Re-examination of the Southern Hemisphere truffle genus () and characterization of the sister genus gen. nov.

is a genus of ectomycorrhizal truffles within that is known from Australia and contains only two described species, and . Species of are closely related to truffles (, ) and cup fungi () from the Northern Hemisphere. Here we reevaluate the species diversity of and related taxa from southern South America and Australia based on new morphological and molecular data. We identify previously undocumented diversity and morphological variability in ascospore color, ascospore ornamentation, hymenial construction, epithecium structure and the amyloid reaction of the ascus in Melzer's reagent. We redescribe two species from Australia and describe seven new species, five from South America and two from Australia. This is the first report of species from South America. We also describe the new South American genus as sister lineage to the clade (including , , , and ). We obtained ITS sequences of mitotic spore mats from gen. & sp. nov. and four of the seven newly described species, providing the first evidence of mitotic spore mats in . Additional ITS sequences from mitotic spore mats reveal the presence of nine additional undescribed and one species that do not correspond to any sampled ascomata. We also identify three additional undescribed species based on environmental sequences from the feces of two grounddwelling bird species from Chile, and . Our results indicate that ascomata from and species are rarely collected, but molecular data from ectomycorrhizal roots and mitotic spore mats indicate that these species are probably common and widespread in southern South America. Finally, we present a time-calibrated phylogeny that is consistent with a late Gondwanan distribution. The time since the most recent common ancestor of: 1) the family had a mean of 276 Ma (217-337 HPD); 2) the clade had a mean of 79 Ma (60-100 HPD); and 3) the clade had a mean of 50 Ma (38-62 HPD). The crown age of had a mean of 39 Ma (25-42 HPD) and had a mean age of 28 Ma (20-37 HPD), falling near the Eocene-Oligocene boundary and the onset of the Antarctic glaciation (c. 35 Ma). : Healy RA, Truong C, Castellano MA, et al. 2023. Re-examination of the Southern Hemisphere truffle genus Amylascus (Pezizaceae, Ascomycota) and characterization of the sister genus Nothoamylascus gen. nov. Persoonia 51: 125-151. doi: 10.3767/persoonia.2023.51.03.