Evolutionary history of the sequestrate genus () reveals a new genus and highlights the utility of ITS minisatellite-like insertions for molecular identification.

The sequestrate (truffle-like) basidiomycete genera , , and are closely related to the epigeous mushroom genera and . In order to elucidate the properties and placement of several undescribed sequestrate taxa in the group and to reveal the evolutionary history of and its allies, we conducted phylogenetic analyses based on three nuclear (ITS, nLSU, ) and two mitochondrial DNA loci ( and mtSSU) as well as precise morphological observations. Phylogenetic analyses of three nuclear loci suggest a complex evolutionary history with sequestrate fruiting bodies present in several clades, including a previously unrecognized sister clade to . Here we propose a new sequestrate genus, , with four new species and one new subspecies as well as two new species of . The three-locus nuclear phylogeny resolves species-level divergence within the lineage, whereas a separate phylogeny based on two mitochondrial genes corresponds to geographic distance within each species-level lineage and suggests incomplete lineage sorting (ILS) and gene introgression within several intraspecific lineages of . Furthermore, topological incongruence among the three nuclear single-locus phylogenies suggests that ancient speciation within probably involved considerable ILS. We also found an unusually long, minisatellite-like insertion within the ITS2 in all and species. A barcode gap analysis demonstrates that the insertion is more informative for discrimination at various taxonomic levels than the rest of the ITS region and could therefore serve as a unique molecular barcode for these genera.