Comparative Leaf Anatomy of Balkan Representatives of Gentiana L. Sect. Ciminalis (Adans.) Dum. (Gentianaceae): Implications for Species Delimitation

The present study investigates the leaf anatomical traits of representatives of Gentiana section Ciminalis in the Balkan Peninsula, focusing on the ecologically and geographically vicariant species Gentiana acaulis, G. clusii, and G. dinarica. These species are distributed across a variety of mountainous habitats, including calcareous and siliceous rocky grounds, and exhibit pronounced morphological similarities that have led to misidentifications in the past. In order to address the challenges in species delimitation, a comparative analysis of leaf anatomical traits was performed on cross-sections of ten rosette leaves from each population. Statistical data analyses were conducted on 18 morphometric traits. A range of statistical techniques were used to assess variability and identify important discriminating traits, including descriptive statistics, principal component analysis, and discriminant analysis. The results indicate that the species can be distinguished based on leaf anatomy, particularly mesophyll thickness and number of cells that contain calcium oxalate crystals. The leaf of G. acaulis has a smaller mesophyll thickness (mean value: 164.31 μm), G. dinarica a larger mesophyll thickness (mean value: 365.85 μm), while G. clusii lies between these two (mean value: 305.35 μm). Crystal-containing cells are most abundant in G. clusii, where they are distributed throughout the entire leaf mesophyll; followed by G. dinarica, where the distribution of these cells are mainly in the upper half of the leaf; while they are sparse or absent in G. acaulis. These results suggest that leaf anatomy is a valuable diagnostic tool for distinguishing taxa within the section Ciminalis of the genus Gentiana.