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Molecular Ecology (2007)
16
, 20912102 doi: 10.1111/j.1365-294X.2007.03266.x
2007 The AuthorsJournal compilation 2007 Blackwell Publishing
Ltd
Blackwell Publishing Ltd
Bread, beer and wine:
Saccharomyces cerevisiae
diversity reflects human history
JEAN-LUC LEGRAS,
*
DIDIER MERDINOGLU,
*
JEAN-MARIE CORNUET
and FRANCIS KARST
*
*
INRA/ULP, UMR Sant de la Vigne et Qualit du Vin, 28 rue de
Herrlisheim, BP 20507, 68021 Colmar Cedex, France,
INRA/ENSAM/CIRAD/IRD, UMR Centre de biologie et de gestion des
populations, Campus International de Baillarguet CS 30016, 34988
Montferrier-sur- Lez Cedex France
Abstract
Fermented beverages and foods have played a significant role in
most societies worldwidefor millennia. To better understand how the
yeast species
Saccharomyces cerevisiae
, themain fermenting agent, evolved along this historical and
expansion process, we analysedthe genetic diversity among 651
strains from 56 different geographical origins, worldwide.Their
genotyping at 12 microsatellite loci revealed 575 distinct
genotypes organized in sub-groups of yeast types, i.e. bread, beer,
wine, sake. Some of these groups presented unex-pected relatedness:
Bread strains displayed a combination of alleles intermediate
betweenbeer and wine strains, and strains used for rice wine and
sake were most closely related tobeer and bread strains. However,
up to 28% of genetic diversity between these technologicalgroups
was associated with geographical differences which suggests local
domestications.Focusing on wine yeasts, a group of Lebanese strains
were basal in an
F
ST
tree, suggestinga Mesopotamia-based origin of most wine strains.
In Europe, migration of wine strainsoccurred through the Danube
Valley, and around the Mediterranean Sea. An approximateBayesian
computation approach suggested a postglacial divergence (most
probable period10 00012 000
BP
). As our results suggest intimate association between man and
wine yeastacross centuries, we hypothesize that yeast followed man
and vine migrations as a commensalmember of grapevine flora.
Keywords
: domestication, fermentation, microsatellite, population
genetics,
Saccharomyces cerevisiae
,wine
Received 13 July 2006; revision received 22 October 2006;
accepted 11 December 2006
Introduction
In most societies, fermented beverages and foods have aunique
place because of their economical and culturalimportance and the
development of fermentation tech-nologies is deeply rooted in their
history. Archaeologistshave found evidence for the production of a
fermentedbeverage in China at 7000
bc
(McGovern
et al
. 2004), and ofwine in Iran and Egypt at 6000
bc
and 3000
bc
, respectively(McGovern
et al
. 1997; Cavalieri
et al
. 2003). Since thattime, it is believed that these fermentation
technologiesexpanded from Mesopotamia through the world.
Forexample, the cultivation of grapevine and the productionof wine
has spread all over the Mediterranean Sea towardsGreece (2000
bc
), Italy (1000
bc
), Northern Europe (100
ad
)
and America (1500
ad
) (Pretorius 2000). Beer technology issupposed to be almost as
ancient as wine and was acquiredfrom the Middle East by Germanic
and Celtic tribes around1st century
ad
, whereas lager beer technology appearedmore recently in the
16th century. While the transfer of plantsby man has favoured
pathogen migrations (Galet 1977),the consequences of the spreading
out of fermentationtechnologies on yeast diversity and population
structurehas never been investigated.
In addition, the question of the natural environment for
Saccharomyces cerevisiae
is still controversial. Because strainisolation from nature or
plants is rare (Davenport 1974;Rosini
et al
. 1982; Sniegowski
et al
. 2002), Martini (1993)concluded that wine yeast comes mainly
from cellars anddescribed this species as domesticated. Very
recently, Fay& Benavides (2005) observed a low diversity among
wineyeast as a further argument for domestication and estimateda
2700-year-old divergence within the vineyard yeast
Correspondence: Legras Jean-Luc, Fax: 33 389224989;
E-mail:[email protected]
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group. The same question of origin can also be raised forother
yeast strains such as those used for ale beer or bread.The rise of
the industrialization era for beer, bread or winemaking should have
led to a standardization of yeast flora.However, the numerous works
made on the microfloradiversity of wine (Frezier & Dubourdieu
1992; Querol
et al
.1994; Versavaud
et al
. 1995; ), bread (Pulvirenti
et al
.2001) and others have revealed a fascinating genetic diver-sity
of
S. cerevisiae
strains. Surprisingly, despite its status asa model species
whose genome sequence has been unrav-elled, no large-scale
diversity study of the yeast species
S.cerevisiae
has been performed, and the role of man on thisdiversity is
still unclear.
The biological and genetic characteristics of
S. cerevisiae
have been recently reviewed by Landry
et al
. (2006). Briefly,
S. cerevisiae
is a diplontic yeast with highly clonal repro-duction.
S. cerevisiae
is also homothallic, which confers thepossibility of
regenerating a diploid cell from a haploid,and could be interpreted
as a way of genome renewal(Mortimer
et al
. 1994). This mechanism could be responsiblefor the high rate
(28%) of homozygote strains found invineyards (Mortimer
et al
. 1994). Many studies also pointedout the aneuploidy of wine
(Bakalinsky & Snow 1990;Guijo
et al
. 1997; Nadal
et al
. 1999), beer or bread strains(Codon
et al
. 1998). This could be a way for yeast to adaptto the various
environments by modifying the dosage ofsome genes important in
adaptation (Bakalinsky & Snow1990; Salmon 1997). In addition, a
high level of karyotypepolymorphism has been observed, especially
for wineyeast, resulting from various mechanisms such as mitoticor
ectopic recombination (Nadal
et al
. 1999; Puig
et al
. 2000)mediated by Ty transposons or other repetitive
sequences(Ness & Aigle 1995). As these mechanisms are very
likelyresponsible for a variable sporulation ability and
sporeviability (Querol
et al
. 2003) the evolutionary importanceof mating in yeast is indeed
a matter of controversy.
We propose here to investigate the possible effects ofhuman
history on yeast diversity from a large-scale evalu-ation of yeast
populations. For that purpose, we character-ized 651 strains
originating from 56 distinct sources using12 microsatellite loci,
and quantified the genetic differenti-ation between the most
significant origins of yeast strains.We infer possible phylogenetic
relationships and furtherevaluate the effects of major factors
acting on this diversity:geographical isolation, and sexual
reproduction. Our resultsgive new insights into yeast genetic
diversity and the role ofman in spreading and selecting this fungus
through history.
Material and methods
Strains
Yeast strains were obtained from our own yeast collection,and
from several laboratories and yeast public or private
collections (Table S1, Supplementary material). Most ofthem were
formerly described as
Saccharomyces cerevisiae.
When no published data was available, species identificationwas
checked by ITS restriction with
Hae
III (White
et al
. 1990).Amplifications at all microsatellite loci were only
obtainedwith
S. cerevisiae
. For
Saccharomyces paradoxus,
we obtainedonly amplification at loci SCAAT5 and YKL172w.
Hybridswere not searched in this work, but previous resultsshowed
that only
S. cerevisiae
alleles are detected from
Saccharomyces uvarum
S. cerevisiae
or
Saccharomyceskudriavzevii
S. cerevisiae
hybrids and should not interferewith the analysis
.
The origins of the 651 strains used here are shown inTable 1.
They were isolated from different substrates(wine, beer, bread,
sake, palm wine, rum ). Strains weremaintained in frozen stocks
(glycerol, 15% v/v) at
80
C,or for short-term storage on YPD agar medium (yeastextract, 1%
w/v, peptone, 1% w/v and glucose, 2% w/v)at
+
4
C.
Microsatellite characterization
Yeast cell cultures and DNA extraction were performed
aspreviously described (Legras
et al
. 2005). Some sampleswere directly analysed from the DNA kindly
provided bythe contacted laboratory. The 12 loci used in this study
havebeen described elsewhere (Legras
et al
. 2005). Two multiplexof six primers pairs corresponding to loci
C5, C3, C8, C11,C9, SCYOR267c and YKL172w, ScAAT1, C4, SCAAT5,
C6,YPL009c, were amplified using the QIAGEN multiplex(polymerase
chain reaction) PCR kit according to themanufacturers instructions
(Table S2, Supplementarymaterial). PCRs were run in a final volume
of 12.5
Lcontaining 10250 ng yeast DNA. Amplification was carriedout
using an Stratagene (Amsterdam, The Netherlands)thermalcycler with
a three-phase temperature program:phase one, 1 cycle: 95
C for 15 min; phase two, 34 cycles:94
C for 30 s, 57
C for 2 min, 72
C for 1 min; phase three,1 cycle: 60
C for 30 min.
PCR product analysis
PCR products were sized for 12 microsatellite loci on acapillary
DNA sequencer (ABI 310) using the polyacry-lamide Pop4 and the size
standards HD400ROX. For rarefragments larger than 400 nucleotides,
three DNA fragmentsof 420, 450 and 485 bp amplified from phage M13
wereadded to the sample. Before the analysis, the PCR ampliconswere
first diluted 50 fold and then 1
L of the dilution wasadded to 18.75
L of formamide (Applied Biosystem) and0.25
L of HD400ROX size marker, and the mixture wasdenaturated at
92
C for 3 min. Allele distribution intoclasses was carried out
using
genotyper
2.5 software(Applied Biosystems).
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Table 1
Strain origin and grouping. The number of strains in the
population used for
F
ST
tree is lower as the first group may include severaltime the
same genotype, or because only strains from a well defined
geographical origin are retained for further analysis (i.e.
Tarragonastrains among Spanish strains, or Firenze strains among
Italians)
OriginNumber of analysed strains
Population for
F
ST
treeNo. of strains inthe population
Alpechin, Spain 1Ale beer miscellaneous (France, Belgium,
England The Netherlands )
8 Ale beer 8
Bread, Italy Sicily 20 Bread, Italy, Sicily 19Bread,
miscellaneous (France, Japan, Spain ) 9 Bread miscellaneous
9Cassava and banana, Burundi 2Cheese, France Camembert 2 Fermented
milk 14Cheese, France Cantal 12 Fermented milk 14Cider, France
Brittany 8Distillery, Australia 1Distillery, Brazil 8 Distillery
Brazil 8Distillery, China 8 Distillery, China 7Fermented milk,
Morocco 1 Fermented milk 14Fruit, Indonesia 1Grapes (
Vitis amurensis
), Russia 1Laboratory strains (USA, France ) 8Lager beer
miscellaneous (France, China, USA ) 15Miscellaneous, Japan 1Natural
resources, Vietnam 5Oak exudates, USA 2Palm wine, Ivory Coast 1Palm
wine, Nigeria 20 Palm wine (Nigeria) 19Rice wine, China
miscellaneous 6 Rice wine 10Rice wine, Laos 3 Rice wine 10Rice
wine, Thailand 1 Rice wine 10Rum, France French Indies 15 Rum
French Indies 15Sake, Japan 14 Sake (Japan) 11Sorghum beer, Ghana
4Trout guts, Norway 1Type strain CBS1907 (Italy) 1Wine and fruits,
Turkey 7Wine, Australia 4Wine, Austria 17 Wine Austria 13Wine,
Croatia 5Wine, France Alsace 100 Wine France Alsace, 71
Wine France Alsace 14Central Europe group
Wine, France Beaujolais 3Wine, France Bordeaux 12 Wine France
Bordeaux 9Wine, France Burgundy 17 Wine France Burgundy 16Wine,
France Champagne 2Wine, France Cognac 27 France Cognac wine 27Wine,
France Jura 3Wine, France Montpellier 20 Wine France, Montpellier
19Wine, France Nantes 22 Wine France, Nantes 19Wine, France Rhone
valley 23 Wine France, Rhone valley 21Wine, Germany 13 Wine Germany
11
(Geisenheim)Wine, Hungary 9 Wine Hungary 9Wine, India 1Wine,
Italy (Firenze and misc.) 35 Wine Italy, Florence 18Wine, Japan
3Wine, Lebanon 25 Wine Lebanon 24Wine, miscellaneous industrial
strains 23Wine, Portugal 1Wine, Romania 10 Wine Romania 10Wine,
South Africa 25 Wine South Africa (Cap) 19Wine, Spain (Tarragona,
37 Wine Spain (Tarragona) 18Penedes, and miscellaneous)Wine,
Uruguay 1Wine, USA 27 Wine USA (California) 16Total 651 502
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Statistical analysis
Strain groups.
Strain groups were made from strainsisolated in the same type of
fermentations, and we tried toobtain groups as large as possible
(at least nine for wineyeast). Genotypes were included only once in
a group(Table 1). For wine, groups were chosen from a well-defined
area. We divided among different regions onlywhen we had enough
strains. These groups were hereafterconsidered as populations.
Because of their characteristics,a group of strains called Alsace
Central Europe strainshas been separated from other strains.
Genetic distances and population analysis.
The chord distanceDc (Cavalli-Sforza & Edwards 1967) was
calculatedbetween each strain with a laboratory-made program.
Alltrees were obtained from distance matrices derived with
neighbour
of the
phylip
3.5 package, using
mega
3(Kumar
et al
. 2004) for tree-drawing. All trees were rootedby the midpoint
method. The reliability of the tree topologieswas assayed through a
jackknife procedure. The validity ofnodes was obtained with the
consens
program (
phylip
3.5package).
Wine yeast population genetic features (
F
IS
, linkagedisequilibrium) were evaluated from a subset of
diploidwine yeast strains using the
fstat
2.9.3 software (http://www.unil.ch/izea/softwares/fstat.html).
Population genetictests were also conducted from the
genepop
on the web(http://wbiomed.curtin.edu.au/genepop/).
The genetic distances
F
ST
(Reynolds
et al
. 1983), and DAS(Bowcock
et al
. 1994) between all groups were calculatedusing the program
microsat
1.5d (Minch
et al
. 1995) afterpooling all alleles detected from one group of
strains andconsidering strains as haploids. The reliability of the
treetopologies was assayed through bootstrap analysis
(1000replicates resampling loci), and the validity of nodes
wasobtained with the
consens
program. Isolation by distancewas evaluated with the
isolde
software from the
genepop
on the web, after calculation of
F
ST
distances with
micro-sat
between all groups. The geographical matrix was builtwith the
help of route-finder software for close origins(such as the
European wine groups) or from air distancesfor more distant
countries. Cheese, beer and bread strainswere not included in the
analysis because of their uncleargeographical origin, as well as
South African and Ameri-can wine strains because of human-driven
migration.
Time divergence estimation.
An estimation of wine yeastdivergence was attempted through an
approximate Bayesiancomputation (ABC) approach (Beaumont
et al
. 2002). Thisconsists of three steps, namely: (i) simulation of
data setsaccording to a demographic, historical and mutational
model,with parameter values drawn from prior distributions;(ii)
rejection of simulated data sets based on the Euclidian
distance between standardized summary statistics of theobserved
data set and those of each simulated data set; and(iii) local
linear regression of individual parameters onsummary statistics of
accepted data sets (see Excoffier
et al
.2005 for a more detailed description).
The two successive divergences occurred at
t
1
and
t
2
years in the past. Prior distributions on divergence timewere
set at
t
1
=
2500 (first traces of the culture of grapeswhen Greeks
established Massalia (Marseille), Dion 1959)and
t
2
U[3500, 53500]. A generalized mutation model wasassumed for
microsatellite loci, with mean mutation ratedrawn from a
uniform
[0.0001, 0.001]. The analysis wasperformed twice: once with the
populations
Lebanon
,
Montpellier
and
Central Europe
, and a second time replacing
Montpellier
by
Rhone valley
.For this analysis, nine loci were retained. We removed
Ykl172w locus, because of its specific behaviour (abnor-mally
high
FST, see below), YPL009c (linked to C9) and C4(aneuplody of
several strains).
Results
Genotypes and strain biodiversity
From 651 strains that were assigned genotypes at all 12loci, a
total of 575 multilocus genotypes were established,with 76 strains
showing genotypes identical to others in thesurvey. Among grapevine
and wine isolates, some clonesisolated from various vineyards in
several continentsresulted in the same genotype. In many cases,
these strainscorresponded to well-known industrial strains such as
the522 Davies group (Fig. 1), or the CIVC8130 and Prise demousse
group (Champagne group Fig. 1). This findinghas already been
described before (Legras et al. 2005) forthe champagne strain
CIVC8130. Several clones of sakestrains obtained from different
collections also displayedthe same pattern.
The 12 microsatellite loci recorded from 13 to 54
differentalleles per locus. SCAAT1, C4 and SCYOR267c displayedthe
highest number of alleles in the global population,which was
expected given the length of these repeatedmotifs and their
selection for high polymorphism (Legraset al. 2005). The number of
alleles per locus per strain variedfrom one to four (Table 2). Half
of the bread strains and alebeer strains exhibited four alleles at
several loci, suggestingtetraploidy. In contrast to the results
with bread strains,88% of wine isolates presented two alleles
maximum for allloci, suggesting a diploid state for most wine yeast
strains.In total, 28% of the isolates were homozygous for all
loci.
The neighbour-joining tree calculated from the Dc chorddistance
matrix for all pairs of strains (Fig. 1) reveals a clearclustering
linked to the technological origin of the strains(Table 1). In
particular, yeast strains used for palm wine,sake, fermented milk
and cheese, beer (ale and lager), are
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Fig. 1 Neighbour-joining tree showing the clustering of 651
yeast strains isolated from different sources. The tree was
constructed from thechord distance between strains based on the
polymorphism at 12 loci and is rooted according to the midpoint
method. Branches arecoloured according to the substrate from which
strains have been isolated. The percentage of occurrence of nodes
obtained through aJackknife procedure is given at the basic node of
main groups. Type strain CBS1171 is given in black. Color code:
wine, dark green; cider,light green; bread, yellow; beer, orange;
fermented milk, pink; sake from Japan, dark blue; Chinese rice wine
and distillery from Vietnamand Thailand, light blue; sorghum beer
or palm wine from Africa, brown; oak tree from America, blue-green;
distillery from South Americaand rum from French Indies, purple;
laboratory strains, red. Misc., miscellaneous.
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gathered in clusters. Bread strains analysed here are
dividedinto two clades; one main clade including strains from
variouscountries (Japan, Spain, France, and Sicily) as well as
industrialbread strains, and a second clade including only
isolatesfrom Sicily. The main group contains mostly tetraploid
strainsand is located at the border of the wine group. It
containsalso some wine yeast and amazingly four strains that
arewidely used for Beaujolais nouveau wine-making.
Almost 95% of wine yeast strains are found in the upperpart of
the tree, which also includes cider strains. Industrialor grape
strains are scattered all over this clade so that it isnot possible
to differentiate them from other wine strains.Several subgroups are
visible in the wine yeast clade, thelargest of which contains
strains isolated in Germany,Alsace (France), Hungary, and Romania;
we called it theCentral Europe wine group. This group also
containssome strains from Lebanon, as well as Spanish flor
yeaststrains. The group containing the Prise de mousse
strain,further called Champagne group, is also related to
theCentral Europe group. Most American vineyard strainsbelong to
the wine yeast groups, except three isolates.Among the few strains
not included in the wine part of thetree, four Austrian and two
Californian strains, are foundin a separate cluster and five other
isolates are found sep-arately. The American strain UCD13, is found
close to twoAmerican oak litter isolates obtained by Sniegowski et
al.in 2002. Similarly, one Russian vine strain (CBS 5287)isolated
from grapes of wild endemic vine Vitis amurensis
in the Russian Far East was not related to other winestrains.
French Indies rum and Southern America distillerystrains are found
together either among wine yeast or dis-persed among other
origins.
The next main specific group is the fermented milk iso-lates
group. These strains have been isolated on cheesemainly in France
but also in Morocco. It is noteworthy thatdespite its French
origin, this group is positioned far fromFrench wine or cider
isolates but close to the beer strainsgroup. Interestingly, all
beer strains (ale and lager) but oneappear clearly different from
bread strains. Despite theirdifferent genetic characteristics
(tetraploid for ale strains,and allotetraploid for lager strains)
these two types of beerstrains are also found in the same
clade.
The geographical effect on the structure of the individualtree
can hardly be proven as some strains are specific toone type of
fermentation such as African Palm wine yeast(Nigeria and Ivory
Coast). However, for Asian strains, wehave two clades that combine
strains from different coun-tries (China, Laos, Thailand, and
Japan), isolated fromdifferent fermentations (rice wine, or
distillery isolates)as well as a Vietnam sugar cane natural isolate
suggestingdomestication from a local origin.
Wine yeast population analysis
As most of our strains were obtained from vineyards,we tried to
investigate the population structure of wine
Table 2 Classification of strains according to the maximum
number of allele encountered per locus for some origins. (One
allele maximummeans homozygosity; two, heterozygosity, three
suggest aneuploidy, and four, tetraploidy)
OriginNo. of different clones 1 allele 2 alleles 3 alleles 4
alleles
Vine and wineAustria Klosterneuburg 17 4 10 2 1France Alsace 86
14 54 9 9France Cognac 27 14 12 1 0France Montpellier 19 9 10 0
0France Nantes 19 8 9 2 0France Rhne 21 1 16 3 1Spain (Taragonna
and Penedes) 36 9 23 3 1Germany 11 2 8 0 1Italy 23 6 15 1 1Japan 3
1 1 1Lebanon 25 10 11 1 3USA (UC Davis) 26 10 10 2 4
Cider (France, Bretagne) 8 2 6 0 0Ale beer (miscellaneous) 8 0 1
2 5Bread (miscellaneous) (group 1) 9 0 3 1 5Fermented milk and
cheese (France + Morocco) 14 0 7 2 5Palm Wine (Nigeria) 19 4 6 5
4Rum (Antilles) 15 3 8 4 0Rice wine and distillery Asia 13 3 1 9
0Sake Japan 11 2 7 2 0
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S. cerevisiae diversity. The 17 most important groups werekept
and considered then as pseudopopulations, corres-ponding to 277
diploid strains. The few aneuploids werenot taken into account in
the analysis. We first checkedassociations within loci to determine
if genotype frequencieswere those expected under HardyWeinberg
equilibrium.The high number of homozygote strains observed for
mostgroups turned out to be excessive when opposed toHardyWeinberg
expectations.
In a second step, we tried to estimate how thegenetic variation
was partitioned within and betweenpopulations using F-statistics
(Table S3, Supplementarymaterial). One locus, YKL172w, presented an
FST valuethree times higher than the other loci, that might
beconnected to the function of this essential gene so thatwe have
not taken this loci into account for the FST analysis.The average
of FIS values over all loci is very high formost groups and can be
explained by inbreeding andmay be related here to the effect of
homothallism onpopulation genetic structure.
An exact test for association of alleles across locibased on
permutation was employed. For wine yeastpopulations, linkage
disequilibrium was not observedfor 26% of comparisons among all
populations (P < 0.05)and 77% inside each population (P <
0.05). This sug-gests that the overall population structure is
clonal butwith some recombination. Also this can result partlyfrom
genetic drift occurring among distantly relatedpopulations.
Population relationships
Two trees were built from the FST and the DAS matrixamong groups
(Fig. 2, Fig. S1 and Table S4, Supplementarymaterial): both give a
structure in agreement with theindividual strain tree (Fig. 1).
All wine strain groups gather within the same clade(89% and 90%
of FST and DAS trees obtained by bootstrapanalysis) and are
separated from other groups. This analysisreveals a clear
difference between Mediterranean vineyardsand the Central Europe
branch (Romania, Hungary, Germany,and some strains of Alsace) that
we correlate to the occur-rence of strains of the Central Europe
group. The Lebanongroup is found at the root of the wine group. A
furtherstructure can also be observed for Rhone valleyBurgundyand
Nantes strains (79% bootstrap) to which is connectedthe Alsace
group (47% bootstrap), and for Cognac andItalian strains or
Montpellier and Spanish strains with alower bootstrap score (44%
and 33%, respectively).
Two groups of strains are found close to the wine yeastgroups,
the French Indies rum and Southern America dis-tillery group
suggesting that these groups are related towine yeasts. In
contrast, Asian strains (Chinese and sake) aswell as beer strains
have a distant position to wine strains.
The analysis of the geographical effect on this yeastdiversity
was attempted for groups of strains for which wecould identify a
clear geographical origin: European wine,African palm wine,
Southern America distillery strainsand French Indies rum, sake,
China rice wine and distillery.
Fig. 2 Consensus tree of populations basedon FST genetic
distances obtained after 1000replicates (resampling loci). The tree
wasbuilt using the neighbour-joining method,and the root was
defined by midpointrooting.
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Bread, beer, and cheese strains were discarded because oftheir
nonspecific geographical origin. The FST distancematrix calculated
from microsat software had been testedand plotted against
geographical distance. PermutationMantel test calculated from
genepop revealed a globalhighly significant value (P < 0.001),
and 28% of the variabilitycould be explained by geographical
isolation (Fig. 3).
Time divergence estimations
Assuming Lebanon as a putative origin for all three
groups(Central Europe, Montpellier and Rhne valley) and a
2500-year-old divergence with Rhone or Montpellier populations,we
obtained two estimations of 10 500 and 11 750 (bp)(Fig. 4),
respectively [confidence intervals (4500, 32 000)and (4750, 36
000)] between Central Europe and Lebaneseyeast strains. As a
consequence, yeast migration seems tohave occurred after the last
ice period.
Discussion
We used both individual and population-based approachesto
analyse Saccharomyces cerevisiae biodiversity.
Multilocusmicrosatellite typing of strains of different origins
revealeda strong structure of yeast strains according to
theirtechnological origins. This structuring can be clearly
seenfrom individual as well as population analysis.
Population analysis of wine yeast
The ploidy of wine strains is a remarkable feature of thewine
group: almost 84% of strains were deduced to bediploid. The
population analysis of these diploid wineyeast groups revealed
several original aspects. First of all,as expected, S. cerevisiae
has a mainly clonal reproduction,as seen from linkage
disequilibrium observed betweenloci. However, a significant
proportion of loci are still
nonsignificantly linked (26% among all populationsand 75% within
each group) which suggests that somerecombination still exists. A
purely clonal populationevolving only under mutation would indeed
lead to arapid disappearance of homozygous strains, whereas aclonal
population evolving under mitotic recombinationwould lead to a
decrease in heterozygous strains. The highratio of homozygous
strains (30%) and the high FIS positivevalues suggest that
homothallism has a high impact onyeast diversity. A similar pattern
has been observed byFundyga et al. (2002) for Candida albicans but
our resultsindicate that S. cerevisiae has a lower rate of
sexualreproduction. Indeed, FIS and linkage disequilibrium are
Fig. 3 Evaluation of isolation by distance.Distances between
groups were calculatedusing the microsat program and theisolation
by distance evaluated with thesoftware isolde from the genepop
website(http://wbiomed.curtin.edu.au/genepop/).
Fig. 4 Distribution of divergence time estimations between
theCentral Europe and Montpellier (black line) and the
CentralEurope and Rhone valley (blue line) populations. The dashed
linecorresponds to the a priori distribution of time
divergence.
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higher and there is a larger proportion of homozygousstrains in
our species.
Further parallels can be drawn with C. albicans, as wealso
detected a macrogeographical differentiation ofstrains between
Asian, European and African yeast demes.Isolation by distance
accounts for 28% of genetic variation(Fig. 3), which is slightly
lower as the 39% estimated forC. albicans (Fundyga et al. 2002).
These genetic differencesbetween yeast groups suggest an ancient
divergence lead-ing to local natural populations (in Asia,
Mesopotamia,Africa) from which multiplication may have been
favouredby humans. This implies also that there must be a
naturalhabitat for yeast which allowed a wide expansion of
thisspecies.
Population relationships inferred from individual and population
trees
The FST and DAS consensus trees on populations confirmthe global
genetic structure of the tree on individuals, witha clear
separation between most wine yeast groups andother technological
groups (89% bootstrap score for the FSTtree).
This structuring has partially been observed with ampli-fied
fragment length polymorphism (AFLP; Azumi &Goto-Yamamoto 2001),
microsatellites (Hennequin et al.2001), and very recently by
multilocus sequence analysis(MLST) for wine and sake origins by Fay
& Benavides(2005) and Ayoub et al. (2006) or single nucleotide
poly-morphism (Ben-Ari et al. 2005).
The reference laboratory strain S288C obtained from anAmerican
isolate is found very close to Nigerian palmwine strains (Fig. 1),
whereas three other American isolates(two oak tree exudates and one
Californian wine isolateUCD13) are much closer to CBS 5287 (Asian
Russia) andClib 414 from Japan. This position far from wine yeast
is inagreement with data of De Barros Lopes et al. (1999) fromAFLP,
Fay & Benavides (2005) from MLST, and Winzeleret al. (2003)
from micro-array karyotyping. It must bepointed out, however, that
we did not characterize anywine yeast isolate close to S288C as
described by Aa et al.(2006).
Three main Asian yeast groups of strains were alsofound: the
sake yeast group and two groups includingmainly rice wine and some
Chinese distillery strains. Sakestrains and the two other groups
are surprisingly not asclose to each other in the individuals tree
as would beexpected from sake technology having originated
fromKorea (Teramoto et al. 1993). But the DAS tree suggests
thatthese groups are related and the global position of
Asianstrains is in agreement with what was described by Azumi&
Goto-Yamamoto (2001), Fay & Benavides (2005), and byAyoub et
al. (2006). The Nigerian palm wine group repre-sents another
well-characterized group including an Ivory
Coast strain. However, Ghana sorghum beer strains arecloser to
beer strains than to palm wine. They are alsodistinct from Burundi
fermented cassava and bananastrains, which suggests that genetic
differentiation betweenAfrican yeast populations exists in a
similar way to whathas been described for wine or bread yeasts.
The wine yeast group is well separated from yeaststrains of
other technological origins. It contains straingroups from ancient
vine areas (Lebanon, Europe) as wellas new world recent vineyards,
which suggests a migra-tion of wine yeast all over the world, which
is revealed bythe structure of the FST tree. In addition to the
historicalhuman transport across the Mediterranean Sea, this
treeclearly supports the hypothesis of a migration pathwayalong the
Danube valley. The occurrence of some specialwine strains outside
the main wine yeast group (UCD13,CBS 5287, and Arka) suggests that
some autochthonousstrains in new world or European vineyards can
still beisolated that do not represent the standard wine strains.In
a similar manner, half of the strains of the groups ofFrench Indies
rum and Brazilian distillery strains foundamong wine strains are
very likely the result of such ahuman-provoked migration whereas
the second half aremore distant.
For bread and beer, Azumi & Goto-Yamamoto (2001),and De
Barros Lopes et al. (1999) found evidence fromAFLP data of a close
relatedness with wine strains. However,Ayoub et al. (2006) found
contradictory results for typestrain CBS1171 originating from beer.
Our results from theindividual tree suggest that bread strains are
close to winestrains, and far from beer strains. In contrast, the
FST andDAS trees indicate on the contrary that beer and
bread(tetraploids) strains are related to each other but are
distantfrom wine yeast. The analysis of the proportion of
allelesshared by each group of strains may give a clue: 79% and67%
of alleles of bread strains (main group) are shared bybeer and wine
strains, respectively, and 96% of breadstrains alleles can be found
either among beer or winestrains. We propose that the main group of
bread strainscould originate from a tetraploidization event between
anale beer and a wine yeast strain, which may explain the
dis-crepancy between published data. It must also be pointedout
that actual S. cerevisiae strains used for making breadcome from
different geographical origins.
Genetic data vs. historical features
The relative positions of most yeast groups are in goodagreement
with historical data. The sake technology issupposed to have
originated from Korean rice winetechnology (Teramoto et al. 1993),
wine tradition fromMesopotamia (Pretorius 2000), and lager beer
from ale bre-wery knowledge (Corran 1975). The above suggestion
thata group of bread strains resulted from a tetraplodization
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event between an ale beer strain and a wine strain alsoimplies
that bread technology appeared after beer andwine technology.
However, as beer strains are obviouslyfar from wine yeast, our
results do not support the classicalhypothesis of wine technology
as an origin for beer(McGovern 2003). Our results suggest more
likely that beeras well as bread have a more oriental origin, which
is inagreement with several results of MLST analysis of bread(or
bread-related wine strains such as 71B or LevulinePrimeur) (Fay
& Benavides 2005; Ayoub et al. 2006). Theposition of some beer
strains found among bread strains isalso logical, considering the
historical exchange of strainsbetween beer and bread yeast makers
since the end of the19th century.
The existence of a wine yeast group including 95% ofstrains,
with a Lebanon group close to the root of the FSTtree (Fig. 2)
suggest a migration from Mesopotamia withthe event of vine
domestication and is compatible withknown vine migration
(Arroyo-Garcia et al. 2006). Butmore strikingly, the substructure
inside the wine yeastcluster is also in agreement with historical
knowledge. Thedifferent wine yeast group locations are consistent
withvine migrations routes (Danube valley, Rhone valleyBurgundy
Alsace and Nantes, or ItalyCognac; This et al.2006). Indeed, Ugni
blanc, main Cognac grape varieties,originated from Italy,
wine-making tradition arrived inBurgundy from the Rhne valley, and
Muscadet (Nantes)was imported from Burgundy at the 15th century
(Viala &Vermorel 1901). With a time divergence estimation
ofabout 11 000 years (bp) between Lebanon and the CentralEurope
group, we can assess a very early divergence pos-terior to the last
glaciations area but we cannot concludewhether this divergence is
connected to a postglaciationcolonization route of wild vine
(Taberlet et al. 1998) or tothe culture of vine. The most probable
estimation suggesta period corresponding to the advent of wine
making asthe oldest archaeological site displaying remains of
winetechnology is 8000 bp. For some more distant yeast strainsfound
in Austria, we can assume that some local strainshave been
domesticated from the wild local Vitis sylvestriswhich are the
progenitors of the actual vine varieties(Levadoux 1956).
Tamed or domesticated yeast?
The question that must be raised is that of yeastdomestication
as proposed by Martini (1993) or Fay &Benavides (2005). The
concept of a domesticated species isoften used with different
meanings. In a recent reviewabout plant and animal domestication,
Diamond (2002)proposed the following definition: species bred in
captivityand thereby modified from its wild ancestors in waysmaking
it more useful to humans who control itsreproduction and (in the
case of animals) its food supply.
Because they have been almost continuously cultivatedsince very
ancient times, rice wine, beer and bread strainsare clearly
fulfilling these criteria of culture and selection,so that we have
at least two different domestication eventswhich occurred in Asia
for rice wine and somewhere elsefor beer.
The way in which wine strains are naturally propagatedis,
however, poorly understood: flor yeasts which growalmost
continuously on the surface of wine during thesherry wine process
are very likely an example of domes-tication. However, for other
types of wine strains, we can-not infer such a continuous human
control of their culture.Mortimer & Polsinelli (1999) have
shown that a populationof yeast exists on grapes. However, whether
these strainsparticipate in the alcoholic fermentation in the
cellar is stillcontroversial: some authors (Rosini et al. 1982;
Ciani et al.2004) observed that only cellar strains were
responsible forthe alcoholic fermentation in the vats, whereas
others showthat grapevine strains can be partially responsible for
thealcoholic fermentation (Constanti et al. 1997; Gutirrezet al.
1999; Le Jeune et al. 2006). We agree with the latterpoint of view
from our own data (Legras, unpublisheddata). The correlation
between grapevine migration andyeast diversity, as well as our time
divergence estimation,suggest clearly that wine yeast biology is
closely con-nected to grapevine, which is compatible with the idea
ofS. cerevisiae as a potential pathogen of the vine (Gognieset al.
2001) or at least a member of the vine commensal flora.However, the
adaptation of yeast observed through theevolution of the SSU1 gene
leading to SO2 resistance(Perez-Ortin et al. 2002; Aa et al. 2006)
demonstrates theadaptation of yeast to the winery environment.
Altogether,these results suggest that yeast have adapted to both
vineand winery environment.
In conclusion, our data show that yeast genetic diversityhas
been highly influenced by human technology throughhistory, as well
as by natural genetic drift and migration ina similar way to other
microorganisms and pathogens,leading to progressively
differentiated populations.
For decades, studies on beverages history have relied onvessels
comparison, and very recently on chemical ana-lysis. Our results
also show that a comprehensive explora-tion of yeast diversity
could provide some new featuresabout the origin of fermentation
technology and humanhistory. The ongoing programs using yeast
populationgenomics could give us new clues to these questions.
AcknowledgementsThe authors would like to thank A. Alais Perot
and G. Butterlin fortheir helpful technical assistance. We are
grateful to C. Schneiderfor answering vine history questions, and
to Dr J. Masson andPr. R. Gardner for critically reading the
manuscript.
We also express gratitude to all the different researchersand
Institutes who provided kindly yeast strains: Dr M.J. Ayoub,
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University of Beyrouth, Lebanon, Dr S. Berger, Weinbau
InstitutKlosterneubourg, Austria; Pr. L. Bisson, UC Davis; USA; Dr
M.Burcea, University of Bucarest, Roumania; Dr S. Colas,
InterRhone,Avignon, France; Dr S. Dequin, UMRSPO INRA Montpellier;
DrJ.F. Drilleau, SRC INRA Rennes, France; Dr H. Erten,
CukurovaUniversity, Turkey; Pr. O. Ezeronye, DBS MOUA, Umudile,
Nigeria;Dr L. Fahrasmane, URTPV INRA Antilles, France; Pr. J. Fay,
Univer-sity of Washington, Saint Louis, USA; Dr N Goto-Yamamoto,
NRIB,Japan, Dr L. Granchi, University of Firenze, Italy; Pr.
Grossmann,State Research Institute Geisenheim, Germany; Dr I.
MasneufPommade, Institut dOenologie de Bordeaux, France; Dr
MC.Montel, URF, INRA Aurillac, France; Dr H.-V. Nguyen, CLIB,INRA
INAPG, France; Dr V. Petravic; University of Zagreb,Croatia; Dr A.
Poulard, ITV, Nantes, France; Dr C Roulland,BNIC, Cognac, France;
Dr N. Rozes, Universitat Rovira i Virgili,Tarragona, Spain; Pr. M.
Sipiczki, University of Debrecen,Debrecen, Hungary; Pr. P.
Sniegowski, University of Chicago,USA; Pr. Van Rensburg, University
of Stellenbosch, South Africa.
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JL Legras is interested in understanding how yeast diversity
isgenerated and affects the technological properties of wine
yeast.F Karst is a yeast geneticist who studies isoprenoid
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Supplementary material
The following supplementary material is available for this
article:
Fig. S1 Consensus tree of populations based on DAS
geneticdistances obtained after 1000 replicates (resampling loci).
The treewas built using the neighbour-joining method, and the root
wasdefined by midpoint rooting.
Table S1 Strains used in the study.
Table S2 Microsatellite loci description and primers.
Table S3 F-statistics in diploid wine yeast populations
(computedwith Genepop).
Table S4 FIS per locus and wine yeast population (computed
withFstat).
Table S4 FST between pairs of yeast populations (computed
withMicrosat 1.5d).
This material is available as part of the online article from:
http://www.blackwell-synergy.com/doi/abs/10.1111/j.1365-294X.2007.03266.x(This
link will take you to the article abstract).
Please note: Blackwell Publishing are not responsible for the
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