From populations to pan-genomes: investigating the role of ecology and genomic architecture in maintaining species boundaries in the porcini mushroom, Boletus edulis

The forces that maintain stable boundaries between closely related groups are poorly characterized in the Fungi . In other organisms prezygotic barriers, such as assortative mating, are sufficient to reinforce local adaptation and prevent hybridization, yet assortative mating and other prezygotic barriers are largely absent in most fungi. Postzygotic barriers, such as large structural changes across the genome, can also maintain lineage segregation through hybrid incompatibility. The role of structural variation in speciation has been well characterized in a wide array of animals and plants, but not in the Fungi , where it may be relatively uncommon. Here, we use a pan-genomic approach in the globally distributed ectomycorrhizal fungus Boletus edulis Bull. with 310 whole genome sequences and 22 newly generated reference genomes to identify whether large genomic changes are necessary to maintain lineage segregation in the presence of ongoing gene flow. In addition, we evaluate the extent to which different lineages possess unique repertories of saprotrophic and effector gene families to determine whether lineages exhibit distinct ecological capacities that contribute toward lineage segregation. We show that B. edulis possesses one of the most diverse pan-genomes of eukaryotes, with the lowest proportion of core proteins identified in the Fungi . Pan-genomic clustering correlates more with geography than with gene flow or phylogenetic relatedness. We also show that structural variation is abundant within B. edulis , but is rarely fixed within a lineage or even shared between two individuals. Moreover, we find that a similar degree of genomic synteny exists between genomes of different lineages as between genomes of the same lineage. Functional protein analysis shows that a new lineage of B. edulis from tropical Guatemala has significant contractions in most saprotrophic gene families and possesses a distinct saprotrophic enzyme repertoire. This lineage also a carries a clear signal of a demographic bottleneck, has very low genome-wide heterozygosity, and an inflated genome size, consistent with a substantial shift in ecological niche. In addition, we reveal preliminary evidence for subtle differences in host preference among European B. edulis . Altogether, our results provide clear evidence that large changes in genome structure or proteome content are not necessary or perhaps even sufficient to maintain long-term lineage fidelity in the presence of ongoing gene flow. Instead, it appears that subtle differences in ecological tendencies can maintain stable boundaries through time. ### Competing Interest Statement The authors have declared no competing interest.