show Abstracthide AbstractBarleria is a genus of approximately 300 species of herbs and shrubs with a wide distribution across the paleotropics; the genus is especially diverse in Angola, Tanzania, and Madagascar. Among Acanthaceae, Barleria can be recognized by a combination of characters: four sepals composed of a small inner pair and larger outer pair; double cystoliths in epidermal cells; an abaxial pair of stamens with filaments that twist through 180 degrees and cross just distal to the synstapetal zone; and globose, honeycombed pollen. Historically, natural groupings in the genus have proven elusive due to rich morphological variation among species of Barleria, including of traits that are homogeneous at the genus level in other Acanthaceae. Previous morphological studies classified Barleria into two subgenera and seven sections. More recently, a molecular study that sampled 53 Barleria species for four chloroplast loci and the nuclear locus nrITS supported the subgenera and four of the seven recognized sections. The aim of our study was to estimate relationships in the genus through a phylogenetic analysis that samples 190 accessions representing 184 taxa, including varieties and subspecies of 167 species of Barleria (56% of total species diversity of ~300). This sample represents all sections and encompasses the geographic range of Barleria. We also included ten outgroups sampled from related genera (i.e., Andrographis, Crabbea, Lepidagathis, Justicia, Neuracanthus, Whitfieldia). Single nucleotide polymorphism data were generated using double-digest restriction-site associated DNA sequencing. The RADseq phylogeny corroborated the topology estimated from the chloroplast and nrITS data, but with greatly increased resolution and support of fine-scale relationships. Importantly, the RADseq phylogeny found six major lineages in subg. Barleria and resolved a polytomy that included the type species of the genus, B. cristata. The topology suggests at least four independent dispersal events to Madagascar with subsequent radiation. Our results are a step towards a stable subgeneric classification and broadly inform our understanding of diversity and evolution in one of the largest genera of Acanthaceae.