Abstract
Despite 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.
Highlights
The continued development of high-throughput sequencing approaches enables time and cost efficient generation of massive amounts of genomic data and has facilitated the expansion of genomic datasets for phylogenetic- and population-level studies from single- and multi-locus matrices to genetic data sampled across entire genomes[1]
Since lichens are symbiotic associations consisting of evolutionarily independent lineages and DNA extracted from a lichen thallus is comprised of genomes from all associated symbionts, we separated the metagenomic data of each sample with a mapping approach to a reference fungal genome from a R. melanophthalma culture
For the restriction associated DNA sequencing (RADseq) simulation, the reference genome sequence of R. melanophthalma was in silico digested by six different restriction enzymes (SbfI, PstI, NsiL, BclI, BstYI, and ApeKI)
Summary
The continued development of high-throughput sequencing approaches enables time and cost efficient generation of massive amounts of genomic data and has facilitated the expansion of genomic datasets for phylogenetic- and population-level studies from single- and multi-locus matrices to genetic data sampled across entire genomes[1]. For many organisms growing in intimate symbiotic associations, such as lichen-forming fungi, it is difficult to physically separate the symbiotic partners and growth of axenic cultures may be prohibitively slow[30, 31] In these cases, a reference-guided approach is crucial to separate the metagenomic pool of sequences that was derived from all symbiotic partners of the holobiont. Developing time and cost-effective approaches for generating genome-scale datasets is crucial to infer robust diversification histories and gain novel insight into evolutionary processes in these symbiotic fungi. This study was able to infer robust phylogenetic relationships, the genome skimming approach was relatively costly and limited to few dozen individuals It remains unclear how other reduced representation approaches for generating phylogenomic datasets can effectively be applied to metagenomic DNA pools isolated from lichen holobionts
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
