RESEARCH ARTICLE
Saccharomyces eubayanus and Saccharomyces uvarum
associated with the fermentation of Araucaria araucana seeds
in Patagonia
rez-Trave
s3, Marcela P. Sangorrın1, Eladio Barrio3,4 &
M. Eugenia Rodrıguez1,2, Laura Pe
1,5
Christian A. Lopes
Correspondence: Christian A. Lopes, Grupo
de Biodiversidad y Biotecnologıa de
Levaduras, Instituto Multidisciplinario de
n y Desarrollo en Ingenierıa de
Investigacio
procesos, Biotecnologıa y Energıas
Alternativas (PROBIEN, Consejo Nacional de
Investigaciones Cientıficas y Tecnicas de la
blica Argentina – Universidad Nacional
Repu
del Comahue), Facultad de Ingenierıa, UNCo,
Buenos Aires 1400 (8300) Neuquen,
Argentina. Tel.: +54 299 4490300 int. 682;
fax: +54 299 4490300;
e-mail: clopes@conicet.gov.ar
Received 20 March 2014; revised 29 June
2014; accepted 7 July 2014. Final version
published online 04 August 2014.
YEAST RESEARCH
DOI: 10.1111/1567-1364.12183
Editor: Cletus Kurtzman
Abstract
Mudai is a traditional fermented beverage, made from the seeds of the Araucaria araucana tree by Mapuche communities. The main goal of the present
study was to identify and characterize the yeast microbiota responsible of Mudai fermentation as well as from A. araucana seeds and bark from different
locations in Northern Patagonia. Only Hanseniaspora uvarum and a commercial bakery strain of Saccharomyces cerevisiae were isolated from Mudai and all
Saccharomyces isolates recovered from A. araucana seed and bark samples
belonged to the cryotolerant species Saccharomyces eubayanus and Saccharomyces uvarum. These two species were already reported in Nothofagus trees from
Patagonia; however, this is the first time that they were isolated from A. araucana, which extends their ecological distribution. The presence of these species
in A. araucana seeds and bark samples, led us to postulate a potential role for
them as the original yeasts responsible for the elaboration of Mudai before the
introduction of commercial S. cerevisiae cultures. The molecular and genetic
characterization of the S. uvarum and S. eubayanus isolates and their comparison with European S. uvarum strains and S. eubayanus hybrids (S. bayanus
and S. pastorianus), allowed their ecology and evolution us to be examined.
Keywords
cryotolerant yeast; hybrids; Saccharomyces
bayanus; yeast diversity.
Introduction
Aboriginal communities in Andean Patagonia (Argentina
and Chile) used to prepare fermented beverages from several raw sources, including cereals and fruits. The Mapuche community, also known as Araucanians, was the
most important aboriginal group inhabiting the temperate forests in Andean Patagonia (de M€
osbach, 1992; Donoso & Lara, 1996). This typical gatherer community used
several available wild fruits, such as beach strawberries
(Fragaria chiloensis), ‘maqui’ or Chilean wineberry (Aris-
ª 2014 Federation of European Microbiological Societies.
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totelia chilensis), ‘calafate’ or Magellan barberries (Berberis
spp.), and others, to produce fermented beverages (Pardo
& Pizarro, 2005).
One of the most interesting cases for study is a traditional fermented beverage, called Mudai, generally used in
religious ceremonies by Mapuche communities. This soft
beverage is made from the seeds, ng€
ulliw in the Mapuche
language, of the Araucaria araucana tree, called Pehuen,
which is a gymnosperm endemic of the lower slopes of
the Chilean and Argentinian south-central Andes, typically above 1000 m of altitude. In Argentina, it occupies a
FEMS Yeast Res 14 (2014) 948–965
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1
n y Desarrollo en Ingenierıa de procesos,
Grupo de Biodiversidad y Biotecnologıa de Levaduras, Instituto Multidisciplinario de Investigacio
blica Argentina - Universidad
Biotecnologıa y Energıas Alternativas (PROBIEN, Consejo Nacional de Investigaciones Cientıficas y T
ecnicas de la Repu
edicas, Universidad Nacional
en, Argentina; 2Facultad de Ciencias M
Nacional del Comahue), Facultad de Ingenierıa, UNCo, Buenos Aires, Neuqu
del Comahue, Comahue, Neuquen, Argentina; 3Departamento de Biotecnologıa, Instituto de Agroquımica y Tecnologıa de los Alimentos, CSIC,
encia, Spain; and 5Facultad de Ciencias Agrarias, Universidad
encia, Val
Paterna, Val
encia, Spain; 4Departament de Genetica, Universitat de Val
Nacional del Comahue, Neuquen, Argentina
949
Saccharomyces in Patagonia
FEMS Yeast Res 14 (2014) 948–965
ferentiate these two species were proposed by Nguyen
et al. (2011) and Pengelly & Wheals (2013).
The aim of the present study was to identify and characterize fermentative yeasts present during fermentation
performed with A. araucana seeds, according to the traditional elaboration procedures, in different locations in
Northern Patagonia. Additionally, the fermentative yeast
biota present in seed and bark samples from the A. araucana tree, from which Mapuche communities obtain the
seeds used in Mudai elaboration, was also sampled and
isolated using selective media.
The genetic characterization of Saccharomyces strains
was performed by PCR-RFLP (polymerase chain reaction
restriction fragment length polymorphism) and sequencing of different nuclear genes, and sequencing of the
mitochondrial gene COX2. The phylogenetic relationships, at the inter- and intra-specific levels, between
native isolates were obtained to determine their origins.
The presence of commercial bakery yeasts in artisanal
traditional beverages as well as the presence of natural
populations of S. eubayanus and S. uvarum associated
with A. araucana trees is described for the first time in
this study.
Materials and methods
Sampling areas
Samples from A. araucana seed fermentation were
obtained from three different areas in Northwestern Patagonia (Neuquen province): Villa Pehuenia (38°540 00″S,
71°190 5800 W, altitude: 1200 m), Junın de los Andes
(39°570 0300 S, 71°040 1500 W, altitude: 902 m) and Huechulafquen (39°790 9000 S, 71°220 5700 W, altitude: 875 m)
(Fig. 1). Fermentations were performed from April to
May, during the Southern Hemisphere autumn.
Araucaria araucana bark and seed samples were collected from three different sampling areas in the same
region: Caviahue (37°520 4400 S, 71°030 5300 W, altitude:
1600 m), Tromen (39°350 0300 S, 71°250 3300 W, altitude:
1250 m) and Huechulafquen (Fig. 1). Sampling in these
areas was carried out during the summer. Annual average
precipitation and temperatures in the different localities
are as follows: Caviahue, 600–1000 mm, ≤ 10 °C; Tromen,
350 mm, 13 °C; Huechulafquen, ≥ 800 mm, ≤ 10 °C.
Isolation of fermentative yeasts
Sampling from Mudai fermentation
Musts were obtained by trituration of A. araucana seeds,
boiling and addition of commercial sucrose according to
traditional methodologies. Musts were transported to the
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narrow strip on the Patagonian Andes ranging from
37°500 to 39°200 S in Neuquen province. Pehuen seeds
have also constituted an important source of carbohydrates for the Mapuche peoples from this area, who are
in fact called Pehuenche (Pehuen people). Pehuen seeds
are eaten raw, boiled or roasted and often ground into
flour to be used as an ingredient in soups and to make
bread and Mudai (Herrmann, 2005).
No literature on the microbial biota present during
Mudai fermentation is available, probably due to the difficulties of obtaining samples from the fermentation performed by Mapuche communities. However, it is well
known that yeasts belonging to Saccharomyces species,
particularly Saccharomyces cerevisiae, are related to diverse
processes including baking, brewing, distilling, winemaking, cider production, and are used in different traditional
fermented beverages and foods around the world (Nout,
2003). In Patagonia, the species S. cerevisiae has been
associated with winemaking environments (Lopes et al.,
2002; Saez et al., 2011) and fruit surfaces during postharvest cold storage (Robiglio et al., 2011); however, other
species of the genus, such as Saccharomyces bayanus var.
uvarum (or Saccharomyces uvarum) and the newly
described species Saccharomyces eubayanus, were isolated
from Patagonian natural habitats in association with Nothofagus trees (Libkind et al., 2011). Two recent studies
(Bing et al., 2014; Peris et al., 2014) extended the geographic range in which S. eubayanus has been isolated.
These authors reported for the first time the presence
S. eubayanus strains from different oaks in the Tibetan
Plateau in Far East Asia and from Fagus and Acer trees in
Wisconsin, USA, respectively.
Nowadays, 10 species are included in the Saccharomyces
genus: Saccharomyces arboricolus, S. bayanus, Saccharomyces cariocanus, S. cerevisiae, S. eubayanus, Saccharomyces
kudriavzevii, Saccharomyces mikatae, Saccharomyces paradoxus, Saccharomyces pastorianus and S. uvarum. However, the discovery of S. eubayanus reignited discussion in
the scientific community about the taxonomic position of
S. bayanus and S. pastorianus. Libkind et al. (2011)
demonstrated that S. bayanus is a taxon composed by
heterogeneous hybrid strains between S. uvarum and
S. eubayanus with minor contributions from S. cerevisiae
in some cases, and S. pastorianus is an hybrid between
S. cerevisiae and S. eubayanus. In a recent work by
Gonzalez et al. (2006), and modified by Perez-Traves et al.
(2014), a rapid method was proposed to differentiate both
‘uvarum’ and ‘eubayanus’ alleles based in the gene
sequences obtained from the fully sequenced strains CBS
7001 (also known as MCYC 623, considered the reference
strain of S. uvarum) and S. pastorianus Weihenstephan
34/70, as well as from sequences obtained for S. bayanus
reference strain NBRC 1948. Additional techniques to dif-
950
M.E. Rodrıguez et al.
laboratory and fermented at 20 °C. Yeast isolates were
obtained from different fermentation stages (initial, middle and end). Additionally, samples of musts prepared
and fermented totally (end stages) following traditional
procedures in the place of origin were also analyzed.
Aliquots of appropriate dilutions (0.1 mL each) were
spread onto GPY agar (w/v: 2% glucose, 0.5% peptone,
0.5% yeast extract, 2% agar) supplemented with chloramphenicol (50 mg L1). After incubation at 20 °C for 2–
3 days, 20 colonies from each fermentation stage were
isolated according to their macroscopic features and frequencies and preserved at 20 °C in a glycerol solution
(20% v/v) and conserved in the NPCC (North Patagonian
Culture Collection) in Neuquen, Argentina. The fermentations were carried out in duplicate and their evolution
was daily followed by weight loss until the same weight
was recorded in two consecutive measures.
Sampling from A. araucana trees
Yeasts were isolated from both bark and seeds of A. araucana trees following the methodology proposed by Sampaio & Goncßalves (2008). Araucaria araucana bark
samples (2 g) and seeds (12 g) were collected aseptically
and introduced into 20-mL sterile flasks containing
10 mL of selective enrichment medium consisting in YNB
(yeast nitrogen base; Difco) supplemented with 1% (w/v)
raffinose and 8% (v/v) ethanol and incubated at 30 °C or
10 °C without agitation. Samples exhibiting yeast growth
(checked microscopically) were plated onto GPY agar and
incubated at the same temperature as the bark or seed
samples (10 °C or 30 °C). A representative number of
yeast colonies were selected according to their frequency
and morphology, and were preserved at 20 °C in glycerol solution (20% v/v) in the NPCC.
ª 2014 Federation of European Microbiological Societies.
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Yeast identification
Yeasts were identified by PCR-RFLP of the region
encompassing the ITS1, 5.8S rRNA and ITS2 (5.8S-ITS
region) as described in Lopes et al. (2010). PCR-RFLP
patterns obtained for each isolate were compared with
those of reference strains available in the www.yeast-id.
org database. Yeast identifications were confirmed by
sequencing both the 5.8S-ITS region and the D1/D2
domain of the 26S rRNA gene (Kurtzman & Robnett,
2003).
Sporulation and spore viability analyses
Sporulation was induced by incubating cells on sodium
acetate medium (w/v: 1% sodium acetate, 0.1% glucose,
0.125% yeast extract and 2% agar) for 5–7 days at 26 °C.
Following preliminary digestion of the ascus walls with
zymoliase (Seikagaku Corporation, Japan) adjusted to
2 mg mL1, spores were dissected using a Singer MSM
Manual micromanipulator in GPY agar plates. After incubation at 26 °C during 3–5 days, the spore viability
analysis was performed and the developed colonies were
transferred to the same sporulation medium in order to
determine the homo/heterothallism of the monosporic
cultures.
Mitochondrial DNA restriction analysis
mtDNA-RFLP patterns were analyzed for all isolates identified as belonging to Saccharomyces. Total DNA extraction was performed according to Querol et al. (1992).
Total yeast DNA was subsequently digested with HinfI
restriction enzyme (Roche Diagnostics, Mannhein, Germany) according to the supplier’s instructions and the
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Fig. 1. Location of the sampling areas –
Caviahue, Tromen, Huechulafquen, Villa
Pehuenia and Junın de los Andes – in
Northwestern Patagonia (Neuqu
en province).
Left top corner, from light to dark gray: South
America, Argentina and Patagonia.
951
Saccharomyces in Patagonia
fragments separated in 1% w/v agarose gels containing
TAE (Tris-acetate-EDTA).
PCR-RFLP analysis of nuclear genes
Restriction site maximum parsimony trees
From the restriction site gains and losses required to
explain the RFLP patterns present in native S. eubayanus
and S. uvarum, two binary matrices were constructed to
codify the presence/absence of restriction sites in the
native S. eubayanus and S. uvarum strains, respectively
(Tables S1 and S2). These matrices were used to construct
most-parsimonious trees that minimize the number of
steps required to connect all the S. eubayanus strains and
all S. uvarum strains. These parsimony trees were
obtained with the MIX program included in the PHYLIP
3.695 package (Felsenstein, 2005) by considering restriction site changes as reversible events (Wagner criterion).
Trees were rooted by including genotypes from the reference strain S. uvarum CBS 7001 and hybrid S. pastorianus
W34/70 (eubayanus subgenome).
FEMS Yeast Res 14 (2014) 948–965
Four nuclear gene regions – BRE5 and EGT2, the D1/D2
domain of the 26S gene and ITS1-5.8s-ITS2 – as well as
the mitochondrial gene COX2 were amplified and
sequenced for phylogenetic study.
Nuclear genes were amplified by PCR as described
above and the gene COX2 was amplified using primers
and conditions described in Belloch et al. (2000). PCR
products purification and sequencing were also performed
as described above. These sequences were submitted to
the GenBank database under accession numbers KJ187251
– KJ187304.
Sequences from all different ‘uvarum’ and ‘eubayanus’
alleles from the fully sequenced strains S. uvarum CBS
7001, S. pastorianus Weihenstephan 34/70 (Nakao et al.,
2009) and S. cerevisiae S288c were used for comparative
purposes. Each set of homologous sequences was aligned
with the CLUSTAL method (Thompson et al., 1994) available in the program MEGA5 (Tamura et al., 2011). The
sequence evolution model that fits our sequence data best
was optimized using the maximum-likelihood Bayesian
information criterion (BIC) for model comparison, also
implemented in MEGA5. The BIC measures the relative
support that sequence data give to different models of
evolution and can be used to compare nested and nonnested models. It is defined as follows: BICi = Cdels.eLi +
Nilogen, where n is the sample size (sequence length),
Ni is the number of free parameters in the evolution
model, and Li is the maximum likelihood value of the
data in the model. The smaller the BIC, the better the fit
of the model to the data (Posada & Crandall, 2001).
The best fitting models were the Tamura & Nei
(1993) model for BRE5 sequences, the Tamura (1992)
three-parameter model for EGT2 sequences, and the
Tamura 3-parameter model, with a gamma distribution
of substitution rates with a shape parameter a = 0.07,
for COX2 gene sequences. Nucleotide distances were
corrected according to the corresponding models, estimated in the previous analysis, and were used to obtain
phylogenetic trees with the neighbor-joining method
(Saitou & Nei, 1987). Tree reliability was assessed using
non-parametric bootstrap re-sampling of 1000 replicates.
All these phylogenetic and molecular evolutionary analyses were also conducted using MEGA5 (Tamura et al.,
2011).
In the case of COX2 sequences, due to evidence of
recombination obtained from sequence comparisons,
neighbor-net network analyses were also performed using
the program SPLITSTREE4 (Huson & Bryant, 2006). Neighbor-net network reliability was also assessed using nonparametric bootstrap analysis based on 1000 replicates.
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The different ‘uvarum’ and ‘eubayanus’ alleles was
detected by PCR amplification and subsequent restriction
analysis of 33 protein-encoding nuclear genes according
to Gonzalez et al. (2006) and Perez-Traves et al. (2014).
PCR amplifications were carried out in a Progene Thermocycler (Techne, Cambridge, UK) as follows: initial
denaturing at 95 °C for 5 min, then 40 PCR cycles with
the following steps: denaturing at 95 °C for 1 min,
annealing at 55 °C for 1 min, and extension at 72 °C for
2 min; and a final extension at 72 °C for 10 min. In the
case of genes ATF1, DAL1, EGT2, KIN82, MNT2, MRC1,
RRI2 and UBP7, annealing was performed at 50 °C. Agarose gel preparation and staining were carried out as
mentioned above. Restriction endonucleases AccI, AspI,
Asp700I, CfoI, DdeI, EcoRI, HaeIII, HindIII, HinfI, MspI,
PstI, RsaI, SacI, ScrFI, TaqI and XbaI (Fermentas, Lituania) were used according to the supplier’s instructions.
The PCR-RFLP profiles were compared with those
reported by Perez-Traves et al. (2014; summarized in
their Supporting Information Tables S2 and S3).
When new profiles were detected, their PCR amplifications were sequenced to confirm that they corresponded
to new alleles. These PCR products were cleaned using
the AccuPrep PCR purification kit (Bioneer, Inc.) and
both strands of the DNA were directly sequenced using
the BigDyeTM Terminator v3.0 Cycle Sequencing Kit
(Applied Biosystems, Warrington, UK), following the
manufacturer’s instructions, in an Applied Biosystems
automatic DNA sequencer Model ABI 3730.
Sequencing and phylogenetic analysis
952
Tree topologies obtained with the 50 and 30 regions of
COX2 were compared with the nonparametric Shimodaira & Hasegawa (1999) test based on maximum likelihood, implemented in the program PAML 4.4 (Yang,
2007). This test is used to simultaneously compare sets of
alternative phylogenetic topologies with the same
sequence dataset.
M.E. Rodrıguez et al.
(a)
1
2
3
4
5
6
7
4
5
6
M
8
CB
M
Results
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
(b) 1
2
3
CB
CB
CB
Fig. 2. (a) mtDNARFLP patterns of some Saccharomyces cerevisiae
isolates from Mudai fermentation (lines 1–8) and the commercial
bakery yeast (CB). (b) Different isolates of S. cerevisiae from
fermented apple juice (lines 1–6) used as control to demonstrate the
variability obtained with mtDNA-RFLP method and the commercial
bakery yeast (CB). M, DNA size marker corresponding to lambda DNA
digested with HindIII.
samples from two different substrates (60 samples from
seeds and 60 from bark). Yeasts were obtained in 20%
and 26.6% of the seed samples incubated at 10 and
30 °C, respectively (Table 1). Lower percentages of yeast
recovery were obtained for bark samples at the two temperatures, 16.6% at 10 °C and 10% at 30 °C. According
to the yeast macroscopic morphology and its frequency
in GPY agar plates, a representative number of colonies
were selected and identified using 5.8S-ITS PCR-RFLP
and confirmed by sequencing the D1/D2 domain of
the 26S rRNA gene. All the isolates were identified as
S. eubayanus and S. uvarum, except for those obtained
from the seed sample from Huechulafquen incubated at
30 °C (Table 1), which corresponded to the species Kazachstania servazzii. Both S. eubayanus and S. uvarum were
detected in samples from Tromen, whereas only one of
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The must obtained from mixing ground A. araucana
seeds, water and sugar was prepared in the traditional
way in Villa Pehuenia (North-Patagonia, Argentina) but
was fermented in our laboratory to assess the fermentation kinetics, the biomass production and the sampling of
the yeast biota associated with this fermentation. Three
independent fermentations were carried out from musts
prepared in the place of origin. The fermentations were
complete in 20 days and the maximum yeast population
densities were 1.5 9 108 CFU mL1.
A very low morphological diversity was observed
among the yeast colonies isolated at the beginning of fermentation. The molecular identification, by PCR-RFLP
analysis of the 5.8S-ITS region PCR-RFLP and 26S rRNA
D1/D2 domain sequencing, of representative yeasts confirmed the presence of a low species diversity; only two
species, Hanseniaspora uvarum and Saccharomyces cerevisiae, were present in 80% and 20% of the total biomass,
respectively. In subsequent stages of fermentation, the
yeast biota corresponded exclusively to S. cerevisiae in the
three analyzed fermentations. Musts that had already been
fermented were then obtained from two additional locations from North Patagonia, Junın de los Andes and Huechulafquen, to get a more complete picture of the
possible yeast biota responsible for the fermentation of
this beverage. In these cases, all isolates obtained were
identified as S. cerevisiae. The intraspecific analysis of all
S. cerevisiae isolates from the five kinds of fermentation
by means of mtDNA-RFLP showed a unique restriction
pattern (Fig. 2). Given this unexpected result and considering a possible cross-contamination with commercial
yeasts used for bread elaboration, the mitochondrial DNA
of commercial bakery yeast was analyzed. The mtDNARFLP pattern obtained for the commercial baker yeast
was identical to that detected in our S. cerevisiae isolates
(Fig. 2). This result led us to search for fermentative yeast
populations in the natural environment from where the
raw material for the elaboration of this beverage comes
from. Seeds and bark samples of A. araucana trees were
collected aseptically from three different sampling areas:
Caviahue, Huechulafquen and Tromen (Fig. 1). Following
the methodology for fermentative yeast isolation proposed by Sampaio & Goncßalves (2008), we evaluated 120
953
Saccharomyces in Patagonia
Table 1. Number of bark and seed samples showing yeast growth at 10 and 30 °C and yeast species detected
Samples with
yeast (%)*
Yeast species
Sampling area
Substrate
10 °C
30 °C
10 °C
Caviahue
Bark
Seed
Bark
Seed
Bark
Seed
2
2
1
0
2
4
0
4
2
1
1
3
Saccharomyces
Saccharomyces
Saccharomyces
–
Saccharomyces
Saccharomyces
Huechulafquen
Tromen
(20)
(20)
(10)
(20)
(40)
(40)
(20)
(10)
(10)
(30)
30 °C
eubayanus
eubayanus
uvarum
uvarum/Saccharomyces eubayanus
eubayanus
–
Saccharomyces eubayanus
Saccharomyces uvarum
Kazachstania servazzii
S. eubayanus
S. uvarum/S. eubayanus
these two species was detected in the two other locations
(S. eubayanus in Caviahue and S. uvarum in Huechulafquen) (Table 1). The two yeast species were obtained at
both isolation temperatures and in bark and seed substrates (Table 1).
All the Saccharomyces isolates identified were subsequently subjected to mtDNA restriction analysis to evaluate the existence of one or more different molecular
patterns, i.e. different strains, in the natural populations
of S. eubayanus and S. uvarum. A total of five (U1m–
U5m) and 13 (E1m–E13m) mtDNA profiles were
detected among the S. uvarum and S. eubayanus isolates,
respectively (Table 2). Each sampling area exhibited
unique profiles; however, a shared profile was detected in
several samples from the same area, e.g. the E2m profile
was detected in different seed and bark samples from
Caviahue but was not found in Huechulafquen or in Tromen (Table 2). The greatest profile diversity was observed
in Tromen area, showing three and seven different profiles for S. uvarum and S. eubayanus, respectively. Only
one species, S. eubayanus or S. uvarum, was found in
each separate seed and bark sample, although in some
cases more than one mitochondrial profile was observed
among the isolates obtained from the same sample
(Table 2).
To evaluate the pure nature of the S. uvarum and S. eubayanus yeast isolates, as well as the potential presence of
natural hybrids between these two sympatric species,
isolates representative of each mtDNA restriction profile
were subjected to PCR amplification and subsequent
restriction analysis of 33 nuclear gene regions located on
different chromosomes. This methodology permits the differentiation of ‘uvarum’ and ‘eubayanus’ alleles along the
genome based on the restriction patterns deduced from
the complete genome sequences of the reference strains
S. uvarum CBS 7001 and S. pastorianus (S. eubayanus 9 S. cerevisiae hybrid) Weihenstephan 34/70 (PerezTraves et al., 2014). In most cases, the RFLP patterns
found in native isolates for the 33 gene regions were identical to those found in the non-cerevisiae (i.e. S. eubayanus) subgenome of S. pastorianus Weihenstephan 34/70
or in S. uvarum CBS 7001. These alleles were indicated as
E1 or U1, respectively, in Table 3. However, new patterns
(corresponding to new alleles) differing in one restriction
site gain or loss were also found for some particular genes
(Table 3). These new alleles, named E2 and E3, were
detected in nine gene regions of native S. eubayanus isolates: MET6, GSY1, PEX2, CBP2, DAL1, UBP7, CBT1,
PPR1 and ORC1 (Table 3). The nuclear genes GAL4 and
KIN82 were successfully amplified and digested from all
Table 2. mtDNA-RFLP genetic characterization of Saccharomyces eubayanus and Saccharomyces uvarum native isolates obtained from bark and
seed samples of Araucaria araucana from three different areas
mtDNA-RFLP profile*
Bark samples
Seed samples
Sampling area
T (°C)
1
2
1
2
3
4
Caviahue
10
30
10
30
10
30
E1m E2m
–
U1m
U1m
E7m
E8m
E3m
–
E4m E2m E5m
E2m
–
–
E9m
U4m U5m
E2m E6m
E2m
–
–
E10m E11m
E13m
–
E2m
–
–
E12m
E12m E8m E9m
–
E2m E3m
–
–
E10m E12m
–
Huechulafquen
Tromen
U2m
U3m
–
*Saccharomyces eubayanus and S. uvarum mtDNA profiles are indicated as Em and Um, respectively. The associated numbers indicate the
different profiles.
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*Number and percent of the 10 samples analyzed at 10 and 30 °C of each substrate and sample area (120 samples in total).
954
M.E. Rodrıguez et al.
Table 3. Restriction patterns detected among Saccharomyces eubayanus and Saccharomyces uvarum indigenous yeast. Chromosome order is
based on that described for S. uvarum CBS 7001
S. eubayanus strains (mtDNA pattern)
S. uvarum
Chr.
I
III
VII
IX
XI
XII
XIII
XVI
VIIItXV
XVtVIII
VItX
XtVI
XIVtIItIV
IVtIItII
IItIItXIV
XIVtIItIV
Gene
NPCC
1283 (E2m)
1284 (E5m)
1285 (E6m)
1287 (E3m)
NPCC
1294 (E13m)
1297 (E9m)
1302 (E11m)
NPCC
1282
(E4m)
NPCC
1286
(E1m)
NPCC
1291
(E12m)
NPCC
1292
(E8m)
NPCC
1296
(E7m)
NPCC
1301
(E10m)
NPCC
1288
(U5m)
CYC3
KIN82
MRC1
MET6
NPR2
KEL2
MNT2
DAL1†
UBP7
BAS1
CBT1
MAG2
PPR1
CAT8
ORC1
GAL4
JIP5
CBP2
ATF1
RRI2
EPL1
GSY1
PEX2
CYR1
EUG1
PKC1
RPN4
UGA3
APM3
OPY1
EGT2
BRE5
E1
E1*
E1
E1
E1
E1
E1
E3
E2
E1
E1
E1
E2
E1
E1
E1*
E1
E1
E1
E1
E1
E2
E2
E1
E1
E1
E1
E1
E1
E1
E1
E2
E1
E1
E1
E1
E1
E1
E1
E1
E2
E1
E1
E1
E2
E1
E2
E1
E1
E2
E1
E1
E1
E1
E3
E1
E1
E1
E1
E1
E1
E1
E1
E2
E1
E1
E1
E1
E1
E1
E1
E3
E2
E1
E1
E1
E2
E1
E1
E1
E1
E1
E1
E1
E1
E2
E2
E1
E1
E1
E1
E1
E1
E1
E1
E2
E1
E1
E1
E2
E1
E1
E1
E3
E2
E1
E1
E1
E2
E1
E1
E1
E1
E1
E1
E1
E1
E2
E2
E1
E1
E1
E1
E1
E1
E1
E1
E2
E1
E1
E1
E1
E1
E1
E1
E1
E2
E1
E1
E1
E2
E1
E1
E1
E1
E1
E1
E1
E1
E2
E2
E1
E1
E1
E1
E1
E1
E1
E1
E2
E1
E1
E1
E1
E1
E1
E1
E1
E3
E1
E2
E1
E2
E1
E1/E2
E1
E1
E2
E1
E1
E1
E3
E3
E1
E1
E1
E1
E1
E1
E1
E1
E2
E1
E1
E1
E1
E1
E1
E1
E1
E2
E1
E1
E1
E2
E1
E1
E1
E1
E1
E1
E1
E1
E2
E3
E1
E1
E1
E1
E1
E1
E1
E1
E2
E1
E1
E1
E1
E1
E1
E1
E1
E2
E1
E1
E1
E2
E1
E1
E1
E1
E2
E1
E1
E1
E1
E3
E1
E1
E1
E1
E1
E1
E1
E1
E2
U1
U1
U1
U1
U1
U1
U2
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U2
U1
U1
U1
U1
U1
U1
U1
U1
U2
NPCC
1289
(U1m)
NPCC
1290
(U4m)
1298
(U3m)
NPCC
1293
(U2m)
U1
U1
U1
U1
U1
U1
U2
U2
U2
U2
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U2
U1
U1
U1
U1
U1
U1
U1
U1
U2
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U2
U1
U1
U1
U1
U1
U1
U1
U1
U2
U1
U1
U1
U1
U1
U1
U2
U2
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U1
U2
U1
U1
U1
U1
U1
U1
U1
U1
U2
E1 and U1 correspond to RFLP patterns exhibited by the reference strains S. pastorianus W34/70 and S. uvarum CBS 7001, respectively.
E1*refers to Saccharomyces eubayanus alleles lost in the reference hybrid strain Saccharomyces pastorianus Weihenstephan 34/70, but described
for European S. eubayanus 9 S. uvarum hybrids (Perez-Trav
es et al., 2014).
E2, E3 and U2 (with black background) correspond to new allele patterns reported in the present study.
E2 and U2 (with white background) are alternative PCR-RFLP patterns not reported in the reference strains but described in other S. pastorianus
and European S. uvarum strains, respectively (Perez-Traves et al., 2014).
†
In the case of DAL1 there is an E2 pattern described in a European S. eubayanus 9 S. uvarum hybrid (P
erez-Trav
es et al., 2014), therefore the
new pattern exhibited by the Patagonian S. eubayanus strains is called E3.
NPCC, North Patagonian Culture Collection, Neuquen, Argentina.
the native isolates of S. eubayanus; however, these genes
are lost in the non-cerevisiae subgenome of S. pastorianus
W34/70. The comparison between the sequences of GAL4
and KIN82 of S. eubayanus native isolates and those present in S. uvarum CBS 7001 showed a low nucleotide similarity, 90.99% for GAL4 and 92.30% for KIN82 (Table 4),
of the same level as other S. eubayanus gene regions,
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
which confirms the S. eubayanus origin of the GAL4 and
KIN82 genes present in these isolates.
Among native S. uvarum strains, restriction patterns
different from those present in the reference strain
S. uvarum CBS 7001 were found for six gene regions:
PEX2, MNT2, DAL1, UBP7, BAS1 and BRE5 (Table 3).
From them, only the new alleles for DAL1 and PEX2 were
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V
S. uvarum strains (mtDNA pattern)
955
Saccharomyces in Patagonia
Table 4. Restriction patterns and nucleotide similarities of new alleles found in native isolates Saccharomyces eubayanus (E2 and E3) and
Saccharomyces uvarum (U2). Restriction fragment lengths are given in base pairs. Reference patterns correspond to those of the alleles present in
the reference strain S. uvarum CBS 7001 and in the ‘eubayanus’ genome fraction of the Saccharomyces pastorianus Weihenstephan 34/70
hybrid
Nucleotide similarity (%) between new alleles and
RE
Reference patterns
CBP2
CfoI
HinfI
HaeIII
MspI
HaeIII
HaeIII
PPR1
TaqI
605 180
370 255 120
360 120
430 50
470 210 80
345 170 100
75 65 5
450 315
765
340 340
680
430 350 100
295 240 155
60 55
270 190 150
60 45
230 210 115
45 10
260 240 140
UBP7
XbaI
HaeIII
HinfI
710
460† 440 95
405 400 171† 20
CBT1
DAL1
GSY1
MET6
ORC1
PEX2
EcoRI
HaeIII
AspI
Asp700 I
HaeIII
TaqI
HaeIII
HaeIII
S. pastorianus Weihenstephan
34/70
S. uvarum CBS 7001
E2
96.56
85.05
Restriction patterns of new alleles
E1
35
E1
E1
U1
E1
E1
E1
75
E1
100
U1
70
E1
E1
785
595
360
240
335
345
150
120
190
210
235
315 240
540 225
340 340
440 240
780 100
295 240
60 55
340 230
45 40
330 210
45 10
260 180
70 60
710
909† 95
405 320
80 20
35
E2
98.11
92.65
50
135 80
100 75
E3*
U2
99.85
93.58
93.12
98.78
210
E2
E2
99.06 (E2)
99.19 (E3)
99.35
95.42 (E2)
95.29 (E3)
96.52
E2
98.99
87.47
93.45 (E2)
92.59 (E3)
99.72
450 315
540 225
E3
155 75
60
E2
115
U2
98.15 (E2)
99.15 (E3)
92.59
140
E2
99.03
94.96
94.83 (E2)
95.06 (E3)
90.25 (E2)
92.24 (E3)
E2
180†
340 270
60 45
891† 95
405 320 162†
80 20
E3
E3
*In the case of DAL1, there is an E2 pattern described in a European S. eubayanus 9 S. uvarum hybrid (P
erez-Trav
es et al., 2014), therefore the
new pattern exhibited by the Patagonian S. eubayanus strains is called E3.
†
These UBP7 fragments show 3-bp microsatellite variations corresponding to CAA/CAG codons for Gln.
not previously reported by Perez-Traves et al. (2014) for
S. uvarum strains. The new alternative alleles were also
confirmed by sequence analysis.
Nucleotide sequence similarities between the new alleles
reported for the first time in this work and those present
in the reference strains are described in Table 4. We
found high similarities between the sequences of the new
alleles in native S. eubayanus isolates (E2 or E3) and the
non-cerevisiae sequences from S. pastorianus W34/70.
These similarity percentages were higher than 98% in
most cases. Conversely, the nucleotide similarities were
significantly lower when these sequences were compared
with those present in the reference strain S. uvarum CBS
7001, with percentages lower than 95%, indicating that
they correspond to S. eubayanus. In the same way, the
new ‘uvarum’ allele sequence was compared with the reference strains, confirming its S. uvarum origin (Table 4).
No hybrid strains were found among native isolates of
S. eubayanus and S. uvarum; however, S. eubayanus strain
NPCC 1292 is a particularly interesting native isolate
FEMS Yeast Res 14 (2014) 948–965
since it exhibits eight new allelic variants, four of them
unique (genes GSY1, UBP7, CBT1 and ORC1). Moreover,
this strain is heterozygous for ORC1, the only heterozygosity observed among our isolates (Table 3).
From the allele sequences for each gene, the restriction
site gains/losses required to connect the different restriction patterns present in native S. eubayanus and S. uvarum can be deduced (see Tables S1 and S2, respectively).
Each restriction site may be treated as a discrete ‘character’ or trait that is present or absent in any given strain.
Most-parsimonious trees that minimize the number of
steps required to connect all the S. eubayanus strains and
all S. uvarum strains can then be constructed (Fig. 3a and
b, respectively). Microsatellite variation in UBP7 was not
considered in these analyses. These trees were rooted by
including genotypes from the reference strain S. uvarum
CBS 7001 and hybrid S. pastorianus W34/70 (its eubayanus subgenome), depicted in Fig. 3 as black circles.
These trees show a phylogeographic structure in the
native S. eubayanus and S. uvarum strains. In this way,
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Gene
956
M.E. Rodrıguez et al.
(a) S. eubayanus
Tromen B
Caviahue
NPCC
1286
MET6
NPCC
1282
1283
1284
1285
1287
Tromen A
NPCC
1291
DAL1
NPCC
1292
CBT1
GSY1
NPCC
1296
PEX2
NPCC
1301
GSY1
CBP2
ORC1 ORC1
NPCC
1294
1297
1302
UBP7
PPR1
PEX2
BRE5
W
34/70
Tromen
Huechulafquen
NPCC
1289
NPCC
1293
UBP7 BAS1
NPCC
1288
DAL1
MNT2
NPCC
1290
1298
CBS
7001
PEX2 BRE5
S. eubayanus strains from Caviahue and Tromen can be
differentiated by their DAL1 pattern, and Tromen strains
can be divided into two subgroups, A and B, by their
CBP2 and GSY1 patterns. Saccharomyces uvarum strains
can also be differentiated into two populations, Huechulafquen and Tromen, by their DAL1 patterns.
We also evaluated the sporulation capability of the
complete set of native isolates. All of them produced
abundant tetraspore asci after 15 days of incubation in
sodium acetate agar medium and, in general, they showed
high spore viability, ranging from 75% to 99% (Table 5).
The only exception was S. eubayanus NPCC 1302, which
showed a spore viability of 55%. F1 cultures obtained
from viable spores were subsequently seeded in sporulation medium and all the monosporic cultures were able
to sporulate, evidence of the homothallic nature of all
S. eubayanus and S. uvarum native strains.
To obtain a more reliable picture of the relations
among the native isolates from different Patagonian
regions, we performed a phylogenetic analysis of partial
sequences of the nuclear genes BRE5 and EGT2 and the
mitochondrial gene COX2. Sequences of ‘eubayanus’ and
‘uvarum’ alleles from S. pastorianus W34/70 and S. uvarum CBS 7001, respectively, as well as sequences of the
reference S. cerevisiae strain S288c were included in the
phylogenetic analyses for comparative purposes.
Phylogenetic trees obtained for the nuclear genes
clearly confirmed the species assignation of the native
strains (Fig. 4). Those identified as belonging to S. eubayanus clearly clustered with the sequences of the ‘eubayanus’ alleles of S. pastorianus W34/70 and those classified
as S. uvarum grouped with the reference S. uvarum CBS
7001, in all cases with high bootstrap values (100%).
In the case of BRE5 (Fig. 4a), no correlation was found
between nucleotide sequences exhibited by native strains
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
Table 5. Spore viability of Saccharomyces
Saccharomyces uvarum indigenous strains
Yeast species
Sampling area
S. eubayanus
Caviahue
Tromen
S. uvarum
Huechulafquen
Tromen
eubayanus
Strains NPCC
(mtDNA-RFLP pattern)
1282
1283
1284
1285
1286
1287
1291
1292
1294
1296
1297
1301
1302
1293
1289
1298
1288
1290
(E4m)
(E2m)
(E5m)
(E6m)
(E1m)
(E3m)
(E12m)
(E8m)
(E13m)
(E7m)
(E9m)
(E10m)
(E11m)
(U2m)
(U1m)
(U3m)
(U5m)
(U4m)
and
Spore
viability (%)
86
86
95
86
87
80
81
98
98
75
90
80
55
75
98
89
89
99
NPCC, North Patagonian Culture Collection, Neuqu
en, Argentina.
and their origin (Caviahue, Tromen and Huechulafquen).
In particular, S. eubayanus strains from Caviahue and
Tromen grouped together exhibiting four allelic variants,
two of them being the most frequent in the two locations
studied. Due to the low number of native strains belonging to the species S. uvarum, it was difficult to observe a
geographical segregation within this species for this or the
other genes. Four different allelic variants were found for
this gene among S. uvarum strains; one of them (NPCC
1289) showed an identical allele to that found in the reference strain CBS 7001, whereas the remaining corresponded to three new alleles.
FEMS Yeast Res 14 (2014) 948–965
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(b) S. uvarum
Fig. 3. Minimum number of restriction site
changes needed to connect the different
genotypes, represented by white circles,
exhibited by the Patagonian Saccharomyces
eubayanus (a) and Saccharomyces uvarum
strains (b). Genotypes from the reference
strains are represented by black circles.
Restriction site changes can be reversible
(gain/loss) and are represented by short lines.
The gene regions involved are also indicated
(for a description, see Tables 3 and 4).
Microsatellite variation was not considered.
Dotted squares show strain groups according
to their geographic origins.
957
Saccharomyces in Patagonia
(a) BRE5
0.05
(b) EGT2
Fig. 4. Neighbor-joining trees obtained with
partial sequences of the genes BRE5 (a) and
ETG2 (b) from Saccharomyces eubayanus and
Saccharomyces uvarum native isolates and
reference strains of Saccharomyces. Nucleotide
distances were corrected with the best fitting
models according to the maximum-likelihood
Bayesian information criterion for model
comparison. The best models were the
Tamura & Nei’s (1993) for BRE5 and Tamura’s
(1992) three-parameter model for EGT2. All
these analyses were performed with the
program MEGA5 (Tamura et al., 2011).
Numbers at the nodes correspond to
bootstrap values based on 1000 pseudoreplicates. The scale is given in nucleotide
substitutions per site. The geographic origin of
the strains is indicated by: (C) Caviahue, (H)
Huechulafquen and (T) Tromen.
NPCC 1286 (C)
NPCC 1282 (C)
NPCC 1283 (C)
NPCC 1284 (C)
51 NPCC 1285 (C)
NPCC 1287 (C)
NPCC 1291 (T)
S. eubayanus
94
NPCC 1296 (T)
60
NPCC 1292 (T)
NPCC 1297 (T)
100
79 NPCC 1302 (T)
NPCC 1294 (T)
69 NPCC 1301 (T)
S. pastorianus W34/70
NPCC 1288 (T)
NPCC 1289 (H)
NPCC 1290 (T)
S. uvarum
99 NPCC 1293 (H)
NPCC 1298 (T)
S. uvarum MCYC 623
S. cerevisiae S288c
0.05
The phylogenetic analysis of gene EGT2 evidenced a
higher genetic variability among S. eubayanus native isolates (seven different alleles) (Fig. 4b). In this case, there
was a possible correlation between ‘eubayanus’ allele
sequences and the origin of the isolates (Caviahue or Tromen), because no common alleles were detected in the
two areas, with the same groupings observed in the maximum parsimony tree based on restriction site variation
(Fig. 3a). In contrast, no genetic variability was observed
among native S. uvarum; all isolates showed the same
FEMS Yeast Res 14 (2014) 948–965
allelic variant, highly similar to that found in the reference strain S. uvarum CBS 7001.
Finally, the analysis of the mitochondrial gene COX2
showed six different alleles among S. eubayanus and five
among S. uvarum native strains. In these phylogenetic
analyses, we included reference strains representative of
the COX2 haplotypes described in a previous study for
S. uvarum, S. bayanus and S. pastorianus (Perez-Traves
et al., 2014). In the COX2 neighbor-joining tree (not
shown), the ‘eubayanus’ haplotype cluster appeared to be
ª 2014 Federation of European Microbiological Societies.
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S. pastorianus W34/70
94 NPCC 1286 (C)
NPCC 1292 (T)
NPCC 1294 (T)
93 NPCC 1297 (T)
NPCC 1301 (T)
NPCC 1302 (T)
74
S. eubayanus
NPCC 1282 (C)
NPCC 1283 (C)
NPCC 1284 (C)
100
NPCC 1287 (C)
NPCC 1291 (T)
NPCC 1296 (T)
NPCC 1285 (C)
NPCC 1288 (T)
NPCC 1298 (T)
98 NPCC 1289 (H)
S. uvarum
100
S. uvarum MCYC 623
65 NPCC 1290 (T)
93 NPCC 1293 (H)
S. cerevisiae S288c
958
Discussion
Loss of yeast diversity in traditional
fermentation
Mudai is a traditional beverage elaborated from A. araucana seeds by indigenous Mapuche communities who
have inhabited the cold regions in southwestern Argentina since the 18th century. Although there are historical
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
records about its preparation and cultural connotations
within the native communities, nothing is known about
the microbiota associated to this fermentation process.
However, only two species, H. uvarum and S. cerevisiae,
were isolated from different Mudai fermentation stages in
this work. Both species have been widely described in different fermentative processes. The apiculate yeast species
H. uvarum and other related species of the same genus
Hanseniaspora have long been associated with the fermentation of different sugar-rich raw materials including
grapes (Barata et al., 2008; Saez et al., 2011), apples
(Morrissey et al., 2004; Suarez-Valles et al., 2007),
oranges (Las Heras-Vazquez et al., 2003) and cocoa beans
(Nielsen et al., 2007), particularly in the initial stages.
Later, these apiculate yeasts are replaced by S. cerevisiae,
which is generally the dominant species during the middle and final stages of most fermentation processes
(Romano et al., 2006).
The molecular characterization of the S. cerevisiae isolates obtained in this work from fermentation elaborated
in different regions, exhibited a genetic homogeneity frequently observed in inoculated fermentation, in which the
selected yeast starter dominates the fermentation process,
but not in non-inoculated processes (Lopes et al., 2007).
Traditional natural production of Mudai does not
involve the use of commercial yeasts; however, the use of
commercial bakery yeasts in breadmaking by people from
the Mapuche communities has been reported (Pardo &
Pizarro, 2005). Therefore, the environment in which this
homemade product is nowadays elaborated, is in constant
contact with commercial yeast cultures used in baking.
MtDNA-RFLP analysis of a commercial bakery strain
showed the same molecular pattern detected in our fermentation, evidencing a clear cross-contamination in this
traditional fermented product.
The use of commercial strains in the industrial production of traditional beverages from Latin America is a
common practice to ensure fast and reproducible fermentation. The use of wine or bakery yeasts has been
reported in the fermentation of agave juice to produce
Tequila (Aguilar-Uscanga et al., 2007) and in the fermentation of sugarcane juice to produce Cachacßa (Marini
et al., 2009). The use of commercial bakery or wine yeasts
in the industrial production of traditional beverages
results not only in lower quality products properties with
less desirable sensory attributes (Marini et al., 2009), but
also in a modification of the yeast microbiota by means
of a replacement of the native Saccharomyces strains or
the formation of intraspecific hybrids between native and
wine yeasts (Badotti et al., 2014).
However, the present work is the first evidence from an
ecological-molecular point of view of the impact of commercial yeast in very traditional fermentation, resulting in
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a ‘paraphyletic’ ancestral group from which the S. pastorianus COX2 and the monophyletic ‘uvarum’ haplotype
cluster derived. In this tree the non-cerevisiae COX2 haplotype P1, present in five S. pastorianus strains, was
located in an intermediate position between the ‘eubayanus’ and ‘uvarum’ haplotype clusters. This intermediate
position may be due to the presence of a recombinant
COX2 haplotype in this strain. A detailed analysis of the
COX2 sequence alignment suggested the possibility of
reticulate evolution due to recombination (Table S3). This
way, the S. pastorianus COX2 haplotype P1 (called E-I in
Perez-Traves et al., 2014) appears to be a chimerical
sequence with a 50 region (nucleotides 1–369) very similar
to that of Patagonian S. eubayanus COX2 haplotypes,
showing only three to four differences, but 11–13 compared with S. uvarum; and a 30 region (nucleotides 370–
553) highly similar to that of Patagonian S. uvarum COX2
haplotypes U6 and U7, and European haplotype U4 (UrE
for Perez-Traves et al., 2014), with only one difference,
but 12–16 differences with respect to S. eubayanus.
As the presence of recombinant COX2 haplotypes in
Saccharomyces hybrids was already described in a previous
study (Peris et al., 2012), we performed a neighbor-net
network phylogenetic analyses of the whole COX2 gene
and the 50 and 30 end regions separately (Fig. 5). In the
complete COX2 network, the S. pastorianus COX2 haplotype again occupies an intermediate position; however, in
the 50 region, network clusters with the S. eubayanus
sequences and in the 50 region, phylogeny appears within
the S. uvarum group, confirming the chimerical nature of
the S. pastorianus COX2 haplotype. Tree topologies
depicted in Fig. 5b and c were compared with the nonparametric Shimodaira & Hasegawa (1999) test based on
maximum likelihood, and proved to be significantly different (P = 0.003 for the COX2 50 region sequences, and
P = 0.000 for the COX2 30 region sequences).
Therefore, this chimerical sequence could be the result
of a recombination in the COX2 gene during a hybridization event between a S. uvarum and a S. eubayanus
strains bearing haplotypes not sampled yet, or could correspond to a chimerical sequence derived from an ancestral recombinant form after nucleotide substitution
fixations.
M.E. Rodrıguez et al.
959
Saccharomyces in Patagonia
(a) COX2
E1
E2
E3
E4
E5
E6
NPCC 1283 (C)
NPCC 1284 (C)
NPCC 1292 (T)
NPCC 1294 (T)
NPCC 1301 (T)
NPCC 1285 (C)
NPCC 1287 (C)
NPCC 1296 (T)
NPCC 1302 (T)
NPCC 1282 (C)
NPCC 1297 (T)
NPCC 1291 (T)
NPCC 1286 (C)
E5 E1 S.
E3
E4
E2
E6
NPCC 1290 (T)
U2 NPCC 1298 (T)
U5 NPCC 1288 (T)
U6 NPCC 1289 (H)
U7 NPCC 1293 (H)
eubayanus
S. pastorianus
99.2
P1(E-I)
88.9
100
99.0
U7
U6
U4(UrE)
U5
U3(U-III)
U1(U-I)
U2(U-II)
100
0.01
S. mikatae IFO1815T
S. cerevisiae S288c
S. uvarum
S. kudriavzevii IFO1802T
E2-E5-E6
E3
E1
E4
(b) COX2 5’ end
(1–369)
S. pastorianus P1
S. eubayanus
83.4
Fig. 5. Phylogenetic neighbor-net networks
obtained with complete (a) and partial 50 -end
(b) and 30 -end (c) sequences of the
mitochondrial COX2 gene from
Saccharomyces eubayanus and Saccharomyces
uvarum native isolates and reference strains of
Saccharomyces. The different COX2 sequence
haplotypes are named by the initial of the
species name of the closest parental (U for
S. uvarum, E for S. eubayanus, P for
Saccharomyces pastorianus) followed by a
number. The COX2 haplotype references used
by P
erez-Trav
es et al. (2014) are given in
parentheses. Strains sharing the same
haplotype are given at the upper left corner.
The S. pastorianus haplotype P1 is highlighted
by a square to indicate its changing positions
in the phylogenetic networks due to its
chimerical nature; its 50 and 30 ends are closely
related to S. eubayanus and S. uvarum COX2
haplotypes, respectively. Numbers located on
the branches correspond to bootstrap values
based on 1000 replicates for those branches
crossed by the dashed lines.
S. mikatae IFO1815T
S. paradoxus
NRRL Y-17217T
U7
U6
90.6
S. cerevisiae S288c
100
U1 to U5
0.01
S. uvarum
E1-E3-E4-E5
S. kudriavzevii IFO1802T
E2
S. eubayanus
E6
(c) COX2 3’ end
(370–553)
98.7
90.3
U7 U4
82.6
S. kudriavzevii
IFO1802T
97.4
0.01
S. cerevisiae S288c
U1
U2
P1
U6
U5
U3
S. pastorianus
S. uvarum
99.7
S. paradoxus
NRRL Y-17217T
a radical substitution of the natural yeast diversity. In fact,
no indigenous Saccharomyces isolates were present in the
Mudai samples.
These results show the difficulties faced in studying traditional fermentation, and led us to reformulate our
methodological approach. We decided to isolate native
fermentative yeasts from A. araucana seeds, the raw
material from which Mudai is prepared. To our great surprise, all Saccharomyces isolates recovered from A. araucana seed and bark samples belonged to the closely
FEMS Yeast Res 14 (2014) 948–965
99.7
96.6
S. mikatae IFO1815T
related species S. eubayanus and S. uvarum. There is no
previous report on the occurrence of S. uvarum or S. eubayanus in traditional fermentation from Latin America,
but their presence in A. araucana seeds allows us to postulate a potential role for these strains in the production
of Mudai. Saccharomyces eubayanus, a recently described
taxon (Libkind et al., 2011), has only been isolated so far
from natural environments in Patagonia, but strains of
this species, as well as of S. uvarum, can be used to make
Mudai and carry out other traditional fermentation under
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
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S. paradoxus
NRRL Y-17217T
70.5
93.2
960
Ecology and distribution of S. eubayanus and
S. uvarum in Northern Patagonia
Information on the natural occurrence of S. uvarum and
S. eubayanus is really scarce. As mentioned, S. uvarum
has mainly been found associated with low-temperature
fermentation processes in regions of oceanic or continental climates. Only a few isolates have been recovered from
insects (Mesophylax adopersus and Drosophila spp.), bark
from Quercus, Arbutus and Prunus trees, and exudates
from Ulmus, Carpinus and Nothofagus trees and mushrooms (Sampaio & Goncßalves, 2008; Libkind et al., 2011;
Naumov et al., 2011).
Saccharomyces eubayanus is a new taxon described from
isolates obtained from Nothofagus trees, including Nothofagus pumilio and Nothofagus antarctica, as well as from
stromata of their parasitic fungi Cyttaria in Northern Patagonia (Libkind et al., 2011). The areas from which we
obtained our isolates are characterized by mixed woodlands containing different species of Nothofagus, as well as
A. araucana trees. Very recently, new populations of
S. eubayanus were discovered from Fagus and Acer trees
in Wisconsin, USA (Peris et al., 2014) as well as from
oak bark samples in Tibetan Plateau in the Far East Asia
(Bing et al., 2014). Both the present study and these new
reports are evidence that S. eubayanus is not host-specific,
as proposed by Libkind et al. (2011).
The Saccharomyces genus contains both cryotolerant
and non-cryotolerant species. Saccharomyces kudriavzevii,
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
S. uvarum and S. eubayanus species are considered
cryotolerant and have been successfully isolated from bark
samples by means of selective isolation methods at low
temperature (Sampaio & Goncßalves, 2008; Lopes et al.,
2010; Libkind et al., 2011). In spite of this, all Saccharomyces isolates obtained in this work at both 10 and 30 °C
temperatures corresponded to the cryophilic species
S. uvarum and S. eubayanus. This result agrees with those
reported by Libkind et al. (2011) and supports the idea
that the cold forests from Patagonia may be an unfavorable ecosystem for the non-cryotolerant Saccharomyces
species such as S. cerevisiae or S. paradoxus, which is easily isolated in other regions using the same methodology
(Sampaio & Goncßalves, 2008; Lopes et al., 2010; Naumov
et al., 2013). The climatic conditions of the sampling
areas in this study, characterized by temperatures between
4 and 11 °C, are highly selective and make Northwestern Patagonia a region suitable only for the cryotolerant
species S. eubayanus and S. uvarum. Interestingly, S. kudriavzevii, the other cryotolerant species of the genus isolated from European and Asian areas of similar climatic
conditions (Sampaio & Goncßalves, 2008; Lopes et al.,
2010; Naumov et al., 2013), was not isolated in this study
or in the previous study by Libkind et al. (2011), perhaps
due to competitive exclusion, as suggested by Sampaio &
Goncßalves (2008).
Of the three areas under study, only Tromen showed
the sympatric distribution of both species; however, only
a single species was isolated from each seed and bark
sample. These results suggest that the two species could
be unable to coexist in the same microhabitat. The fact
that we only obtained isolates belonging to one species in
samples from Caviahue and Huechulafquen (S. eubayanus
and S. uvarum, respectively) may also suggest that these
locations are exclusive habitats for each particular species;
however, further sampling in these areas and different
hosts would be needed to support this claim.
Therefore, the present study is of utmost significance
since this is a second report confirming the sympatric
coexistence of the two cryotolerant species in a same
region, Northwestern Patagonia, but in areas and on a
plant species different from those previously reported
(Libkind et al., 2011). In particular, this work extends the
habitat of this species northward in the South Hemisphere, from 40°090 500 S reported by Libkind et al. (2011)
to 37°520 4400 S (Caviahue area in this work).
Hybridization and introgression in
S. eubayanus and S. uvarum
The genetic characterization of Northern Patagonian
S. eubayanus and S. uvarum native isolates showed that
they are homothallic and homozygous for most analyzed
FEMS Yeast Res 14 (2014) 948–965
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laboratory conditions with promising results (Rodrıguez
ME, Barbagelata RJ, Origone AC and Lopes CA manuscript in preparation). In the case of S. uvarum, this species is a better candidate because it is frequently found in
low-temperature fermentation processes from European
regions of oceanic or continental climates, in which it
coexists and even replaces S. cerevisiae as the main yeast
responsible for wine (Naumov et al., 2000b, 2002; Rementerıa et al., 2003; Demuyter et al., 2004; Lopandic
et al., 2008; Csoma et al., 2010) and cider (Naumov
et al., 2001; Coton et al., 2005; Suarez-Valles et al., 2007)
fermentation.
Nonetheless, we cannot discard that S. uvarum or
S. eubayanus could be replaced by native S. cerevisiae during Mudai fermentation, in the same way that a commercial bakery S. cerevisiae strain has recently done.
However, the absence of native S. cerevisiae strains in
A. araucana seeds and bark samples, as well as the
absence of hybrids between commercial S. cerevisiae and
native S. eubayanus or S. uvarum strains may be indicative of a recent substitution of native S. uvarum or S. eubayanus strains by commercial S. cerevisiae strains for
Mudai fermentation.
M.E. Rodrıguez et al.
961
Saccharomyces in Patagonia
FEMS Yeast Res 14 (2014) 948–965
et al., 2011; Perez-Traves et al., 2014) and in native wild
environments (Liti et al., 2006; Wei et al., 2007; Doniger
et al., 2008; Muller & McCusker, 2009; Dunn et al.,
2012). However, introgressions are due to unstable
hybridization followed by successive backcrossing with
one or the other parental species, resulting in introgressed
but fertile strains (Naumova et al., 2011).
In conclusion, these observations suggest that even
though hybridization may be occurring in nature, stable
hybrids seem to be only successful in fermentative environments, under conditions different from those present
in the ancestral habitat of the parental species.
Origin of European S. eubayanus hybrids
Restriction analysis and sequencing of nuclear genes
revealed that Patagonian S. uvarum and S. eubayanus share
alleles with the European S. uvarum strains and S. pastorianus hybrids, respectively. However, specific allelic variants
were exhibited by Patagonian S. eubayanus and, to a lesser
extent, by Patagonian S. uvarum. This new variation
allowed us to differentiate Patagonian strains and detect a
certain degree of population structure based on the association between their geographic origin and the molecular
variation of the native S. eubayanus strains.
Libkind et al. (2011) suggested that, because S. eubayanus has not been found in Europe, S. pastorianus
hybrids may have appeared in the lager brewery environments of Central Europe by hybridization between ale
S. cerevisiae strains and immigrant S. eubayanus yeasts
arriving from America after the advent of trans-Atlantic
trade. The major caveat to the validity of this assessment
is that although lager brewing is more or less contemporary with the arrival of Europeans to America (Hornsey,
2003), the Patagonian region was not colonized until the
late 19th century, during the Chilean occupation of Araucanıa and the Argentine Conquest of the Desert, mainly
due to the fierce resistance of the Mapuche (Araucanian)
peoples (Bengoa, 2000). However, the presence of S. eubayanus has been recently reported in other regions of
America (Peris et al., 2014) that were colonized earlier,
which may explain its presence in Patagonia. In addition,
lager brewing may have originally been performed using
other cryophilic yeasts also present in brewery environments,
e.g.
S. cerevisiae 9 S. kudriavzevii
hybrids
(Gonzalez et al., 2008) and only later have been replaced
by the newly generated S. pastorianus (S. cerevisiae 9 S. eubayanus) hybrids.
In their recent study, Peris et al. (2014), reported the
presence of two diverse and highly differentiated populations in Nothofagus from Patagonia and the rare isolation
of S. eubayanus strains in Wisconsin, USA, which represent a recent mixture of the two Patagonian populations.
ª 2014 Federation of European Microbiological Societies.
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genes (the only exception is ORC1 from S. eubayanus
NPCC 1292), showing high sporulation ability and spore
germination. Most Saccharomyces strains of enological
origin are characterized by being homothallic, with a low
level of heterozygosity (Mortimer et al., 1994; Johnston
et al., 2000; Pretorius, 2000; Bradbury et al., 2006; Legras
et al., 2007). High homozygosity levels but a low ascospore viability was also observed among natural isolates
of S. kudriavzevii (Lopes et al., 2010). These features are
important for the survival of yeasts in nature and may be
involved in the removal of deleterious alleles and chromosome rearrangements (Mortimer et al., 1994), and are
indicative of the non-hybrid nature of these S. eubayanus
and S. uvarum native isolates. The absence of natural
hybrids between S. uvarum and S. eubayanus in the Patagonian sampling regions was also confirmed by their
molecular characterization based on the restriction analysis and sequencing of different gene regions.
The absence of hybrids among native S. eubayanus and
S. uvarum isolates contrasts with the very complex situation found in Europe, where hybridization is very common among strains related to S. eubayanus and S. uvarum
(Rainieri et al., 2006; Nguyen et al., 2011; Perez-Traves
et al., 2014). Among them, we found strains belonging to
the S. uvarum species that contain a single type of genome
related to that sequenced for the strain CBS 7001 (also
called MCYC 623), although some small introgressed
regions from S. cerevisiae can be present (Naumova et al.,
2005, 2011; Perez-Traves et al., 2014). There is also a panoply of hybrid strains containing different portions of the
S. uvarum and S. eubayanus genomes (included in the
S. bayanus taxon), S. cerevisiae and S. eubayanus genomes
(included in the S. pastorianus taxon), and S. cerevisiae
and S. uvarum genomes (Gonzalez et al., 2006; Le Jeune
et al., 2007; Perez-Traves et al., 2014), as well as hybrids
with portions of the genomes from S. cerevisiae, S. uvarum
and a third species, S. kudriavzevii (Peris et al., 2012).
COX2 gene sequencing has shown useful to decipher
the origin of mitochondria in Saccharomyces hybrid strains
(Gonzalez et al., 2008; Peris et al., 2012; Perez-Traves
et al., 2014). The presence of a chimeric COX2 haplotype
in the European S. pastorianus hybrids may be due to the
presence of a recombinant COX2 haplotype in strains
belonging to this species. The presence of recombinant
COX2 haplotypes in Saccharomyces hybrids was already
described in our previous studies (Peris et al., 2012; PerezTraves et al., 2014) and may also be the result of the interspecies hybridization during the evolution of these strains.
In the recent study by Peris et al. (2014), recombination
in the mitochondrial COX2 sequences from S. eubayanus
and S. pastorianus strains has also been found.
Introgressed strains have also been described both in
fermentation processes (Naumova et al., 2011; Nguyen
962
Acknowledgements
This work was supported by grants PICT 2007-1449 and
PICT 2011-1738 to C.L. from the National Agency for
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
Scientific and Technical Promotion, PI04-173 to C.L.
from National Comahue University, as well as by grant
AGL2012-39937-C02-01 and 02 to A.Q. and E.B., respectively, from the Spanish Ministry of Economy and Competitiveness. We thank David Lazaro and Marian
Calder
on Borra for his technical assistance, and Silvana
del M
onaco for collecting bark samples. We are also
grateful to the Spanish Type Culture Collection (CECT),
University of Valencia and CSIC, for kindly providing
online access to the yeast identification database (http://
www.yeast-id.org).
References
Aguilar-Uscanga B, Arriz
on J, Ramırez J & Solıs-Pacheco J
(2007) Effect of Agave tequilana juice on cell wall
polysaccharides of three Saccharomyces cerevisiae strains from
different origins. Antonie Van Leeuwenhoek 91: 151–157.
Badotti F, Vilacßa ST, Arias A, Rosa CA & Barrio E (2014) Two
interbreeding populations of Saccharomyces cerevisiae strains
coexist in cachacßa fermentations from Brazil. FEMS Yeast
Res 14: 289–301.
Barata A, Gonzalez SS, Malfeito-Ferreira M, Querol A &
Loureiro V (2008) Sour rot-damaged grapes are sources of
wine spoilage yeasts. FEMS Yeast Res 8: 1008–1017.
Belloch C, Querol A, Garcıa MD & Barrio E (2000) Phylogeny
of the genus Kluyveromyces inferred from mitochondrial
cytochrome-c oxidase II gene. Int J Syst Evol Microbiol 50:
405–416.
Bengoa J (2000) Historia del pueblo mapuche: siglos XIX y XX,
7th edn. LOM ediciones, Santiago.
Bing L, Han P-J, Liu W-Q, Wang Q-M & Bai F-Y (2014)
Evidence for a Far East Asian origin of lager beer yeast.
Curr Biol 24: R380.
Bradbury J, Richards K, Niederer H, Lee S, Rod Dunbar P &
Gardner R (2006) A homozygous diploid subset of
commercial wine yeast strains. Antonie Van Leeuwenhoek 89:
27–37.
Coton E, Coton M, Levert D, Casaregola S & Sohier D (2005)
Yeast ecology in French cider and black olive natural
fermentations. Int J Food Microbiol 108: 130–135.
Csoma H, Zakany N, Capece A, Romano P & Sipiczki M
(2010) Biological diversity of Saccharomyces yeasts of
spontaneously fermenting wines in four wine regions:
comparative genotypic and phenotypic analysis. Int J Food
Microbiol 140: 239–248.
de M€
osbach EW (1992) Botanica indıgena de Chile. Museo
Chileno de Arte Precolombino. Andres Bello, Santiago, Chile.
Demuyter C, Lollier M, Legras JL & Le Jeune C (2004)
Predominance of Saccharomyces uvarum during spontaneous
alcoholic fermentation, for three consecutive years, in an
Alsatian winery. J Appl Microbiol 97: 1140–1148.
Doniger SW, Kim HS, Swain D, Corcuera D, Williams M,
Yang SP & Fay JC (2008) A catalog of neutral and
deleterious polymorphism in yeast. PLoS Genet 4: e1000183.
FEMS Yeast Res 14 (2014) 948–965
Downloaded from https://academic.oup.com/femsyr/article-abstract/14/6/948/521956 by guest on 12 June 2020
This new Patagonian population (so-called B) seems to
be closer to the S. eubayanus subgenome of the hybrid
lager strains, although the populations reported by Bing
et al. (2014) in Asia, may be even closer.
The genetic differentiation between our Patagonian
S. eubayanus population from A. araucana and the S. eubayanus-genome fraction of the European S. pastorianus
and S. bayanus hybrids suggests that these A. araucana
strains belong to the Nothofagus population first
described (so-called population A). Although the Peris
et al. (2014) results suggest that European hybrids were
generated by an ancestor of the Patagonian B population,
a possible origin in an unknown European S. eubayanus
population, not sampled yet, as proposed by Gibson et al.
(2013), cannot be discarded, in a similar way to the species S. kudriavzevii, which was first described from Japanese isolates (Naumov et al., 1995, 2000a) but whose
hybrids with S. cerevisiae were found in fermentation
environments from Europe (Gonzalez et al., 2006) before
any European strain of this species were isolated from
nature (Sampaio & Goncßalves, 2008; Lopes et al., 2010).
Both possibilities would solve the problem of the origin
of the lager yeast S. pastorianus mentioned above. Finally,
Bing et al. (2014) strongly suggest that the new Tibetan
population of S. eubayanus is the direct donor of the
non-S. cerevisiae subgenome of lager yeast due to the
proximity between Europe and Asia and the trade history
between the two continents.
In contrast, nobody has posited colonization events to
explain the distribution of S. uvarum and the origin of its
hybrids. However, as pointed out, European and Patagonian S. uvarum populations share more alleles than Patagonian S. eubayanus and their European hybrids. This
species is considered to have a world-wide distribution
because it has recurrently been isolated in Europe, mainly
in industrial environments (for a review see Naumov
et al., 2011) and from natural environments in Far East
Asia (Naumov et al., 2003) and in America (Naumov
et al., 1996, 2006; Sampaio & Goncßalves, 2008; Libkind
et al., 2011; present study). However, although it seems
to be quite frequent in wild environments of Patagonia
(Naumov et al., 2006; Libkind et al., 2011; present study),
it is very rare in non-fermentative environments of Europe (only three cases, Table 3 in Naumov et al., 2011). It
is clear that additional environmental sampling is necessary to understand the geographic distribution and niche
occupation of these cryophilic sibling species in relation
to other Saccharomyces species.
M.E. Rodrıguez et al.
Saccharomyces in Patagonia
FEMS Yeast Res 14 (2014) 948–965
Liti G, Barton DBH & Louis EJ (2006) Sequence diversity,
reproductive isolation and species concepts in
Saccharomyces. Genetics 174: 839–850.
Lopandic K, Tiefenbrunner W, Gangl H et al. (2008)
Molecular profiling of yeasts isolated during spontaneous
fermentations of Austrian wines. FEMS Yeast Res 8: 1063–
1075.
Lopes CA, van Broock M, Querol A & Caballero AC (2002)
Saccharomyces cerevisiae wine yeast populations in a cold
region in Argentinean Patagonia. A study at different
fermentation scales. J Appl Microbiol 93: 608–615.
Lopes C, Rodrıguez M, Sangorrın M, Querol A & Caballero A
(2007) Patagonian wines: the selection of an indigenous
yeast starter. J Ind Microbiol Biotechnol 34: 539–546.
Lopes CA, Barrio E & Querol A (2010) Natural hybrids of
Saccharomyces cerevisiae x Saccharomyces kudriavzevii share
alleles with European wild populations of S. kudriavzevii.
FEMS Yeast Res 10: 412–421.
Marini MM, Gomes FCO, Silva CLC, Cadete RM, Badotti F,
Oliveira ES, Cardoso CR & Rosa CA (2009) The use of
selected starter Saccharomyces cerevisiae strains to produce
traditional and industrial cachacßa: a comparative study.
World J Microbiol Biotechnol 25: 235–242.
Morrissey WF, Davenport B, Querol A & Dobson ADW
(2004) The role of indigenous yeasts in traditional Irish
cider fermentations. J Appl Microbiol 97: 647–655.
Mortimer RK, Romano P, Suzzi G & Polsinelli M (1994)
Genome renewal – a new phenomenon revealed from a
genetic study of 43 strains of Saccharomyces cerevisiae
derived from natural fermentation of grape musts. Yeast 10:
1543–1552.
Muller LAH & McCusker JH (2009) A multispecies-based
taxonomic microarray reveals interspecies hybridization and
introgression in Saccharomyces cerevisiae. FEMS Yeast Res 9:
143–152.
Nakao Y, Kanamori T, Itoh T, Kodama Y, Rainieri S,
Nakamura N, Shimonaga T, Hattori M & Ashikari T (2009)
Genome sequence of the lager brewing yeast, an interspecies
hybrid. DNA Res 16: 115–129.
Naumov GI, Naumova ES & Louis EJ (1995) Two new
genetically isolated populations of the Saccharomyces sensu
stricto complex from Japan. J Gen Appl Microbiol 41: 499–
505.
Naumov GI, Naumova ES & Sancho ED (1996) Genetic
reidentification of Saccharomyces strains associated with
black knot disease of trees in Ontario and Drosophila species
in California. Can J Microbiol 42: 335–339.
Naumov GI, James SA, Naumova ES, Louis EJ & Roberts IN
(2000a) Three new species in the Saccharomyces sensu stricto
complex: Saccharomyces cariocanus, Saccharomyces
kudriavzevii and Saccharomyces mikatae. Int J Syst Evol
Microbiol 50: 1931–1942.
Naumov GI, Masneuf I, Naumova ES, Aigle M & Dubourdieu
D (2000b) Association of Saccharomyces bayanus var.
uvarum with some French wines: genetic analysis of yeast
populations. Res Microbiol 151: 683–691.
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
Downloaded from https://academic.oup.com/femsyr/article-abstract/14/6/948/521956 by guest on 12 June 2020
Donoso C & Lara A (1996) Utilizaci
on de los bosques nativos
en Chile: pasado, presente y futuro. Ecologıa de los Bosques
Nativos de Chile (Armesto JJ, Villagran C & Arroyo MK,
eds), pp. 367–387. Editorial Universitaria, Santiago.
Dunn B, Richter C, Kvitek DJ, Pugh T & Sherlock G (2012)
Analysis of the Saccharomyces cerevisiae pan-genome reveals
a pool of copy number variants distributed in diverse yeast
strains from differing industrial environments. Genome Res
22: 908–924.
Felsenstein J (2005) PHYLIP: Phylogeny Inference Package. v.
3.69. University of Washington, Seattle.
Gibson BR, Storg
ards E, Krogerus K & Vidgren V (2013)
Comparative physiology and fermentation performance of
Saaz and Frohberg lager yeast strains and the parental
species Saccharomyces eubayanus. Yeast 30: 255–266.
Gonzalez SS, Barrio E, Gafner J & Querol A (2006) Natural
hybrids from Saccharomyces cerevisiae, Saccharomyces
bayanus and Saccharomyces kudriavzevii in wine
fermentations. FEMS Yeast Res 6: 1221–1234.
Gonzalez SS, Barrio E & Querol A (2008) Molecular
characterization of new natural hybrids between
Saccharomyces cerevisiae and Saccharomyces kudriavzevii
from brewing. Appl Environ Microbiol 74: 2314–2320.
Herrmann TM (2005) Knowledge, values, uses and
management of the Araucaria araucana forest by the
indigenous Mapuche Pewenche people: a basis for
collaborative natural resource management in southern
Chile. Nat Resour Forum 29: 120–134.
Hornsey IS (2003) A History of Beer and Brewing, pp. 1–742.
The Royal Society of Chemistry, Cambridge.
Huson DH & Bryant D (2006) Application of phylogenetic
networks in evolutionary studies. Mol Biol Evol 23: 254–267.
Johnston JR, Baccari C & Mortimer RK (2000) Genotypic
characterization of strains of commercial wine yeasts by
tetrad analysis. Res Microbiol 151: 583–590.
Kurtzman CP & Robnett CJ (2003) Phylogenetic relationships
among yeasts of the ‘Saccharomyces complex’ determined
from multigene sequence analyses. FEMS Yeast Res 3: 417–
432.
Las Heras-Vazquez FJ, Mingorance-Cazorla L,
Clemente-Jimenez JM & Rodrıguez-Vico F (2003)
Identification of yeast species from orange fruit and juice by
RFLP and sequence analysis of the 5.8S rRNA gene and the
two internal transcribed spacers. FEMS Yeast Res 3: 3–9.
Le Jeune C, Lollier M, Demuyter C, Erny C, Legras JL, Aigle M
& Masneuf-Pomarede I (2007) Characterization of natural
hybrids of Saccharomyces cerevisiae and Saccharomyces
bayanus var. uvarum. FEMS Yeast Res 7: 540–549.
Legras JL, Merdinoglu D, Cornuet JM & Karst F (2007) Bread,
beer and wine: Saccharomyces cerevisiae diversity reflects
human history. Mol Ecol 16: 2091–2102.
Libkind D, Hittinger CT, Valerio E, Goncßalves C, Dover J,
Johnston M, Goncßalves P & Sampaio JP (2011) Microbe
domestication and the identification of the wild genetic
stock of lager-brewing yeast. P Natl Acad Sci USA 108:
14539–14544.
963
964
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
of S. cerevisiae x S. kudriavzevii hybrid yeasts unveils a high
genetic diversity. Yeast 29: 81–91.
Peris D, Sylvester K, Libkind D, Goncalves P, Sampaio JP,
Alexander WG & Hittinger CT (2014) Population structure
and reticulate evolution of Saccharomyces eubayanus and its
lager-brewing hybrids. Mol Ecol 23: 2031–2045.
Posada D & Crandall KA (2001) Selecting the best-fit model of
nucleotide substitution. Syst Biol 50: 580–601.
Pretorius IS (2000) Tailoring wine yeast for the new
millennium: novel approaches to the ancient art of
winemaking. Yeast 16: 675–729.
Querol A, Barrio E & Ram
on D (1992) A comparative study
of different methods of yeast-strain characterization. Syst
Appl Microbiol 15: 439–446.
Rainieri S, Kodama Y, Kaneko Y, Mikata K, Nakao Y &
Ashikari T (2006) Pure and mixed genetic lines of
Saccharomyces bayanus and Saccharomyces pastorianus and
their contribution to the lager brewing strain genome. Appl
Environ Microbiol 72: 3968–3974.
Rementerıa A, Rodrıguez JA, Cadaval A, Amenabar R,
Muguruza JR, Hernando FL & Sevilla MJ (2003) Yeast
associated with spontaneous fermentations of white wines
from the ‘Txakoli de Bizkaia’ region (Basque Country,
North Spain). Int J Food Microbiol 86: 201–207.
Robiglio A, Sosa MC, Lutz MC, Lopes CA & Sangorrın M
(2011) Yeast biocontrol of fungal spoilage of pears stored at
low temperature. Int J Food Microbiol 147: 211–216.
Romano P, Capece A & Jespersen L (2006) Taxonomic and
ecological diversity of food and beverage yeasts. Yeasts in
Food and Beverages (Querol A & Fleet GH, eds), pp. 13–53.
Springer-Verlag, Berlin.
Saez JS, Lopes CA, Kirs VE & Sangorrın M (2011) Production
of volatile phenols by Pichia manshurica and Pichia
membranifaciens isolated from spoiled wines and cellar
environment in Patagonia. Food Microbiol 28: 503–509.
Saitou N & Nei M (1987) The neighbor-joining method: a
new method for reconstructing phylogenetic trees. Mol Biol
Evol 4: 406–425.
Sampaio JP & Goncßalves P (2008) Natural populations of
Saccharomyces kudriavzevii in Portugal are associated with
oak bark and sympatric with S. cerevisiae and S. paradoxus.
Appl Environ Microbiol 74: 2144–2152.
Shimodaira H & Hasegawa M (1999) Multiple comparisons of
log-likelihoods with applications to phylogenetic inference.
Mol Biol Evol 16: 1114–1116.
Suarez-Valles B, Pando-Bedri~
nana R, Fernandez-Tasc
on N,
Querol A & Rodrıguez-Madrera R (2007) Yeast species
associated with the spontaneous fermentation of cider. Food
Microbiol 24: 25–31.
Tamura K (1992) Estimation of the number of nucleotide
substitutions when there are strong transition-transversion
and G+C-content biases. Mol Biol Evol 9: 678–687.
Tamura K & Nei M (1993) Estimation of the number of
nucleotide substitutions in the control region of
mitochondrial DNA in humans and chimpanzees. Mol Biol
Evol 10: 512–526.
FEMS Yeast Res 14 (2014) 948–965
Downloaded from https://academic.oup.com/femsyr/article-abstract/14/6/948/521956 by guest on 12 June 2020
Naumov GI, Nguyen HV, Naumova ES, Michel A, Aigle M &
Gaillardin C (2001) Genetic identification of Saccharomyces
bayanus var. uvarum, a cider-fermenting yeast. Int J Food
Microbiol 65: 163–171.
Naumov GI, Naumova ES, Antunovics Z & Sipiczki M (2002)
Saccharomyces bayanus var. uvarum in Tokaj wine-making
of Slovakia and Hungary. Appl Microbiol Biotechnol 59: 727–
730.
Naumov GI, Gazdiev DO & Naumova ES (2003) The finding
of the yeast species Saccharomyces bayanus in Far East Asia.
Microbiology 72: 738–743.
Naumov GI, Serpova E & Naumova ES (2006) A genetically
isolated population of Saccharomyces cerevisiae in Malaysia.
Microbiology 75: 201–205.
Naumov GI, Naumova ES, Martynenko NN &
Masneuf-Pomarede I (2011) Taxonomy, ecology, and
genetics of the yeast Saccharomyces bayanus: a new object
for science and practice. Microbiology 80: 735–742.
Naumov GI, Lee CF & Naumova ES (2013) Molecular genetic
diversity of the Saccharomyces yeasts in Taiwan:
Saccharomyces arboricola, Saccharomyces cerevisiae and
Saccharomyces kudriavzevii. Antonie Van Leeuwenhoek 103:
217–228.
Naumova ES, Naumov GI, Masneuf-Pomarede I, Aigle M &
Dubourdieu D (2005) Molecular genetic study of
introgression between Saccharomyces bayanus and S.
cerevisiae. Yeast 22: 1099–1115.
Naumova ES, Naumov GI, Michailova YV, Martynenko NN &
Masneuf-Pomarede I (2011) Genetic diversity study of the
yeast Saccharomyces bayanus var. uvarum reveals
introgressed subtelomeric Saccharomyces cerevisiae genes. Res
Microbiol 162: 204–213.
Nguyen H-V, Legras J-L, Neuveglise C & Gaillardin C (2011)
Deciphering the hybridisation history leading to the lager
lineage based on the mosaic genomes of Saccharomyces
bayanus strains NBRC1948 and CBS380T. PLoS ONE 6:
e25821.
Nielsen DS, Teniola OD, Ban-Koffi L, Owusu M, Andersson
TS & Holzapfel WH (2007) The microbiology of Ghanaian
cocoa fermentations analysed using culture-dependent and
culture-independent methods. Int J Food Microbiol 114:
168–186.
Nout MJR (2003) Traditional fermented products from Africa,
Latin America and Asia. Yeasts in Food: Beneficial and
Detrimental Aspects (Boekhout T & Robert V, eds), pp. 451–
473. Woodhead Publishing Ltd, Cambridge.
Pardo LO & Pizarro JL (2005) La Chicha en el Chile
Precolombino. Sociedad Editorial Mare Nostrum, Santiago.
Pengelly RJ & Wheals AE (2013) Rapid identification of
Saccharomyces eubayanus and its hybrids. FEMS Yeast Res
13: 156–161.
Perez-Traves L, Lopes CA, Querol A & Barrio E (2014) On the
complexity of the Saccharomyces bayanus taxon: hybridization
and potential hybrid speciation. PLoS ONE 9: e93729.
Peris D, Belloch C, Lopandic K, Alvarez-P
erez JM, Querol A &
Barrio E (2012) The molecular characterization of new types
M.E. Rodrıguez et al.
965
Saccharomyces in Patagonia
FEMS Yeast Res 14 (2014) 948–965
Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Table S1. Presence/absence matrix of variable restriction
sites (constant sites are not shown for simplification)
present in different genes of native S. eubayanus strains.
Table S2. Presence/absence matrix of variable restriction
sites (constant sites are not shown for simplification)
present in different genes of native S. uvarum strains.
Table S3. Comparison of COX2 haplotype sequences
from Patagonian S. eubayanus and S. uvarum strains, and
reference strains of S. uvarum and S. pastorianus hybrids
from Perez-Traves et al. (2014).
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
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Tamura K, Peterson D, Peterson N, Stecher G, Nei M & Kumar
S (2011) MEGA5: Molecular Evolutionary Genetics Analysis
using maximum likelihood, evolutionary distance, and
maximum parsimony methods. Mol Biol Evol 28: 2731–2739.
Thompson JD, Higgins DG & Gibson TJ (1994) CLUSTAL W:
improving the sensitivity of progressive multiple sequence
alignment through sequence weighting, position-specific gap
penalties and weight matrix choice. Nucleic Acids Res 22:
4673–4680.
Wei W, McCusker JH, Hyman RW et al. (2007) Genome
sequencing and comparative analysis of Saccharomyces
cerevisiae strain YJM789. P Natl Acad Sci USA 104:
12825–12830.
Yang Z (2007) PAML 4: a program package for
phylogenetic analysis by maximum likelihood. Mol Biol
Evol 24: 1586–1591.