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.

Prunus is an economically important genus widely distributed in the temperate Northern Hemisphere. Previous studies on the genus using a variety of loci yielded conflicting phylogenetic hypotheses. Here, we generated nuclear reduced representation sequencing data and plastid genomes for 36 Prunus individuals and two outgroups. Both nuclear and plastome data recovered a well-resolved phylogeny. The species were divided into three main clades corresponding to their inflorescence types, - the racemose group, the solitary-flower group and the corymbose group - with the latter two sister to one another. Prunus was inferred to have diversified initially in the Late Cretaceous around 67.32 million years ago. The diversification of the three major clades began between the Paleocene and Miocene, suggesting that paleoclimatic events were an important driving force for Prunus diversification. Ancestral state reconstructions revealed that the most recent common ancestor of Prunus had racemose inflorescences, and the solitary-flower and corymb inflorescence types were derived by reduction of flower number and suppression of the rachis, respectively. We also tested the hybrid origin hypothesis of the racemose group proposed in previous studies. Prunus has undergone extensive hybridization events, although it is difficult to identify conclusively specific instances of hybridization when using SNP data, especially deep in the phylogeny. Our study provides well-resolved nuclear and plastid phylogenies of Prunus, reveals substantial cytonuclear discord at shallow scales, and sheds new light on inflorescence evolution in this economically important lineage.

Purpose

To evaluate the safety of oral fluorescein angiography (FA) and to compare its efficacy in detection of macular edema (ME) with spectral-domain optical coherence tomography (SD-OCT).

Methods

Results of imaging studies for 1,928 eyes of 1,019 patients who had simultaneously undergone both oral FA and SD-OCT by a confocal laser ophthalmoscope were reviewed. Sensitivity in detecting ME, discrepancy rate, and "kappa" agreement were determined for both the techniques and with eyes stratified by disease diagnosis.

Results

No allergic reactions occurred after oral FA. Mild gastric discomfort was noted in <1% of the patients; 1,840 eyes (95.4%) showed concordance between the two techniques, and kappa agreement was 90.3%. For ME, oral FA showed an overall sensitivity of 0.97 and SD-OCT of 0.91. Equivalent sensitivity was found in cases of wet age-related macular degeneration (0.99). Oral FA was more sensitive than SD-OCT in cases of retinovascular diseases. The SD-OCT showed higher sensitivity in cases of macular holes. Detection of ME by SD-OCT was significantly higher in cases of intense leakage on oral FA (P < 0.001).

Conclusion

Oral FA proved to be a safe and an adequate technique to evaluate ME. It is more sensitive than SD-OCT in detection of ME in cases of retinovascular diseases but can fail to detect ME in cases of macular holes. A noninvasive examination with simultaneous oral FA and SD-OCT may be considered to obtain a comprehensive evaluation of the presence of ME from different pathologies.

Pulsatilla (Ranunculaceae) consists of about 40 species, and many of them have horticultural and/or medicinal value. However, it is difficult to recognize and identify wild Pulsatilla species. Universal molecular markers have been used to identify these species, but insufficient phylogenetic signal was available. Here, we compared the complete chloroplast genomes of seven Pulsatilla species. The chloroplast genomes of Pulsatilla were very similar and their length ranges from 161,501 to 162,669 bp. Eight highly variable regions and potential sources of molecular markers such as simple sequence repeats, large repeat sequences, and single nucleotide polymorphisms were identified, which are valuable for studies of infra- and inter-specific genetic diversity. The SNP number differentiating any two Pulsatilla chloroplast genomes ranged from 112 to 1214, and provided sufficient data for species delimitation. Phylogenetic trees based on different data sets were consistent with one another, with the IR, SSC regions and the barcode combination rbcL + matK + trnH-psbA produced slightly different results. Phylogenetic relationships within Pulsatilla were certainly resolved using the complete cp genome sequences. Overall, this study provides plentiful chloroplast genomic resources, which will be helpful to identify members of this taxonomically challenging group in further investigation.