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Abstract PremiseThe agaricomycete order Cantharellales contains approximately 1000 species of fungi characterized by diverse morphological forms, ecological guilds, and nutritional modes. Examples include coralloid lichens that form symbioses with unicellular green algae, bulbil‐forming lichenicolous species, corticioid free‐living fungi that degrade dead sources of organic carbon, pathogens that cause plant disease, orchid root endosymbionts, and ectomycorrhizal fungi including popular edible mushrooms. However, evolutionary relationships in the Cantharellales remain poorly understood due to conflicting estimates based on ribosomal DNA loci. MethodsWe constructed a five‐gene phylogeny of the Cantharellales using data from 301 specimens to evaluate family‐level relationships. We used penalized likelihood to estimate divergence times and ancestral state reconstruction to test the hypothesis of multiple independent origins of biotrophic ecologies in the order and whether those transitions are younger than the divergence times of associated plant or lichen hosts. ResultsFour monophyletic families were recovered with strong support: Botryobasidiaceae, Ceratobasidiaceae, Hydnaceae s.l., and Tulasnellaceae, with Hydnaceae containing the greatest species richness and morphological diversity. Our results suggest the Cantharellales diverged during the Carboniferous period with subsequent diversification following the Permian‐Triassic extinction. Ancestral state reconstruction supports a saprotrophic most recent common ancestor with at least three transitions to an ectomycorrhizal ecology, multiple transitions to a lichenicolous habit with one or more subsequent transitions to mutualistic nutritional modes, four transitions to an orchid mycorrhizal ecology, and two transitions to a lichenized lifestyle. ConclusionsThis study represents the first comprehensive examination of the evolution of form and function across this ecologically and morphologically diverse order of fungi. 

Abstract Multi‐locus sequence data are widely used in fungal systematic and taxonomic studies to delimit species and infer evolutionary relationships. We developed and assessed the efficacy of a multi‐locus pooled sequencing method using PacBio long‐read high‐throughput sequencing. Samples included fresh and dried voucher specimens, cultures and archival DNA extracts of Agaricomycetes with an emphasis on the order Cantharellales. Of the 283 specimens sequenced, 93.6% successfully amplified at one or more loci with a mean of 3.3 loci amplified. Our method recovered multiple sequence variants representing alleles of rDNA loci and single copy protein‐coding genesrpb1,rpb2 andtef1. Within‐sample genetic variation differed by locus and taxonomic group, with the greatest genetic divergence observed among sequence variants ofrpb2 andtef1 from corticioid Cantharellales. Our method is a cost‐effective approach for generating accurate multi‐locus sequence data coupled with recovery of alleles from polymorphic samples and multi‐organism specimens. These results have important implications for understanding intra‐individual genomic variation among genetic loci commonly used in species delimitation of fungi.