Efficient Identification of Pulsatilla (Ranunculaceae) Using DNA Barcodes and Micro-Morphological Characters

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.

The molecular taxonomy of three endemic Central Asian species of Ranunculus(Ranunculaceae)

“…ITS are widely used for differentiating between the generic and infrageneric levels of plant phylogenetics [16] and are one of the most powerful barcode DNA markers available [17]. ITS markers have been confirmed to be useful for clarifying the phylogenetic relationships between closely related species and genera in Ranunculaceae [18][19][20][21]. However, a complete study of the taxonomy of the genus was based on using both DNA markers and morphological data, with the division of 226 taxa into two subgenera and 17 sections [14].…”

Phylogenetic relationships and status of taxa of Pulsatilla uralensis and P. patens s.str. (Ranunculaceae) in north-eastern European Russia

“…and P. uralensis belong to a single clade and some groups within it arose with moderate statistical support, which makes it impossible to draw more definitive conclusions. Such low resolution is generally associated with Pulsatilla species ( Li et al 2019 ) and with specific factors related to the evolution of the species (by hybridisation and polyploidisation). Therefore, our discussion will be carefully constructed, given the low resolution that we obtained.…”

“…Sequences of plastid DNA ( rbc L and mat K) and nuclear DNA (ITS2) have been successfully used for plant identification and DNA barcoding in various taxonomic groups ( Cai et al 2010 , Wattoo et al 2016 , Saddhe Kumar 2017), including the genus Pulsatilla ( Li et al 2019 ). ITS2 is considered to be the most effective barcode for the identification of more than half of Pulsatilla species ( Li et al 2019 ). Therefore, we tested the ability of DNA barcoding to distinguish between the taxa of P. patens s.lat.…”

“…In addition, there are several questions regarding the identification and size of taxa within the P. patens s.lat. complex, despite several recent studies on molecular phylogenetic relationships in the genus ( Li et al 2019 ; Sramko et al 2019 ).…”

“…(e.g. Hoot et al 1994 , 2012 ), comprises more than 30 taxa which, as a rule, form intricate species complexes with a high degree of morphological variability ( Stepanov 2014 ; Li et al 2019 ; Sramko et al 2019 ). The general classification, boundaries and the number of species and lower taxa have been revised several times, but no consensus has yet been reached (e.g.…”