Abstract
Pulsatilla (Ranunculaceae) comprises about 40 species, many of which have horticultural and/or medicinal importance. However, the recognition and identification of wild Pulsatilla species is difficult due to the presence of complex morphological characters. DNA barcoding is a powerful molecular tool capable of rapidly and accurately distinguishing between species. Here, we assessed the effectiveness of four commonly used DNA barcoding loci—rbcL (R), trnH-psbA ( T ), matK (M), and ITS (I)—to identify species of Pulsatilla from a comprehensive sampling group. Among the four barcoding single loci, the nuclear ITS marker showed the highest interspecific distances and the highest rate of correct identification. Among the eleven combinations, the chloroplast multi-locus R+T and R+M+T combinations were found to have the best species discrimination rate, followed by R+M. Overall, we propose that the R+M+T combination and the ITS marker on its own are, respectively, the best multi- and single-locus barcodes for discriminating among species of Pulsatilla. The phylogenetic analysis was able to distinguish species of Pulsatilla to the subgenus level, but the analysis also showed relatively low species resolution. This may be caused by incomplete lineage sorting and/or hybridization events in the evolutionary history of the genus, or by the resolution limit of the candidate barcodes. We also investigated the leaf epidermis of eight representative species using scanning electronic microscopy. The resulting micro-morphological characters were valuable for identification of related species. Using additional genome fragments, or even whole chloroplast genomes combined with micro-morphological data may permit even higher resolution of species in Pulsatilla.
Highlights
The Ranunculaceae is a large and complex plant family, including approximately 59 genera and 2,500 species (Tamura, 1995)
Within subgenus Pulsatilla, our analyses found that P. camanella and P. ambigua were resolved as sister to one another with high support
We selected nuclear ITS and three chloroplast barcodes to evaluate their suitability for use in classifying a comprehensive array of Pulsatilla samples
Summary
The Ranunculaceae is a large and complex plant family, including approximately 59 genera and 2,500 species (Tamura, 1995). Plants of Pulsatilla species are often covered with long, soft hairs. Their flowers are solitary and bisexual, with three bracts forming a bell-shaped involucre. Miller (1754), Adanson (1763), and Wang et al (2001) have supported a model that separates Pulsatilla from Anemone as an independent genus. Pulsatilla can be distinguished from Anemone s.s., since species of the former have a long, plumose beak on the achenes formed by the persistent style and stamens (Tamura, 1995; Wang et al, 2001) whereas species of the latter do not. Because the primary goal of the present study is to test the use of DNA barcodes for species in the Pulsatilla clade, we here follow the treatment of Wang et al (2001) and Grey-Wilson (2014), regarding Pulsatilla as a distinct genus
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