bread beer wine

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


, 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,


,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


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]

2092

J . - L . L E G R A S

E T A L .


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).

Y E A S T D I V E R S I T Y R E F L E C T S H U M A N H I S T O R Y

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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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J . - L . L E G R A S

E T A L .


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

Y E A S T D I V E R S I T Y R E F L E C T S H U M A N H I S T O R Y 2095


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.

2096 J . - L . L E G R A S E T A L .


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



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.

2098 J . - L . L E G R A S E T A L .


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.



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

2100 J . - L . L E G R A S E T A L .


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,



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 biosynthesis.D Merdinoglu is a plant geneticist and has a special interest forgrapevine resistance to its pathogens. JM Cornuet is a populationgeneticist with interest in developing methods and softwares forstatistical inference in population genetics.

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 con-tent or functionality of any supplementary materials supplied bythe authors. Any queries (other than missing material) should bedirected to the corresponding author for the article.

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