An integrative approach clarifies species delimitation and biogeographic history of Debregeasia (Urticaceae).

Integrative data from plastid and nuclear loci are increasingly utilized to resolve species boundaries and phylogenetic relationships within major angiosperm clades. Debregeasia (Urticaceae), an economically important genus, presents challenges in species delimitation due to its overlapping morphological traits and unstable taxonomic assignments. Here, we analyzed 14 morphological traits and generated 12 data matrices from the plastomes and nrDNA using genome skimming from the nine recognized morphospecies to clarify species boundaries and assess barcode performance in Debregeasia. We also used a universal set of 353 nuclear genes to explore reticulate evolution and biogeographic history of Debregeasia. Plastomes of Debregeasia exhibited the typical quadripartite structure with conserved gene content and marginal independent variations in the SC/IR boundary at inter- and intra-specific levels. Three Debregeasia species were non-monophyletic and could not be discerned by any barcode; however, ultra-barcodes identified the remaining six (67%), outperforming standard barcodes (56%). Our phylogenetic analyses placed Debregeasia wallichiana outside the genus and suggested six monophyletic clades in Debregeasia, although the placement between Debregeasia hekouensis and Debregeasia libera varied. There was extensive trait overlap in key morphologically diagnostic characters, with reticulation analysis showing potentially pervasive hybridization, likely influenced by speciation patterns and overlaps between species ranges. We inferred that Debregeasia crown diversification began at ca. 12.82 Ma (95% HPD: 11.54-14.63 Ma) in the mid-Miocene within Australia, followed by vicariance and later long-distance dispersal, mainly out of southern China. Our findings highlight the utility of genomic data with integrative lines of evidence to refine species delimitation and explore evolutionary relationships in complex plant lineages.