Angiosperms-353 target enrichment of Greek orchids

Tuberous orchids are ecologically vulnerable species, threatened by a range of environmental pressures such as overharvesting, grazing and land use change. Conservation efforts require accurate species identification, but are impeded by limited phylogenetic resolution of traditional genetic markers, which is exacerbated by widespread taxonomic conflict regarding the classification of orchids. Target enrichment promises to resolve both these challenges by offering a large set of nuclear loci with which to increase phylogenetic resolution and evaluate competing species models.In this study, we evaluate the effectiveness of the Angiosperms353 markers for distinguishing over 50 tuberous orchid species native to Greece and we explore the possibility of narrowing these markers to a smaller set that could function as a minimal probe set. Our methodology consists of a three-tiered approach: 1) generating a species-level phylogeny using all Angiosperms353 loci with sufficient target recovery, 2) evaluating competing species models based on splitter and lumper classifications through Bayes Factor species delimitation, and 3) ranking the potential of Angiosperms353 loci to discriminate representatives of lineages with different divergence times based on their phylogenetic informativeness.While the inferred multi-species coalescent phylogeny overall had high support, Bayes Factor delimitation revealed mixed outcomes, favouring the splitter approach in Serapias, and in Ophrys alternately favouring splitting in basal clades and lumping in more recently diverged clades. A molecular clock analysis of Ophrys showed rapid and recent radiation in clades marked by phylogenetic uncertainty, suggesting that additional loci may be required to resolve certain relationships. Finally, we found a subset of 30 loci to be highly phylogenetically informative across four epochs of orchid evolution, and promising candidates for future marker development.Our findings enhance the Plant and Fungal Tree of Life (PAFTOL) by contributing additional phylogenomic data for species that were previously underrepresented, while shedding light on the splitter-vs-lumper debate and offering new directions for species identification of tuberous orchids, a group with distinct taxonomic and conservation challenges.