cryptococcus saitoi B/H/F 2 – – – 41 – 2 1 100–99 (0–1)

Hanseniaspora opuntiae A/S/Sa 1 – – – 41 – 1 � 100

Aureobasidium pullulans A/Pe/D 4 – – – 41 – 1 ND 100

�Basidiomycetous yeasts classification was based on Bauer et al. (2006). A, Ascomycota; B, Basidiomycota; P, Pucciniomycotina; H, Hymenomycotina;

Pe, Pezizomycotina; S, Saccharomycotina; Sp, Sporidiobolales; C, Cystobasidiales; T, Tremellales; Sa, Saccharomycetales; L, Leucosporidiales; F,

Filobasidiales; Pl, Pleosporales; D, Dothioraceae.wNumbers indicate yeast cell densities in CFU L�1, lakes: V, Verde; A, Azul; T, Toncek; I, Ilon; N, Negra. n, number of isolates.zNovel taxa, BLAST results indicate number of nucleotide differences to closest known species (in descent order): Rhodotorula araucariae CBS 6031T,

Dioszegia hungarica CBS 4214, Filobasidium globisporum CBS 7642T and Cryptococcus festucosus VKM Y-2930T. MYC, mycosporines production. ND,

not determined; C, number of different MSP-PCR classes found for each species; T, identical to respective type strain.



members of the orders Sporidiobolales and Cystobasidiales as

representatives of the Pucciniomycotina.

Photoprotective compounds and UVR

The ability of the isolates to produce photoinducible UV-

absorbing compounds (mycosporines), when grown under

intense light, was investigated. Among pigmented yeasts,

mycosporine-synthesizing species were poorly represented

in high-altitude lakes, an environment exposed to high

UVR, in which the ability to accumulate photoprotective

compounds could be a useful adaptation. Only species not

belonging to the Sporidiobolales (R. minuta, R. laryngis and

Dioszegia spp.) were positive for mycosporine production.

Libkind & van Broock (2006) observed that species of the

Sporidiobolales group had higher constitutive levels of total

carotenoids than species that did produce mycosporine.

These basal levels of pigments may provide enough photo-

protection for the successful colonization of highly UV

exposed habitats such as the ones studied here.

The proportion of nonpigmented species able to produce

mycosporine (54%, the black yeast was excluded) was higher

than that observed for pigmented yeasts (38%). Higher

percentages of mycosporine-positive nonpigmented species

were found in the lakes with higher transparency (i.e. Negra

and Azul lake), as was the case for carotenoid-producing

yeasts. Verde lake, the less transparent water body studied

(see Table 2), did not have mycosporine-synthesizing yeasts

either among pigmented or among nonpigmented species.

Interestingly, all Dioszegia isolates from Patagonian

mountain lakes were mycosporine positive. The synthesis

of these UV screening compounds by Dioszegia yeasts may,

at least in part, explain their occurrence in Patagonian high-

altitude lakes exposed to high UVR. This hypothesis is also

in agreement with the typical phylloplane habitat of Diosze-

gia spp. (Bai et al., 2002; Inacio et al., 2002, 2005; Madhoura

et al., 2005), which is a substrate exposed to high levels of

solar irradiation. Inversely, the absence of Cystofilobasidium

species in mountain lakes (despite their presence in lowland

water bodies) could be related to the lack of photoprotective

compounds in this species (Libkind et al., 2005a, b). How-

ever, a more extensive sampling is necessary to confirm this

hypothesis.

UVB survival

Randomly selected R. mucilaginosa strains from each lake

and the species type strain (CBS 316T) were tested for UVB

tolerance under the same experimental conditions. The

results of this assay showed a significantly (Po 0.001) lower

resistance of the type strain and the strain isolated from

Verde lake than the rest of the tested strains (Fig. 2). The

UVB resistance of the other four R. mucilaginosa strains was

approximately threefold higher. These differences were not

clearly correlated with carotenoid pigment content (data not

shown), probably because this species has alternative photo-

protective and/or antioxidant mechanisms. The fact that the

isolate from the less transparent lake (Verde lake) was the

most UVB sensitive among the native strains suggests that

transparent waters induce additional UVB resistance neces-

sary to survive in high-altitude lakes. The photoprotective

mechanisms responsible for this additional UVB resistance

of R. mucilaginosa strains are being investigated by our

laboratory.

To help understand the presence of Diozegia yeasts in

high-altitude lakes and the simultaneous absence of Cystofi-

lobasidium yeasts despite their presence in lowland water

bodies, the UVB survival of representative strains/species of

both groups was studied. The results of these experiments

are shown in Table 3. Our findings indicate that Dioszegia

yeasts possess an outstanding UVB resistance, having survi-

val rates 4 50% after a 120-min exposure. These values are

even higher than those observed for all R. mucilaginosa

strains, and for R. babjevae and S. ruberrimus, other com-

mon yeast species from high-altitude lakes (data not

0

10

20

30

40

50

Type Verde Azul Ilon Toncek Negra

UV

R-B

sur

viva

l (%

)

a

a

bb

b

b

Fig. 2. UVB survival of Rhodotorula mucilaginosa strains. Different

letters indicate statistical significant differences (Po 0.001).

Table 3. Differential UVB resistance between Dioszegia and Cystofilo-

basidium yeasts

Species CRUB no. Origin

UVB survival (%)

60 min 120 min

D. fristingensis 1150 L. Ilon ND 95.2� 32.7

D. fristingensis 1152 L. Negra ND 89.4� 16.9

Dioszegia sp. 1147 L. Toncek ND 51.1� 8.2

C. capitatum 1111 Minas river 0 0

C. infirmominiatum 1045 L. Fonck 0.1� 0.2 0

C. macerans 1618 Cyttaria hariotii ND 1.9� 0.4

ND, not determined.



shown). Thus, the presence of Dioszegia spp. in highly

exposed UVR environments is well justified by its excellent

ability to cope with high UVB irradiances. The fact that

MGG absorbs mainly UVB (maximum A310 nm), and that it

is accumulated at very high concentrations particularly in

Dioszegia (Libkind et al., 2005a, b), suggests that the synth-

esis of mycosporine may enhance the UVB resistance of

these yeasts.

When exposed to the same UVB conditions as Dioszegia

spp., Cystofilobasidium yeasts showed almost no growth

(0–2% survival), evidencing a high susceptibility to UVB,

which could explain their absence in mountain water

bodies. Although the presence of Cystofilobasidium yeasts in

the environment surrounding mountain lakes has not been

confirmed, their occurrence in such environments is prob-

able because several substrates typically colonized by these

yeasts are shared with lowland lakes (i.e. stromata of

Cyttaria hariotii and soil).

Final remarks

This work is the first description of the yeast diversity of

mountain lakes and its relationship with physical, chemical

and environmental factors, especially UVR. Solar UVR

(290–400 nm) is a crucial environmental factor in mountain

lakes (Sommaruga, 2001); however, unlike most other

aquatic organisms (Zagarese et al., 1999; Sommaruga, 2001;

Fernandez Zenoff et al., 2006), the ecological importance of

UVR in mountain lakes yeast flora has not been studied. We

found an interesting diversity of yeast species, including

novel ones, inhabiting such extreme habitats. Water trans-

parency is one of the factors conditioning UV penetration in

aquatic environments. Our results strongly suggest a rela-

tionship between the ability to produce photoprotective

compounds (carotenoids and mycosporines) and the trans-

parency of mountain lakes. Carotenoid- and/or mycospor-

ine-synthesizing yeasts prevailed in highly transparent

waters, in which UVR probably eliminates susceptible yeasts,

selecting the more resistant ones. An example of highly

UVR-resistant yeasts is Diozsegia spp., which were only

found in high-altitude lakes. Furthermore, UVB-susceptible

yeast groups (i.e. Cystofilobasidium spp.), although common

in lowland lakes, were not found at high altitudes. Thus,

UVR is an important environmental factor affecting yeast’s

community structure in aquatic habitats. We also observed

that, at least for R. mucilaginosa yeasts, UVB resistance may

vary among strains, and that this variation may depend on

the UV underwater climate of the lake. Similar to other

aquatic microorganisms thriving in high-altitude lakes

(Fernandez Zenoff et al., 2006), yeasts have apparently

developed a number of strategies to minimize UV damage.

The widespread synthesis or bioaccumulation of different

compounds that directly (mycosporines) or indirectly (caro-

tenoids) absorb UV energy may be one of these strategies.

The photoprotective role in yeasts of these metabolites is yet

to be experimentally demonstrated. However, the evidence

accumulated to date, and that provided in the present work,

suggests that both carotenoids and mycosporines have a

UVR-protective function.

Acknowledgements

This work was accomplished with financial aid from the

Universidad Nacional del Comahue (Project B121), Consejo

Nacional de Investigaciones Cientıficas y Tecnologicas

(CONICET – Project PIP6536) and ANPCYT (Project

PICT-1176). SECYT-GRICES bilateral cooperation agree-

ment (PO/PA02-BI/002) and the ICSU/TWAS/UNESCO

short fellowship Programme in the Basic Sciences supported

D.L.’s travel and subsistence in Portugal. We would like to

thank the authorities of Parques Nacionales (Argentina), for

providing permission for water sample collection within the

Nahuel Huapi National Park. Special thanks are due to

M. de la Vega, H. Libkind and the Tato’s for their valuable

help during high-altitude water sampling. Thanks are also

due to A. de Negri for map design.

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Yeasts from high-altitude lakes: influence of UV radiation