Multiple origins of sequestrate basidiomes within Entoloma inferred from molecular phylogenetic analyses.

The genus Entoloma comprises diverse trophic modes and basidiome morphologies. Although Entoloma includes some sequestrate species, their origins are not clearly understood in relation to phylogenetic position and trophic status. In this study, we collected 34 sequestrate Entoloma specimens in Japan over a 9-y period. Their identities and phylogenetic positions were determined by molecular analyses using three nuclear loci [internal transcribed spacer (ITS) and large subunit (LSU) regions of rDNA and RNA polymerase II second LSU (rpb2) gene]. Based on species delimitation of 97 % sequence matches in the ITS region, which is a suitable region for species-level identification of higher fungi, we identified four sequestrate Entoloma species. Molecular phylogenetic analyses that included all related sequences in the International Nucleotide Sequence Database revealed that the four sequestrate Entoloma species belonged to two major phylogroups. One of the phylogroups was Inocephalus-Cyanula, which is composed only of saprotrophic species. Three of the Japanese sequestrate species, as well as three previously known sequestrate species from other regions, fell into at least two independent clades in this phylogroup, indicating multiple origins of sequestrate forms within this saprotrophic lineage. Another phylogroup, Rhodopolioid, was also shown to include a sequestrate species for the first time. Because the Rhodopolioid phylogroup is composed exclusively of mycorrhizal species (ectomycorrhizal and tuberculate mycorrhizal species), the sequestrate form may also have evolved from a mycorrhizal ancestor. Our results suggest that sequestrate basidiomes have evolved multiple times, irrespective of their trophic status in Entoloma. Finally, based on molecular and morphological characteristics, here we describe two new sequestrate Entoloma species, i.e., Entoloma prismaticum Sasaki, Kinoshita et Nara, sp. nov. and Entoloma hypogaeum Sasaki, Kinoshita et Nara, sp. nov.