Phylogeny and species delimitation in the Medicago rigidula (L.) All. complex based on nrDNA sequence data

Annual species of the genus Medicago L. (Fabaceae) are ecologically significant components of Mediterranean vegetation. The Medicago rigidula complex, comprising M. rigidula, M. rigiduloides, M. sinskiae, and M. constricta, has undergone substantial taxonomic revision recently, but species boundaries remain unclear due to overlapping morphological traits, recent divergence, and selfing reproductive strategies. To clarify species relationships within the complex, we conducted an integrative analysis of populations sampled from diverse regions of Iran. Floral morphology (۳۴ characters), anatomical features (۱۷ characters), palynology, and seed micromorphology were studied using light and SEM microscopy. Morphological and anatomical differences, particularly in floral characters, pollen aperture types, seed surface vesiculation, and pod vascular bundle shape, helped discriminate among taxa. Phylogenetic analyses based on combined ITS and ETS nuclear ribosomal DNA sequences supported the distinctiveness of M. constricta and M. sinskiae, while also validating the separation of M. rigidula and M. rigiduloides despite partial morphological overlap. A new ETS primer pair, designed specifically for the core Medicago group, improved amplification success and resolution. Cluster and PCA analyses of floral characters further supported species-level distinctions. Although clinal variation was observed in pod morphology, integration of multiple data sources enabled more robust species delimitation. Our findings underscore the importance of combining multilocus molecular data with detailed morphological and anatomical traits to resolve taxonomic complexity in recently diverged, selfing taxa. An updated identification key for the M. rigidula complex is provided, contributing to the systematics within the group.

کلیدواژه ها:

Annual medics ، Iran ، Mediterranean region ، primer design ، Speciation ، Taxonomy