program. no. peris d1,* 1 2 3 1,4 - uv

Population structure, admixture, and migration of Saccharomyces eubayanus and their lager-brewing alloploid hybrids

Peris D1,*, Sylvester K1, Libkind D2, Gonçalves P3, Sampaio JP3, Hittinger CT1,4 1Laboratory of Genetics, DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53706, USA; 2Laboratorio de Microbiología Aplicada y Biotecnología, Instituto de Investigaciones en Biodiversidad y Medio-ambiente, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)-Universidad Nacional del Comahue, 8400 Bariloche, Argentina; 3Centro de Recursos Microbiológicos, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal; 4Genome Center of Wisconsin, Wisconsin Bioenergy Initiative ; *e-mail address: perisnavarro@wisc.edu

INTRODUCTION

RESULTS

Program. No.

A048 362

CBS 7001

CBS380

CBS1546

W34_70

CBS 1503

NBRC 1948

yHCT96

yHCT101

yHCT104

yHCT72

yHCT90

yHCT99

yHCT114

yHKS210

yHKS211

yHKS212

yHCT61 yHCT88 yHCT107

yHCT62 yHCT91

yHCT63 yHCT92

yHCT70 yHCT94

yHCT76 yHCT105

Patagonia

Patagonia

&

Lager

S. eubayanus

S. uvarum

Hybrids

S. eubayanus

x

S. uvarum

A

B CyHCT107

yHCT70

yHCT88

yHCT91

yHCT62

yHCT61

yHCT92

yHCT76

yHCT94

yHCT105

yHCT63

W34_70

CBS 1503

yHCT101

yHCT104

yHCT96

yHCT114

yHCT72

yHCT90

yHCT99

yHKS210

yHKS211

yHKS212

CBS7001

100

77

60

93

56

100

76

71

78

61

71

0.005

PA

TA

GO

NIA NORTH

AMERICA

PA

TA

GO

NIA

-LA

GE

R

0.149

(0-0.375)

0.63

(0.5-0.75)

0.419

(0.25-0.625)

0.149

(0-0.375)

0.31

(0.25-0.5)

70/72/0.99

99/100/1

99/100/1

98/92/1

63/63/0.99

94/92/1

51/53/0.88

yHCT104

yHCT96

yHCT101

yHCT114

yHCT90

yHCT99

yHCT72

0.005

yHKS210

yHKS212

yHKS211

yHCT105

yHCT91

yHCT88

yHCT70

yHCT107

yHCT94

yHCT61

yHCT92

W34_70

CBS 1503

yHCT63

yHCT62

yHCT76

CBS7001

PA

TA

GO

NIA

PA

TA

GO

NIA

-LA

GE

RN

OR

TH

AM

ER

ICA

0.410

(0.25-0.625)

0.36

(0.25-0.5)

0.31

(0.25-0.5)

0.379

(0.25-0.625)

0.364

(0.25-0.5)

0.149

(0-0.375)

0.63

(0.5-0.75)

yHKS211

yHKS212

yHKS210

yHCT92

yHCT94

yHCT76

yHCT62

yHCT61

yHCT63

yHCT105

W34_70

CBS 1503

yHCT107

yHCT70

yHCT88

yHCT91

yHCT101

yHCT104

yHCT96

yHCT114

yHCT72

yHCT90

yHCT99

CBS7001

86

95

100

100

80

99

65

99

99

88

65

57

98

0.01

PA

TA

GO

NIA

NORTH

AMERICA

PA

TA

GO

NIA

-LA

GE

R

0.36

(0.25-0.5)

0.146

(0.125-0.25)

0.419

(0.25-0.625)

0.36

(0.25-0.5)

0.31

(0.25-0.5)

yHCT105W34_70

CBS 1503

CBS 7001 S. cerevisiae

S. paradoxusS. mikatae

S. kudriavzevii

yHCT61

yHCT72

yHCT88

yHCT91

yHCT104

yHCT101

yHKS210

yHKS211

yHKS212

yHCT68

yHCT70

yHCT90

yHCT99

yHCT107

yHCT114

yHCT69

yHCT76

yHCT63

yHCT64

yHCT92 yHCT94yHCT62

S. uvarum

S288c IFO1802

1939T

IFO18015

S. eubayanus

MATERIAL & METHODS

CONCLUSIONS

0.3%

0.2% 0.8%

7%

Figure 1. Phylogenetic supernetwork Figure 2. Phylogenetic trees

Figure 3. Phylogenetic network of COX2

The Saccharomyces genus includes seven species. Hybrids have

been described in biotechnological environments. The most studied

hybrid is the lager brewing S. pastorianus1 (S. cerevisiae x S.

eubayanus). The application of new methodologies for yeast

isolation has allowed the identification of the one unknown wild

genetic stock of S. pastorianus, the new species S. eubayanus2.

However, little is known about the diversity of this species, its

distribution, or how it contributed to the evolution of lager brewing

yeast. As a result of the Wild YEAST (Yeast Exploration and

Analysis Science Team) Program3 and the application of many

phylogenetic methods, we were able to explore the diversity of S.

eubayanus strains and their phylogenetic relationship with their

hybrids.

Phylogenetic supernetwork reconstructed using the neighbor-joining trees of the six nuclear genes

(FSY1, FUN14, GDH1, HIS3, MET2, RIP1) by the Z-closure method implemented in SplitsTree 4.

Incongruent topologies are displayed adding new edges to the network. Filter was set up to two to

represent the splits found in at least two phylogenetic trees. Scale bar represents the edge’s

weights inferred using the tree size weighted means options, a measure similar to those from

branches in a phylogenetic tree. STRUCTURE analysis supported two cluster/population

(Patagonia and Patagonia-Lager (data not shown). Nucleotide diversity of each population is

displayed in red color.

A) Phylogenetic tree reconstruction of the multilocus alignment sequences. The best tree topology inferred in RaxML is

shown. Branch support is represented by bootstrap values performed in RaxML and MEGA after 1000 pseudo-replicates

and posterior probability obtained in Beast: Bs RaxML/Bs MEGA/Pp Beast. Outgroup CBS7001 correspond to a S.

uvarum strain. B) NJ tree reconstructed using the concatenation of nuclear genes (FSY1, FUN14, RIP1, URA3) that

support the clustering of North American strains with Patagonia strains. C) NJ tree using the concatenation of nuclear

genes (DCR1, GDH1, HIS3, MET2) that support the clustering of North American strains with the Patagonia-Lager

group. In red color is represented the average and, in parenthesis, the standard deviation of the concordance factors (a

measure of proportion of trees that support a specific branch) obtained by BUCKy software. Red squares represent

nucleotide divergence between S. uvarum and S. eubayanus, and Patagonia and Patagonia-Lager. Scale is given in

nucleotide substitutions per site.

Phylogenetic Neighbor-Net network reconstructed from partial mitochondrial COX2

gene sequences, which contain a recombination hotspot4. Species specific cluster

is displayed using COX2 gene sequences from type strains.

North

America

Patagonia

Europe

Villa Pehuenia

Lanin

Nahuel Huapi

Chile Argentina

Sheboygan

Wisconsin

LITERATURE 1. Martini, A., and Kurtzman, C.P. (1985) Deoxyribonucleic Acid relatedness among species of the genus Saccharomyces Sensu Stricto. Int. J. Syst. Bact. 35, 508-511.

2. Libkind, D., Hittinger, C.T., Valério, E., Gonçalves, C., Dover, J., Johnston, M., Gonçalves, P. and Sampaio, J.P. (2011) Microbe domestication and the identification of the wild

genetic stock of lager-brewing yeast. Proc. Natl. Acad. Sci. U. S. A. 108, 14539-14544.

3. http://hittinger.genetics.wisc.edu/Outreach/YEAST/index.html

4. Peris, D., Belloch, C., Lopandic, K., Álvarez-Pérez, J.M., Querol, A. and Barrio E. (2012) The molecular characterization of new types of S. cerevisiae x S. kudriavzevii hybrid

yeasts unveils a high genetic diversity. Yeast 29, 81-91.

1. The genetic diversity of S. eubayanus strains are higher in Patagonia,

Argentina than among European brewing isolates, all of which are interspecies

hybrids.

2. Two populations exist in sympatry South America, “Patagonia” and “Patagonia-

Lager”.

3. Phylogenetic incongruence could be due to admixture and hybridization.

4. North American strains appear to be closely related and originated by

admixture between Patagonia and Patagonia-Lager strains.

5. COX2 could indicate ancestral hybridization between S. eubayanus and S.

uvarum.

BEAST

SplitsTree 4

RaxML

450 isolates

(3 S. eub strains)

Structure

jModelTest

ABSTRACT

Lager brewing yeast (Scer x Seub)

Brewing contaminants (Seub x Scer x Suva)

yHCT & yHKS: wild S. eubayanus

Lager beer is brewed with Saccharomyces pastorianus, an alloploid hybrid of S. cerevisiae

and a species we recently discovered in Patagonia, Argentina, called S. eubayanus.

However, little is known about the geographic distribution and diversity of S. eubayanus.

The aim of this study was to isolate S. eubayanus strains from South and North America

and to determine their population structure using a multilocus phylogenetic approach. To

infer their evolutionary relationships with wild S. eubayanus, we compared representative

strains from the Frohberg and Saaz groups of lager-brewing alloploid hybrids, as well as

“S. bayanus” triple-hybrid brewing contaminants. Our findings showed a high efficiency of

isolation of S. eubayanus in Nothofagus trees from Patagonia and, for the first time, a rare

isolation from Wisconsin, USA. Multilocus phylogenetic analyses indicate high genetic

diversity among S. eubayanus from Patagonia and support two differentiated populations,

the “Patagonia” and “Patagonia-Lager” populations. The Patagonia-Lager population is

closely related to lager-brewing alloploid hybrids and to the S. bayanus triple hybrids,

suggesting that European brewing strains and contaminants are derived from crosses

involving this population or a close subpopulation. Finally, the genetic characteristics of the

new strains found in North America are consistent with the admixture of Northern and

Patagonia-Lager populations and the recent expansion to North America by an unknown

vector. In conclusion, the range of S. eubayanus is more widespread than expected, but

the non-Patagonian strains have low genetic diversity and appear to have originated by the

admixture or hybridization of migrant strains.

15 sampling

sites

(200+ S. eub

strains)

Lager brewing yeast (Scer x Seub)

yHCT & yHKS: wild S. eubayanus

Recombinant Type I

Recombinant Type II

AKNOWLEDGMENTS This work was funded in part by the DOE Great Lakes Bioenergy

Research Center (DOE Office of Science BER DE-FC02-07ER64494)

and NSF CAREER Award DEB-1253634