Merging GBS datasets to analyze the phylogeny of western Eurasian lime trees (Tilia) and place the Hyrcanian Forest taxa

Background Morphological uniformity in Tilia (Malvaceae s.l., Tilioideae) and unclear species delimitations resulted in over 500 names associated with the about 30 species of this genus. This is also an issue for the Hyrcanian Forest diversity hotspot of northern Iran, where between one and seven Tilia species were described to occur. We used genotyping-by-sequencing (GBS) to analyze the phylogeny of western Eurasian Tilia species, place the Hyrcanian Tilia populations, and infer their phylogeography. To arrive at a complete taxon sample, we merged our own data (104 samples) with a Tilia GBS dataset available in GenBank (11 samples) and processed and analyzed them together. Results We confirmed GBS data to be additive, i.e. independent datasets can be merged. We found three major groups within the Eurasian Tilia species and our analyses were able to resolve the phylogenetic relationships of the species. Neither T. cordata nor T. platyphyllos , both diploids and previously reported for Iran, are present in the region. Instead, the Iranian Tilia samples were identified as tetraploid by flow cytometry. Phylogenetic analyses clearly separated all species and found the Iranian samples to belong to T. dasystyla . The Hyrcanian populations we analyzed are genetically rather uniform but include a clade formed by individuals conforming mainly T. sabetii . Within the Hyrcanian populations we found a colonization pattern from the northwest to the eastern Hyrcanian forests. Conclusions Merging published GBS datasets from different labs will allow the study of geographically widespread taxa like Tilia , where representative species or populations might not all be accessible to individual researchers. Based on the samples we analyzed, the different taxa described for Iran belong genetically to T . dasystyla , with morphological variation that might be attributed to local environmental factors.