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Species delineation in microorganisms is challenging due to the limited markers available for accurate species assignment. Here, we applied an integrative taxonomy approach, combining extensive sampling, whole-genome sequence-based classification, phenotypic profiling, and assessment of interspecific reproductive isolation. Our work reveals the presence of a distinct Saccharomyces lineage in Nothofagus forests of coastal Patagonia. This lineage, designated Saccharomyces chiloensis sp. nov., exhibits 7% genetic divergence from its sister species S. uvarum, as revealed by whole-genome sequencing and population analyses. The South America-C (SA-C) coastal Patagonia population forms a unique clade closely related to a previously described divergent S. uvarum population from Oceania (AUS, found in Australia and New Zealand). Our species reclassification is supported by a low Ortho Average Nucleotide Identity (OANI) of 93% in SA-C and AUS relative to S. uvarum, which falls below the suggested species delineation threshold of 95%, indicating an independent evolutionary lineage. Hybrid spore viability assessment provided compelling evidence that SA-C and AUS are reproductively isolated from S. uvarum. In addition, we found unique structural variants between S. chiloensis sp. nov. lineages, including large-scale chromosomal translocations and inversions, together with a distinct phenotypic profile, emphasizing their intraspecies genetic distinctiveness. We suggest that S. chiloensis sp. nov diverged from S. uvarum in allopatry due to glaciation, followed by post-glacial dispersal, resulting in distinct lineages on opposite sides of the Pacific Ocean. The discovery of S. chiloensis sp. nov. illustrates the uniqueness of Patagonia's coastal biodiversity and underscores the importance of adopting an integrative taxonomic approach in species delineation to unveil cryptic microbial species. The holotype of S. chiloensis sp. nov. is CBS 18620T., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Peña et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Lager yeasts are limited to a few strains worldwide, imposing restrictions on flavour and aroma diversity and hindering our understanding of the complex evolutionary mechanisms during yeast domestication. The recent finding of diverse S. eubayanus lineages from Patagonia offers potential for generating new lager yeasts with different flavour profiles. Here, we leverage the natural genetic diversity of S. eubayanus and expand the lager yeast repertoire by including three distinct Patagonian S. eubayanus lineages. We used experimental evolution and selection on desirable traits to enhance the fermentation profiles of novel S. cerevisiae x S. eubayanus hybrids. Our analyses reveal an intricate interplay of pre-existing diversity, selection on species-specific mitochondria, de-novo mutations, and gene copy variations in sugar metabolism genes, resulting in high ethanol production and unique aroma profiles. Hybrids with S. eubayanus mitochondria exhibited greater evolutionary potential and superior fitness post-evolution, analogous to commercial lager hybrids. Using genome-wide screens of the parental subgenomes, we identified genetic changes in IRA2, IMA1, and MALX genes that influence maltose metabolism, and increase glycolytic flux and sugar consumption in the evolved hybrids. Functional validation and transcriptome analyses confirmed increased maltose-related gene expression, influencing greater maltotriose consumption in evolved hybrids. This study demonstrates the potential for generating industrially viable lager yeast hybrids from wild Patagonian strains. Our hybridization, evolution, and mitochondrial selection approach produced hybrids with high fermentation capacity and expands lager beer brewing options., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Molinet et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Population-level sampling and whole-genome sequences of different individuals allow one to identify signatures of hybridization, gene flow and potential molecular mechanisms of environmental responses. Here, we report the isolation of 160 Saccharomyces eubayanus strains, the cryotolerant ancestor of lager yeast, from ten sampling sites in Patagonia along 2,000 km of Nothofagus forests. Frequency of S. eubayanus isolates was higher towards southern and colder regions, demonstrating the cryotolerant nature of the species. We sequenced the genome of 82 strains and, together with 23 available genomes, performed a comprehensive phylogenetic analysis. Our results revealed the presence of five different lineages together with dozens of admixed strains. Various analytical methods reveal evidence of gene flow and historical admixture between lineages from Patagonia and Holarctic regions, suggesting the co-occurrence of these ancestral populations. Analysis of the genetic contribution to the admixed genomes revealed a Patagonian genetic origin of the admixed strains, even for those located in the North Hemisphere. Overall, the Patagonian lineages, particularly the southern populations, showed a greater global genetic diversity compared to Holarctic and Chinese lineages, in agreement with a higher abundance in Patagonia. Thus, our results are consistent with a likely colonization of the species from peripheral glacial refugia from South Patagonia. Furthermore, fermentative capacity and maltose consumption resulted negatively correlated with latitude, indicating better fermentative performance in northern populations. Our genome analysis, together with previous reports in the sister species S. uvarum suggests that a S. eubayanus ancestor was adapted to the harsh environmental conditions of Patagonia, a region that provides the ecological conditions for the diversification of these ancestral lineages., Competing Interests: the authors have declared that no competing interests exist.