Tracing Species Boundaries Through Genomic Baits: Diagnostic SNP Panels for Genotyping Orchid Pollinaria

Orchidaceae is one of the largest angiosperm families, with extensive floral morphological diversity, complex pollination systems, and frequent hybridization, which complicate species delimitation. Target capture offers a scalable alternative to whole-genome sequencing for phylogenomic and ecological applications, but operational thresholds for reliable species identification, particularly from reduced or degraded single-nucleotide polymorphism (SNP) datasets, remain poorly quantified. We developed the 20KOrchidbaits, a kit of 20,000 nuclear and 400 plastid baits, specifically designed for Orchidaceae by using already available (Phalaenopsis) and recently sequenced (Ophrys) orchid genomes. We then tested the kit for assessing phylogenetic relationships and pollinaria assignment in Catasetum, a young and fast-evolving neotropical orchid genus. In silico tests of the 20KOrchidbaits kit showed high recovery efficiency for nuclear baits, particularly in the subfamilies Apostasioideae, Orchidoideae, and Epidendroideae. The kit also showed robust plastid performance (85.9% average mapping). Applying 20KOrchidbaits to Catasetum members confirmed its high recovery efficiency and showed a strong ability to disclose phylogenetic relationships and identify species-specific SNPs. Using the 20KOrchidbaits kit for Catasetum pollinaria genotyping, the pollinaria were accurately assigned to their source orchid species, both with the full dataset of species-specific SNPs and with subsets of it. The 20KOrchidbaits provide a versatile toolkit for gathering species resolution even in rapidly radiating orchid clades. This tool also represents the first reliable species-level identification of orchid pollinarium with target capture, surpassing traditional barcode markers.