Different evolutionary trends form the twilight zone of the bacterial pan-genome

The pan-genome is defined as the combined set of all genes in the gene pool of a species. Pan-genome analyses have been very useful in helping to understand different evolutionary dynamics of bacterial species: an open pan-genome often indicates a free-living lifestyle with metabolic versatility, while closed pan-genomes are linked to host-restricted, ecologically specialised bacteria. A detailed understanding of the species pan-genome has also been instrumental in tracking the phylodynamics of emerging drug resistance mechanisms and drug resistant pathogens. However, current approaches to analyse a species pan-genome do not take the species population structure into account, nor do they account for the uneven sampling of different lineages, as is commonplace due to over-sampling of clinically relevant representatives. Here we present the application of a population structure-aware approach for classifying genes in a pan-genome based on within-species distribution. We demonstrate our approach on a collection of 7,500 E. coli genomes, one of the most-studied bacterial species used as a model for an open pan-genome. We reveal clearly distinct groups of genes, clustered by different underlying evolutionary dynamics, and provide a more biologically informed and accurate description of the species pan-genome.