AbstractDespite increasing availability of phylogenomic datasets, strategies to generate genome-scale data from organisms involved in symbiotic relationships remains challenging. Restriction site-associated DNA sequencing (RADseq) can effectively generated reduced representation genomic loci. However, when using metagenomic DNA from inseparable symbiotic organisms, RADseq loci may belong to any number of the organisms involved in these intimate associations. In this study, we explored the potential for a reference-based RADseq approach to generate data for lichen-forming fungi from metagenomic DNA extracted from intact lichens. We simulated RAD data from draft genomes of closely related lichenized fungi to test if RADseq can reconstruct robust evolutionary relationships. Subsequently, we generated empirical RADseq data from metagenomic lichen DNA, with RADseq loci mapped back to a reference genome to exclude loci from other lichen symbionts that are represented in metagenomic libraries. In all cases, phylogenetic reconstructions using RADseq loci recovered diversification histories consistent with a previous study based on more comprehensive genome sampling. Furthermore, RADseq loci were found to resolve relationships among closely related species, which were otherwise indistinguishable using a phylogenetic species recognition criterion. Our studies revealed that a modified, reference-based RADseq approach can successfully be implemented to generate symbiont-specific phylogenomic data from metagenomic reads.