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Abstract: Porcini (Boletus section Boletus: Boletaceae: Boletineae: Boletales) are a conspicuous group of wild, edible mushrooms characterized by fleshy fruiting bodies with a poroid hymenophore that is “stuffed” with white hyphae when young. Their reported distribution is with ectomycorrhizal plants throughout the Northern Hemisphere. Little progress has been made on the systematics of this group using modern molecular phylogenetic tools because sampling has been limited primarily to European species and the genes employed were insufficient to resolve the phylogeny. We examined the evolutionary history of porcini by using a global geographic sampling of most known species, new discoveries from little explored areas, and multiple genes. We used 78 sequences from the fast-evolving nuclear internal transcribed spacers and are able to recognize 18 reciprocally monophyletic species. To address whether or not porcini form a monophyletic group, we compiled a broadly sampled dataset of 41 taxa, including other members of the Boletineae, and used separate and combined phylogenetic analysis of sequences from the nuclear large subunit ribosomal DNA, the largest subunit of RNA polymerase II, and the mitochondrial ATPase subunit six gene. Contrary to previous studies, our separate and combined phylogenetic analyses support the monophyly of porcini. We also report the discovery of two taxa that expand the known distribution of porcini to Australia and Thailand and have ancient phylogenetic connections to the rest of the group. A relaxed molecular clock analysis with these new taxa dates the origin of porcini to between 42 and 54 million years ago, coinciding with the initial diversification of angiosperms, during the Eocene epoch when the climate was warm and humid. These results reveal an unexpected diversity, distribution, and ancient origin of a group of commercially valuable mushrooms that may provide an economic incentive for conservation and support the hypothesis of a tropical origin of the ectomycorrhizal symbiosis. [Copyright &y& Elsevier]

Abstract: Psathyrella is the archetypal little brown mushroom genus with few easily discernable characters causing it to be considered a “clean-up” genus for other small brown-spored saprotrophic species found worldwide. While molecular studies have demonstrated that mushroom genera based on homoplastic morphological characters are artificial, the degree of phylogenetic heterogeneity contained within Psathyrella and Psathyrellaceae has never been appropriately addressed. For this study, 132 ribosomal sequences from approximately one-tenth of the known Psathyrella species worldwide, including representatives of most subgeneric subdivisions, and three closely related coprinoid genera (Parasola, Coprinopsis, Coprinellus) were evaluated using multiple phylogenetic methods, including likelihood, with Agaricaceae as the outgroup. Our results indicated that Psathyrella was polyphyletic. Conservatively, the genus can be separated into 11 clades of which five can be raised to generic status. Most species of Psathyrella, including its type species P. gracilis, formed a large clade with Coprinellus, which appeared to be derived from within Psathyrella. Generic limits of Parasola, Lacrymaria, and Coprinopsis should be reevaluated. Several taxa previously synonymized based on morphological features were phylogenetically distinct. Morphological features traditionally used to subdivide Psathyrella appeared to be mostly convergent (homoplasious) when traced upon the resulting phylogenies, although several had high RI values. These results were interpreted in light of the two major taxonomic treatments of Psathyrella and revealed substantial inconsistencies between the molecular- and morphology-derived inferences of relationships. [Copyright &y& Elsevier]

• The mechanisms of concerted evolution may insufficiently homogenize the rDNA array. • Alleles persist in the ITS regions at low frequencies, and may be undetectable by Sanger Sequencing. • ITS gene relatedness and gene flow in Boletus edulis does not correlate with genomic relatedness. • Significant inbreeding may allow fixation of novel ITS alleles, creating false signals of gene flow. • ITS barcoding for identification of population boundaries and complex species resolution is suspect. Concerted evolution of the ribosomal DNA array has been studied in numerous eukaryotic taxa, yet is still poorly understood. rDNA genes are repeated dozens to hundreds of times in the eukaryotic genome (Eickbush and Eickbush, 2007) and it is believed that these arrays are homogenized through concerted evolution (Zimmer et al., 1980; Dover, 1993) preventing the accumulation of intragenomic, and intraspecific, variation. However, numerous studies have reported rampant intragenomic and intraspecific variation in the rDNA array (Ganley and Kobayashi, 2011; Naidoo et al., 2013; Hughes and Petersen, 2001; Lindner and Banik, 2011; Li et al., 2013; Lindner et al., 2013; Hughes et al., 2018), contradicting our current understanding of concerted evolution. The internal transcribed spacers (ITS) of the rDNA cistron are the most commonly used DNA barcoding region in Fungi (Schoch et al., 2012), and rely on concerted evolution to homogenize the rDNA array leading to a "barcode gap" (Puillandre et al., 2012). Here we show that in Boletus edulis Bull., ITS intragenomic variation persists at low allele frequencies throughout the rDNA array, this variation does not correlate with genomic relatedness between populations, and rDNA genes may not evolve in a strictly concerted fashion despite the presence of unequal recombination and gene conversion. Under normal assumptions, heterozygous positions found in ITS sequences represent hybridization between populations, yet through allelic mapping of the rDNA array we found numerous heterozygous alleles to be stochastically introgressed throughout, presenting a dishonest signal of gene flow. Moreover, despite the signal of gene flow in ITS, our organisms were highly inbred, indicating a disconnect between true gene flow and barcoding signals. In addition, we show that while the mechanisms of concerted evolution are ongoing in pseudo-heterozygous individuals, they are not fully homogenizing the ITS array. Concerted evolution of the rDNA array may insufficiently homogenize the ITS gene, allowing for misleading signals of gene flow to persist, vastly complicating the use of the ITS locus for DNA barcoding in Fungi. [ABSTRACT FROM AUTHOR]