Nucleotide sequences from the internal transcribed spacers (ITS) of nuclear ribosomal DNA and 5.8S gene were used to infer the phylogeny of Tilia species (represented by 13 distinct populations) growing in different geographical areas of Hyrcanian forests in northern Iran. Four well-supported lineages were revealed, including that of a new species, T. hyrcana, with stellate trichomes on both sides of the leaves and petiole. T. hyrcana is a well-supported cladospecies, with the ITS sequence and secondary structure following the diagnosable phylogenetic species concept, and is also characterized by a distinct morphology. A controversial species is Tilia rubra subsp. caucasica, with three different forms—an assemblage of taxa characterized by a lack of stellate trichomes on leaves—while Tilia begonifolia is distinguished by stellate trichomes on the underside of both leaves and petiole. The fourth lineage group, T. dastyla, is characterized by the presence of trichomes on the style. A single taxon found in the west of the Hyrcanian forest region is similar to T. begonifolia, but due to the former being located in a distinct group, a reassessment of the diagnostic morphology is recommended. ITS sequence data also suggested a closer relationship between T. rubra and T. begonifolia. Compensatory base change analysis was not strong enough to separate individual species within the Tilia genus. In general, the study supports the utility of ITS sequence data and secondary structure as accessory taxonomic characteristics with which to help clarify the systematics of the Tilia genus.
