Phylogenomic species delimitation reveals distinct pace and mode of lineage evolution in Amanita jacksonii

Amanita jacksonii s.l. represents a challenging case of species delimitation in mushroom-forming fungi, where traditional morphological characters and standard molecular markers have proven insufficient for clear taxonomic resolution. Using exon-targeted sequencing of over 100 genetic loci from 63 specimens across North America, we provide robust phylogenomic evidence supporting seven distinct species within the closely related group. Our analyses recovered 30,342 high-quality variants and 172 loci averaging 2.5 Kb in length, enabling comprehensive genetic structure and species delimitation analyses. Population structure analyses revealed clear geographic patterns in Mesoamerican highland populations but showed no evident spatial structure among lowland forest taxa in the United States. Multiple coalescent-based species delimitation methods consistently supported previously identified taxa, with the exception of A. sp. jack5 and A. sp. jack6, which were merged into a single structured species. Molecular dating indicates the group began diversifying during the Pliocene (∼4.32 Ma), with most speciation events occurring within a 2-million-year window. Notably, sympatric lowland species showed deeper divergence (6%-8%) compared to allopatric highland taxa (0.4%), suggesting possible prezygotic barriers in the former. Our results also confirm that A. jacksonii s.s. is restricted to the Appalachian region and eastern Canada, likely resulting from post-glacial expansion. This study demonstrates the power of phylogenomic approaches in resolving species boundaries within morphologically similar fungal taxa and provides insights into the diversification patterns of ectomycorrhizal fungi in North America.