Contrasting evolutionary trajectories of nitrate assimilation across Brettanomyces bruxellensis lineages

Brettanomyces bruxellensis is a yeast species associated with diverse fermentation environments and characterized by extensive genetic diversity, including diploid, autotriploid, and allotriploid lineages resulting from independent hybridization events. These lineages are associated with distinct ecological niches and provide a framework for studying metabolic trait evolution in complex genomes. Nitrate assimilation is a relatively uncommon trait among yeasts and has been reported in B. bruxellensis, but its distribution and evolutionary history within the species remain poorly understood. Here, we combined phenotypic characterization of 151 strains with genomic analyses of 946 whole-genome sequences to investigate nitrate assimilation. Growth assays revealed that nitrate assimilation is widespread but unevenly distributed across genetic lineages, with some populations largely retaining the trait whereas others have frequently lost it. Genomic analyses identified extensive variation affecting the nitrate assimilation gene cluster composed of YNR1, YNI1, and YNT1. Nitrate assimilation was strongly associated with both gene copy number and predicted gene functionality, with nitrate-assimilating strains generally carrying more functional copies of the cluster. Leveraging the complex genomic architecture of the species, we independently analyzed primary and acquired genomes in allotriploid lineages and uncovered contrasting evolutionary trajectories following hybridization. While nitrate assimilation genes were generally maintained in primary genomes, acquired genomes showed a higher prevalence of gene loss and predicted loss-of-function variants, revealing asymmetric dynamics between subgenomes. Altogether, our results suggest that nitrate assimilation represents an ancestral trait that has been differentially maintained across B. bruxellensis lineages through a combination of copy number variation, gene degeneration, and genome-specific evolutionary dynamics. These findings provide new insights into how genome architecture and polyploid evolution shape the maintenance and loss of metabolic traits in an industrially relevant yeast species.