Achieving Symbiont-Level Evenness in Nuclear Gene Recovery Using Parallel Dual-Target Capture

Premise: Symbiotic systems underpin major ecological and evolutionary processes, yet many fundamental questions about these interactions remain unresolved because symbiotic partners are physically inseparable and occur in highly asymmetric abundances. This imbalance limits the joint recovery of genomic data from multiple partners from the metagenomic sample, constraining analyses of specificity, co-diversification, community assembly, and ecological interactions. Methods: We developed a dual target capture approach to simultaneously enrich nuclear loci from multiple symbiotic partners using a single metagenomic extraction and library. We evaluated this method in fog lichens (Cenozosia, Niebla, and Vermilacinia) and their green algal photobionts (Trebouxia) using two novel baits sets across 47 specimens, including historical specimens. Results: Dual target capture substantially improved recovery of low-abundance photobiont loci relative to unenriched metagenomic sequencing, while maintaining consistent recovery across taxa and specimen types. Comparable sets of orthologous nuclear loci were recovered from both fungal and algal partners using single libraries. We also demonstrate reuse of libraries for sequential hybridization, increasing efficiency for low-input DNA. Discussion: Dual target capture enables balanced and simultaneous recovery of genomic data from multiple symbiotic partners, facilitating a shift from single-partner phylogenomics toward joint investigations of symbioses where physical separation of the partners or independent sequencing is not feasible.