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Population structure, admixture, and migration of Saccharomyces eubayanus and their lager-brewing alloploid hybrids Peris D 1,* , Sylvester K 1 , Libkind D 2 , Gonçalves P 3 , Sampaio JP 3 , Hittinger CT 1,4 1 Laboratory of Genetics, DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53706, USA; 2 Laboratorio 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; 3 Centro de Recursos Microbiológicos, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal; 4 Genome Center of Wisconsin, Wisconsin Bioenergy Initiative ; *e-mail address: [email protected] 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 C yHCT107 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 PATAGONIA NORTH AMERICA PATAGONIA - LAGER 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 PATAGONIA PATAGONIA - LAGER NORTH AMERICA 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 PATAGONIA NORTH AMERICA PATAGONIA - LAGER 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) yHCT105 W34_70 CBS 1503 CBS 7001 S. cerevisiae S. paradoxus S. mikatae S. kudriavzevii yHCT61 yHCT72 yHCT88 yHCT91 yHCT104 yHCT101 yHKS210 yHKS211 yHKS212 yHCT68 yHCT70 yHCT90 yHCT99 yHCT107 yHCT114 yHCT69 yHCT76 yHCT63 yHCT64 yHCT92 yHCT94 yHCT62 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. pastorianus 1 (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. eubayanus 2 . 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) Program 3 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 hotspot 4 . 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
