Evolutionary history of the sequestrate genus <i>Rossbeevera</i> (<i>Boletaceae</i>) reveals a new genus <i>Turmalinea</i> and highlights the utility of ITS minisatellite-like insertions for molecular identification

The sequestrate (truffle-like) basidiomycete genera <i>Rossbeevera</i>, <i>Chamonixia</i>, and <i>Octaviania</i> are closely related to the epigeous mushroom genera <i>Leccinum</i> and <i>Leccinellum</i>. In order to elucidate the properties and placement of several undescribed sequestrate taxa in the group and to reveal the evolutionary history of <i>Rossbeevera</i> and its allies, we conducted phylogenetic analyses based on three nuclear (ITS, nLSU, <i>EF-1α</i>) and two mitochondrial DNA loci (<i>ATP6</i> 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 <i>Rossbeevera</i>. Here we propose a new sequestrate genus, <i>Turmalinea</i>, with four new species and one new subspecies as well as two new species of <i>Rossbeevera</i>. The three-locus nuclear phylogeny resolves species-level divergence within the <i>Rossbeevera-Turmalinea</i> 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 <i>Rossbeevera</i>. Furthermore, topological incongruence among the three nuclear single-locus phylogenies suggests that ancient speciation within <i>Rossbeevera</i> probably involved considerable ILS. We also found an unusually long, minisatellite-like insertion within the ITS2 in all <i>Rossbeevera</i> and <i>Turmalinea</i> 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.