Evolutionary innovations can occur via several mutational paths but an integrated view of novel gene acquisition at the population level is still lacking. The different mechanisms that generate or introduce new genes in a genome act continuously throughout evolution, resulting in genes of different ages. However, most studies on novel genes were conducted at the interspecific level by comparing a single reference genome per species. Population genomics is presently shifting the field of comparative genomics from single reference genomes to population pangenomes, thereby giving access to individual variations in presence/absence of genes at the population level. The collection of genes present in a population is named the pangenome. The pangenome consists of core genes invariably present in all individuals and accessory genes that are segregating in the population at varying frequencies. Here, we define Novel Accessory Genes (NAGs) as the subset of accessory genes that are not vertically inherited from the species ancestor but were gained or emerged during the diversification of the species. NAGs originate mainly from introgression events, horizontal gene transfers (HGTs) and de novo gene emergence. When novel genes first appear, they are present at very low frequency in a population, likely in a single individual, and subsequently can either disappear or eventually raise in frequency. Therefore, we make the hypothesis that the best evolutionary time scale to investigate gene acquisition mechanisms would be at the population level. We propose to use the yeast Saccharomyces cerevisiae as a model organism to explore, at the species level and at the genome scale, the dynamics of gene acquisition at the time they arise and before they are removed by selection. We will directly benefit both from the high quality population genomic dataset available and the possibility to perform large-scale experimental testing to a level not accessible in any other model organism. The main goal of our proposal is to systematically explore the mechanisms of acquisition of NAGs and their relative contributions to the emergence of evolutionary novelties. First, we will analyse the evolutionary trajectories of NAGs based on high quality population genomics datasets and using state of the art computational approaches. Second, we will measure fitness effect of natural and engineered NAGs by combining genome editing, synthetic biology and high throughput phenotyping. Finally, we will investigate how NAGs functionally wire into cellular networks by measuring transcription, translation and post- translational modifications for both natural and engineered NAGs in various environments in order to quantify their level of i cellular integration. Altogether we plan to generate a large pool of heterogeneous data that we will integrate into a single conceptual framework including information on ecological niches and genetic backgrounds. The outcome of this proposal will provide a multi-layered view of the functional and evolutionary impacts of NAGs in the yeast pangenome revealing general rules leading to the evolution of new genes and new functions in eukaryotes.