Molecular identification and diversity assessment of Tyrrhenian Romulea species (Iridaceae)

Taxonomic assignments based only on morphology are often insufficient for delimiting species, particularly in complexes shaped by hybridization and polyploidy, where species boundaries are unclear. This limitation hinders progress in ecological, biogeographic and conservation research. The genus Romulea, distributed across Africa and the Mediterranean Basin, exemplifies this challenge. Despite its remarkable diversity, Mediterranean Romulea has not received much attention from genetic and molecular studies. Here, we present the first multilocus genotype analysis of Mediterranean Romulea taxa, focusing on the Tyrrhenian biogeographic province. Using target-capture sequencing with the universal Angiosperms353 kit, we generated genomic data for 272 individuals representing 18 putative taxa. Our findings reveal genetic groups that align with current taxonomy, the existence of cryptic divergence, and highlight the role of hybridization. Furthermore, analysis of intra-individual genetic diversity suggests one or several allopolyploid origins for Mediterranean Romulea. Four taxa (R. assumptionis, R. revelieri, R. ligustica, R. rollii) are consistently well differentiated across nuclear and plastid datasets, supporting their recognition as distinct species. In contrast, the widespread species R. ramiflora and R. columnae contain well-differentiated groups that may represent cryptic speciation. Several other taxa, including R. x melitensis, R. corsica, and R. bulbocodium, exhibit genomic signatures consistent with hybrid origins. Plastid and nuclear variation patterns are consistent with a hypothesis of rapid radiation in the Tyrrhenian region. These results provide a primary genomic framework for the integrative taxonomy of Romulea.