Low resolution phylogenetic trees made using the chloroplast regions mentioned above have been reported for other taxa, including Curcuma and Sisyrinchium . The inadequate resolution may be due to the lower substitution rates and lack of variation found in single plastid regions. Thus, we do not recommend single plastid regions as DNA barcodes for this the genus. Among the candidate barcode genes, the Consortium for the Barcode of Life Plant Working Group suggested that rbcL, matK, and the rbcL+matK combination should be sufficient for a plant barcode, and that this combination should be supplemented with additional markers as required . In addition, Kress et al. and Chase et al. proposed that trnH-psbA can be used in two-locus or three-locus barcode systems to improve resolution. For instance, two of the three combinations of the three chloroplast loci tested in this study, rbcL+trnH-psbA and rbcL+matK+trnH-psbA exhibited higher discriminatory performance than any single marker. Consequently, this highlights the need to use chloroplast multi-locus barcodes to improve the resolution of species identification in Pulsatilla. The nuclear ITS region provided the highest inter-and intraspecific divergences and had a higher success rate for the correct identification of species in TAXONDNA . However, as for the treebuilding method,large pot with drainge the discriminatory performance of ITS is not satisfactory, as its highest resolution is 39.02% .
As evidenced by previous studies, the multi-locus barcode is one of the combinations that demonstrated the highest species resolution rate, e.g., Aceraceae , Lysimachia , Oberonia , Rhodiola and Schisandraceae . However, in this study, addition of ITS to different kinds of combinations of chloroplast markers did not increase the resolution rate obviously . The resolution rate based on tree-building analyses was 51.22% for BI and 58.54% for PWG. In addition, we found no distinct barcoding gap. This phenomenon may be due to the one or more of several reasons. first, incomplete lineage sorting and non-homogeneous concerted evolution are likely to occur at the ITS locus . Second, the three chloroplast regions cannot compensate for the drawbacks of ITS because they are sourced from a different genome. Although the nuclear genome is inherited biparentally, the chloroplast genome is inherited uniparentally. Thus, the chloroplast genome experiences more complete lineage sorting than the ITS locus does. Third, hybridizations may cause conflicts between ITS and chloroplast loci, as well as problematic results in ITS phylogeny due to the possibility of homogenization to paternal copies in some lineages and maternal copies in others. A combination of DNA markers from different genomes— which have different modes of inheritance and conflicting phylogenies—can hinder our understanding of species delimitation and the evolutionary processes of speciation. Because of its myriad variable sites that can reliably distinguish species, resulting from a high mutation rate and rapid concerted evolution, we recommend ITS as a good single barcode for the genus Pulsatilla.Phylogenetic identification and species recognition are foundationally important for biology . The results of the phylogenetic analyses performed in this study may shed some light on the identification and taxonomy of the genus Pulsatilla . Here, we found that Pulsatilla formed a monophyletic group with high support. Moreover, the three recognized subgenera — i.e. subg. Pulsatilla, subg. Kostyczewianae, and subg.
Preonanthus— were resolved as distinct monophyletic groups, which is consistent with the recent phylogenetic result . Within subgenus Pulsatilla, our analyses found that P. camanella and P. ambigua were resolved as sister to one another with high support. These two species share many common morphological characters, such as almost fully expanded leaves at anthesis,black plastic planting pots and dense, long trichomes. The flowers of both species nod before anthesis . However, during anthesis, the sepals of P. camanella can easily be distinguished from those of P. ambigua by color . At the same time, the micro-morphological characters of the leaves are also different . Actinocytic and anomocytic stomata exist in both species, but most stomata in P. camanella are actinocytic, whereas most are anomocytic in P. ambigua. Thus, molecular data as well as micromorphological characters can distinguish between these two species relatively well. Both types of evidence may be helpful to accurately identify specimens that are damaged or lack sufficient diagnostic characters. In addition to its use in identifying specimens, DNA barcoding is also useful for resolving taxonomic uncertainty .They did not have distinct barcodes. The micro-morphological characters were also found to be the same, since both plants showed polygonal epidermal cells with striation, a dense distribution of stomata, and glabrous or sparsely short trichomes. In addition, the geographical distribution of these two species overlaps in Inner Mongolia. Taken together, these distinct lines of evidence collectively suggest that P. turczaninovii and P. tenuiloba are the same species. The discovery of hybridization, introgression, and/or incomplete lineage sorting among species is another useful application of DNA barcoding . The chloroplast region is inherited maternally, but the nuclear genome, including the ITS region, is inherited biparentally . Thus, if there are different results in different phylogenetic analyses from chloroplast and nuclear data, we speculate that these differences may be caused by hybridization and/or introgression among species, which could result in a non-monophyletic clade. In subg. Pulsatilla, we found several complex groups. The samples of P. chinensis and P. cernua in cluster III, were indistinguishable. In the Bayesian inference tree based on ITS sequences , the samples of P. cernua clustered in a clade along with sample P. chinensis. However, in the Bayesian inference tree based on the combination of chloroplast sequences , sample P. chinensis clustered in a clade with all other samples of P. chinensis. P. chinensisis a widespread species and has a geographical range that covers that of P. cernua; in addition, sample P. chinensis was collected near populations of P. cernua in Jilin Province, China. Hybridization or introgression might have occurred during the speciation of P. chinensisis and P. cernua. A similar situation was also found for sample P. ambigua108 and cluster I , suggesting hybridization may have occurred between P. ambigua and P. tenuiloba/P. turczaninovii.