Re-examination of the Southern Hemisphere truffle genus Amylascus (Pezizaceae, Ascomycota) and characterization of the sister genus Nothoamylascus gen. nov

Amylascus is a genus of ectomycorrhizal truffles within Pezizaceae that is known from Australia and contains only two described species, A. herbertianus and A. tasmanicus . Species of Amylascus are closely related to truffles ( Pachyphlodes , Luteoamylascus ) and cup fungi ( Plicariella ) from the Northern Hemisphere. Here we reevaluate the species diversity of Amylascus 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 Amylascus species from Australia and describe seven new Amylascus species, five from South America and two from Australia. This is the first report of Amylascus species from South America. We also describe the new South American genus Nothoamylascus as sister lineage to the Pachyphlodes - Amylascus - Luteoamylascus clade (including Amylascus , Luteoamylascus , Pachyphlodes , and Plicariella ). We obtained ITS sequences of mitotic spore mats from Nothoamylascus erubescens gen. & sp. nov. and four of the seven newly described Amylascus species, providing the first evidence of mitotic spore mats in Amylascus . Additional ITS sequences from mitotic spore mats reveal the presence of nine additional undescribed Amylascus and one Nothoamylascus species that do not correspond to any sampled ascomata. We also identify three additional undescribed Amylascus species based on environmental sequences from the feces of two ground-dwelling bird species from Chile, Scelorchilus rubecula and Pteroptochos tarnii . Our results indicate that ascomata from Amylascus and Nothoamylascus 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 Pezizaceae had a mean of 276 Ma (217–337 HPD); 2) the Amylascus - Pachyphlodes - Nothoamylascus - Luteoamylascus clade had a mean of 79 Ma (60–100 HPD); and 3) the Amylascus - Pachyphlodes clade had a mean of 50 Ma (38–62 HPD). The crown age of Pachyphlodes had a mean of 39 Ma (25–42 HPD) and Amylascus had a mean age of 28 Ma (20–37 HPD),