Phylogeographic analysis of a temperate-deciduous forest restricted plant (Bupleurum longiradiatum Turcz.) reveals two refuge areas in China with subsequent refugial isolation promoting speciation.

This study investigates the influence of climate-induced oscillations and complicated geological conditions on the evolutionary processes responsible for species formation in presently fragmented temperate forest habitats, located in continental East Asia. In addition to this, we also investigate the heavily debated issue of whether temperate forests migrated southwards during such glacial periods or, alternatively, whether there existed refugia within north China, enabling localized survival of temperate forests within this region. In order to achieve these, we surveyed the phylogeography of Bupleurum longiradiatum Turcz. (a herbaceous plant solely confined to temperate forests) constructed from sequence variation in three chloroplast (cp) DNA fragments: trnL-trnF, psbA-trnH and rps16. Our analyses show high genetic diversity within species (h(T)=0.948) and pronounced genetic differentiation among groups (yellow and purple flowers) with a significant phylogeographical pattern (N(ST)>G(ST), P<0.05). Forty-three haplotypes were identified and clustered into two lineages (the purple-flowered lineage and the yellow-flowered lineage). Two corresponding refuge areas, one in Qinling and its adjacent regions and one in the Changbai Mountains/eastern China, were revealed across the entire distribution ranges of Bupleurum longiradiatum. These results provide evidence for the hypothesis that independent refugia were maintained across the range of temperate forests in northern China during the last glacial maximum or earlier cold periods. Bupleurum longiradiatum var. porphyranthum formed a single taxon based on molecular data. This specific formation process suggests that the historical vicariance factors, i.e. climate-induced eco-geographic isolation through the biotic displacement of temperate-deciduous forest habitats, enhanced the divergence of the yellow and purple flower lineages at different spatial-temporal scales and over glacial and interglacial periods. Additionally, geological conditions that restricted gene flow might also be responsible for the observed high genetic and geographic differentiation. A nested clade analysis (NCA) revealed that allopatric fragmentation was a major factor responsible for the phylogeographic pattern observed, and also supported a role for historical vicariance factors. Our results therefore support the inference that Quaternary refugial isolation promoted allopatric speciation of temperate plants in East Asia. This may help to explain the existence of high diversity and endemism of plant species in East Asia.