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 totruffles (Pachyphlodes, Luteoamylascus) and cupfungi(Plicariella) from the NorthernHemisphere. 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 ascusin Melzer's reagent. Were describe 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 Americangenus Nothoamylascus as sister lineage to the Pachyphlodes -Amylascus-Luteoamylascus clade (including Amylascus, Luteoamylascus, Pachyphlodes, and Plicariella). We obtained ITS sequences of mitoticsporemats from Nothoamylascus erubescens gen.&sp. nov. and four of the seven newly described Amylascus species, providing the first evidence of mitoticsporematsin Amylascus. Additional ITS sequences from mitoticsporemats reveal the presence of nine additional undescribed Amylascus and one Nothoamylascus species that do not correspond to any sampled as comata. We also identify three additional undescribed Amylascus secies base do nenvironmental sequences from the feces of two ground dwelling bird species from Chile, Scelorchilus rubecula and Pteroptochos tarnii. Our results indicate that as comata from Amylascus and Nothoamylascus speciesare rarely collected, but molecular data from ectomy corrhizal roots and mitoticsporemats indicate that these species are probably common and wide spread 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 (217–337HPD);2) the Amylascus-Pachyphlodes-Nothoamylascus-Luteoamylascus clade had a mean of 79Ma(60–100HPD); and 3) the Amylascus- Pachyphlodes cladehadameanof50Ma(38–62HPD). Thecrownageof Pachyphlodes had a mean of 39Ma(25–42HPD) and Amylascus had a mean age of 28 Ma(20–37HPD), falling near the Eocene Oligocene boundary and the onset of the Antarcticglaciation (c.35Ma).