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

Alex Baumel, Virgile Noble, Cyllène Chatellier, Juan Viruel, Stephen Mifsud, Henri Michaud, Alain Delage, Lisandru Leandri, Andrea Lallai, Gianluca Iiriti, Gianniantonio Domina, Gabriele Casazza, Pere Fraga-Arguimbau, Baptiste Pierre, Anouar Toumi, Frédéric Médail

Taxonomic assignments based only on morphology are often insufficient for delimiting species, particularly in polyploid complexes with extensive morphological overlap. This limitation hinders biogeographic and conservation issues. The genus Romulea (Iridaceae), distributed across Africa and the Mediterranean Basin, exemplifies this challenge. Despite its remarkable diversity, Mediterranean Romulea has remained largely understudied at the molecular level. Here, we present the first multilocus genotype analysis of several taxa of the Mediterranean region, with a sampling focused on the Tyrrhenian biogeographic province. Using the hybseq method with the universal Angiosperms353 target capture kit, we sequence nuclear (on target) and plastid loci (off target) for 272 individuals representing 18 putative taxa. Nuclear SNP genotyping yield 8,168 high-confidence loci, revealing 19 genetic groups. Hind/HE analyses indicate a predominance of allopolyploid inheritance. Analyses of genetic diversity reveal incomplete differentiation, introgression, or plastid capture, which complicate species delimitation. Several taxa, including R. x melitensis, R. corsica, and R. bulbocodium, show genomic signatures consistent with hybrid origins. 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. The widespread species R. ramiflora and R. columnae contain well-differentiated groups that are either isolated or close to other taxa, calling for further analysis to differentiate cryptic speciation from historical introgression. Our findings question the current taxonomic status of several narrow endemic species. Based on robust genetic boundaries, we propose various solutions for further taxonomic studies. The plastid phylogeny reveals a star-like diversification pattern, consistent with rapid radiation in the Tyrrhenian region. These results provide a primary genomic framework for integrative taxonomy in Romulea, highlighting the central role of polyploidy and reticulate evolution in shaping Mediterranean diversity and offering key insights for biogeography, systematics and conservation.