Enrichment Probe Sets Combining Universal and Lineage-Specific Targets Help Resolve Recalcitrant Lineages

The combination of target capture sequencing (TCS) with low-coverage whole genome sequencing (lcWGS), an approach known as Hyb-Seq, has allowed the integration of natural history collections into the genomics revolution, transforming biodiversity research. To implement Hyb-Seq, a collection of genomic targets is needed to design probes. In flowering plants, the Angiosperms353 kit has been proven effective at multiple evolutionary scales, with limitations. Malpighiales is one of the most challenging flowering plant orders to resolve. Within this order, the clusioid clade (~2.200 species, 94 genera, five families) is no exception. To resolve phylogenetic relationships in this recalcitrant clade, we design a custom probe set composed of 39,936 120-mer probes targeting 626 nuclear orthologs. The Clusioids626 kit includes all Angiosperms353 targets and 273 clusioid-specific ones, carefully chosen taking copy-number, length evenness, and phylogenetic informativeness into account. We tested our probe set on 70 accessions representing all clusioid families and tribes. On average, 50.4% reads mapped to our targets, recovering a median of ~600 orthologs/sample. Relationships for all clusioid families are fully resolved for our nuclear targets. A Hypericaceae-Podostemaceae clade is sister to Calophyllaceae, which are all sister to Bonnetiaceae, and then to Clusiaceae. Additionally, we retrieved 105 plastid coding sequences from the lcWGS fraction with a custom target file, evidencing strong incongruence between nuclear and plastid topologies. The Clusioids626 kit performs better than the Angiosperms353 one alone. Our design workflow can be extended to other lineages for which a universal probe set exists but more resolution is needed.