HPLC analysis of leaf surface flavonoids for the preliminary classification of birch species

Flavonoid aglycones found on the surfaces of birch (Betula spp.) leaves may constitute up to 10% of the dry weight of the leaf. A facile extraction and HPLC procedure has been developed that can be used for the preliminary classification of birch species according to the patterns of their leaf surface flavonoids. The procedure involves no complex sample preparation steps, and is able to provide HPLC chromatograms from fresh leaves in less than 30 min. If necessary, leaves do not even need to be removed from the tree. Since the genus Betula is taxonomically complex and separation of different birch species can be problematic, the developed method was applied to 15 Betula species and four sub-species of Betula pendula Seven of the studied species were classified as B. pubescens and eight as B. pendula-type birches. The remaining four species did not belong to either of these two classes on account of their unique pattern of external flavonoids. The difference between the leaf surface flavonoid composition of B. pubescens and B. pendula type birch species was unambiguously clear, and the developed method could reliably distinguish between the two species. Whilst leaf surface flavonoids can be valuable chemotaxonomic markers, they classify birch species differently from morphological markers. Birch species with diploid chromosome sets did not contain any of the flavanones that were present in the leaves of other species. The close relationship between the occurrence of some flavonoid aglycones and the ploidy level of Betula species suggests that these chemotaxonomic markers may be useful both in taxonomic and phylogenetic analyses.