A novel target capture probe set reveals widespread unknown hybridization in North American pondweeds (Potamogetonaceae)

Premise Advancements in phylogenomics and bioinformatics provide opportunities to more robustly characterize hybrid lineages and incorporate them into phylogenetic hypotheses. Target capture remains the most cost‐effective molecular method of hybrid determination at scale. The pondweed family is an ecologically important but taxonomically difficult freshwater plant family due to high rates of hybridization. Historically, hybrid pondweeds have been challenging to characterize with limited molecular data, and hybridization is thought to be underdescribed as a result. Methods We designed a novel target capture probe set for Potamogetonaceae targeting 350 loci (Pondweeds350), combining 132 custom Potamogetonaceae targets, 205 Angiosperms353 loci, and 13 functional genes for aquatic adaptation. The probe set was tested on 285 samples of North American Potamogetonaceae. HybPhaser was used to identify and phase hybrid accessions for phylogenetic analysis. Results Target enrichment was successful for 280 of 285 samples with an average of 336 genes producing contigs per sample. HybPhaser provided evidence for 16 novel hybrid combinations in Potamogeton . Discussion We demonstrate the effectiveness of the Pondweeds350 probe set as well as the utility of HybPhaser in detecting hybrids at a large scale. We provide evidence for undetected Potamogeton hybrids in addition to the most densely sampled and well‐resolved phylogeny of North American Potamogetonaceae to date.