Domestication provides an excellent framework for studying adaptive divergence. Using population genomics and phenotypic assays, we reconstructed the domestication history of the blue cheese mould Penicillium roqueforti. We showed that this fungus was domesticated twice independently. The population used in Roquefort originated from an old domestication event associated with weak bottlenecks and exhibited traits beneficial for pre‐industrial cheese production (slower growth in cheese and greater spore production on bread, the traditional multiplication medium). The other cheese population originated more recently from the selection of a single clonal lineage, was associated with all types of blue cheese worldwide except Roquefort, and displayed phenotypes more suited for industrial cheese production (high lipolytic activity, efficient cheese cavity colonization ability and salt tolerance). We detected genomic regions affected by recent positive selection and putative horizontal gene transfers. This study sheds light on the processes of rapid adaptation and raises questions about genetic resource conservation.

24Domestication provides an excellent framework for studying adaptive divergence. Using population 25 genomics and phenotypic assays, we reconstructed the domestication history of the blue cheese 26 mold Penicillium roqueforti. We showed that this fungus was domesticated twice independently. 27

Studies of food microorganism domestication can provide important insight into adaptation mechanisms and lead to commercial applications. Penicillium roqueforti is a fungus with four genetically differentiated populations, two of which were independently domesticated for blue cheese-making, with the other two populations thriving in other environments. Most blue cheeses are made with strains from a single P. roqueforti population, whereas Roquefort cheeses are inoculated with strains from a second population. We made blue cheeses in accordance with the production specifications for Roquefort-type cheeses, inoculating each cheese with a single P. roqueforti strain, using a total of three strains from each of the four populations. We investigated differences between the cheeses made with the strains from the four P. roqueforti populations, in terms of the induced flora, the proportion of blue color, water activity and the identity and abundance of aqueous and organic metabolites as proxies for proteolysis and lipolysis as well as volatile compounds responsible for flavor and aroma.We found that the population-of-origin of the P. roqueforti strains used for inoculation had a minor impact on bacterial diversity and no effect on the abundance of the main microorganism. The cheeses produced with P. roqueforti strains from cheese populations had a higher percentage of blue area and a higher abundance of the volatile compounds typical of blue cheeses, such as methyl ketones and secondary alcohols. In particular, the Roquefort strains produced higher amounts of these aromatic compounds, partly due to more efficient proteolysis and lipolysis. The Roquefort strains also led to cheeses with a lower water availability, an important feature for preventing spoilage in blue cheeses, which is subject to controls for the sale of Roquefort cheese. The typical appearance and flavors of blue cheeses thus result from human selection on P. roqueforti, leading to the acquisition of specific features by the two cheese populations. These findings have important implications for our understanding of adaptation and domestication, and for cheese improvement.

The study of food microorganism domestication can bring important insights on adaptation mechanisms and have industrial applications. The Penicillium roqueforti mold is divided into four main populations, with two populations domesticated for blue-cheese making and two populations thriving in other environments. While most blue cheeses worldwide are made with the same P. roqueforti clonal lineage, the emblematic Roquefort cheeses are inoculated with a specific population. To study the differences among P.roqueforti populations in the context of domestication for cheesemaking, we compared blue cheeses made with the four fungal populations following Roquefort-type production specifications. We found that the P. roqueforti populations had a minor impact on the cheese bacterial diversity and none on the main microorganism abundance. The cheese P. roqueforti populations produced cheeses with higher percentages of blue area and with different sets and higher quantities of desired volatile compounds. The Roquefort P. roqueforti population in particular produced higher quantities of positive aromatic compounds in cheeses, which was related due to its most efficient proteolysis and lipolysis, and also produced cheeses with lower water activity, thus restricting spoiler microorganisms. Our results show the strong influence of P. roqueforti populations on several important aspects of cheese safety, appearance and flavour. The typical appearance and flavours of blue cheeses are therefore the result of human selection on P. roqueforti, thus constituting domestication, and the two cheese populations have acquired specificities. This has important implications for our understanding of adaptation and domestication processes as well as for improving cheese production.Importance: Several fungi have been domesticated for food fermentation, with selection for traits beneficial for food production. The mold used for making blue cheeses, Penicillium roqueforti, is subdivided into four genetically different populations, two being found in cheese, one being specific of the Roquefort protected designation of origin, and two in other environments. The cheese P. roqueforti populations produced bluer cheeses with higher quantities of desired volatile compounds. The Roquefort P. roqueforti population in particular produced higher quantities of positive aromatic compounds in cheeses, in relation to its most efficient proteolysis and lipolysis, and also produced cheeses with lower water activity, thus restricting spoiler microorganisms. Our results support that the blue cheese typical aspect and flavors are the result of a selection by humans and show the strong influence of P. roqueforti populations for several important aspects of cheese safety, aspect and flavor, paving the way for improving cheese production.

Many fungi have been domesticated for food production, with genetic differentiation between populations from food and wild environments, and food populations often acquiring beneficial traits through horizontal gene transfers. We studied the population structures and phenotypes of two distantly related Penicillium species used for dry-cured meat production, P. nalgiovense, the most common species in the dry-cured meat food industry, and P. salamii, used locally by farms. Both species displayed low genetic diversity, with no differentiation between strains isolated from dry-cured meat and those from other environments. Nevertheless, the strains collected from dry-cured meat within each species displayed slower proteolysis and lipolysis than their wild-type conspecifics, and those of P. nalgiovense were whiter. The phenotypes of the non-dry-cured meat strains were more similar to their sister species than to their conspecific dry-cured meat strains, indicating an evolution of specific phenotypes in dry-cured meat strains. A comparison of available Penicillium genomes from various environments revealed evidence of multiple horizontal gene transfers, particularly between P. nalgiovense and P. salamii. Some horizontal gene transfers involving P. biforme, also found in dry-cured meat products, were also detected. We also detected positive and purifying selection based on amino-acid changes. Our genetic and phenotypic findings suggest that human selection has shaped the P. salamii and P. nalgiovense populations used for dry-cured meat production, which constitutes domestication. Several genetic and phenotypic changes were similar in P. salamii, P. nalgiovense, and P. biforme, providing an interesting case of convergent adaptation to the same human-made environment.