Taxonomic analysis of certain taxa of Epipactis in Slovenia

The predominantly allogamous Broad-leaved Helleborines (Epipactis helleborine) and the predominantly autogamous Narrow-lipped Helleborines (Epipactis leptochila) represent two taxonomically complex groups with great morphological diversity and close genetic relationships at the subspecies level. They are influenced by environment, clinal variability, hybridization between allogamous taxa, and in particularly by speciation through transitions from allogamy to autogamy. We analysed floral traits, genome size and performed molecular analyses (Internal transcribed spacer region, ITS; Amplified Fragment Length Polymorphism, AFLP) of several individuals of E. helleborine and E. leptochila aggregate from different geographical regions in Slovenia, focusing on intermediate traits of E. helleborine subsp. moratoria, E. helleborine subsp. leutei and E. leptochila subsp. neglecta. In order to successfully distinguish studied taxa, the main attention should be paid to the shape of the epichil and the ovary, as well as to the size ratio of sepals and petals. Among the microfloral characters, the shape and size of the lower margin of the stigma and staminodes are found to be supportive. E. neglecta clearly shows intermediate floral characteristics between E. leptochila and E. leutei, suggesting that the transition to autogamy evolved E. neglecta as an intermediate stage in the speciation process, coinciding with the colonisation of forest sites with lower light intensity. Analysis of genome size revealed significant differences, with E. neglecta having the greatest value. Using molecular markers, we were able to determine two main ribotype clusters within the E. helleborine group, which were also determined by AFLP analyses. E. leutei and E. neglecta formed two clearly defined subclusters within the E. leptochila lineage. The results of the presented study suggest that combining molecular markers and genome size with information on how variation in floral morphology is distributed, using multivariate analyses, is the most reliable way to gain improved insight into the relationships among the taxa analysed.