A long-read high-throughput multi-locus sequencing method for fungal systematics and species delimitation
Multi-locus sequence data are widely used in fungal taxonomic and systematic 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, culture isolates, and archival DNA extracts of fungi in the Agaricomycotina 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 genes rpb1, rpb2, and tef1. Within-sample genetic variation varied by locus and taxonomic group, but the greatest genetic divergence was observed among sequence variants of rpb2 and tef1 from corticioid Cantharellales. Our method is a cost-effective approach to generating accurate multi-locus sequence data with the ability to recover alleles from polymorphic samples and compound organisms. These results have important implications for understanding intra-individual genomic variation among genetic loci commonly used in species delimitation of fungi.