Comparative analysis of chloroplast genomes of Pulsatilla species reveals evolutionary and taxonomic status of newly discovered endangered species Pulsatilla saxatilis.

BACKGROUND: Pulsatilla saxatilis, a new species of the genus Pulsatilla has been discovered. The morphological information of this species has been well described, but its chloroplast genome characteristics and comparison with species of the same genus remain to be reported. RESULTS: Our results showed that the total length of chloroplast (cp.) genome of P. saxatilis is 162,659 bp, with a GC content of 37.5%. The cp. genome contains 134 genes, including 90 known protein-coding genes, 36 tRNA genes, and 8 rRNA genes. P. saxatilis demonstrated similar characteristics to other species of genus Pulsatilla. Herein, we compared cp. genomes of 10 species, including P. saxatilis, and found that the cp. genomes of the genus Pulsatilla are extremely similar, with a length of 162,322-163,851 bp. Furthermore, The SSRs of Pulsatilla ranged from 10 to 22 bp in length. Among the four structural regions of the cp. genome, most long repeats and SSRs were detected in the LSC region, followed by that in the SSC region, and least in IRA/ IRB regions. The most common types of long repeats were forward and palindromic repeats, followed by reverse repeats, and only a few complementary repeats were found in 10 cp. genomes. We also analyzed nucleotide diversity and identified ccsA_ndhD, rps16_trnK-UUU, ccsA, and rbcL, which could be used as potential molecular markers for identification of Pulsatilla species. The results of the phylogenetic tree constructed by connecting the sequences of high variation regions were consistent with those of the cp. gene phylogenetic tree, and the species more closely related to P. saxatilis was identified as the P. campanella. CONCLUSION: It was determined that the closest species to P. saxatilis is P. campanella, which is the same as the conclusion based on pollen grain characteristics, but different from the P. chinensis determined based on morphological characteristics. By revealing information on the chloroplast characteristics, development, and evolution of the cp. genome and the potential molecular markers, this study provides effective molecular data regarding the evolution, genetic diversity, and species identification of the genus Pulsatilla.

Breaking the limits - multichromosomal structure of an early eudicot Pulsatilla patens mitogenome reveals extensive RNA-editing, longest repeats and chloroplast derived regions among sequenced land plant mitogenomes.

BACKGROUND: The mitogenomes of vascular plants are one of the most structurally diverse molecules. In the present study we characterize mitogenomes of a rare and endangered species Pulsatilla patens. We investigated the gene content and its RNA editing potential, repeats distribution and plastid derived sequences. RESULTS: The mitogenome structure of early divergent eudicot, endangered Pulsatilla patens does not support the master chromosome hypothesis, revealing the presence of three linear chromosomes of total length 986 613 bp. The molecules are shaped by the presence of extremely long, exceeding 87 kbp repeats and multiple chloroplast-derived regions including nearly complete inverted repeat. Since the plastid IR content of Ranunculales is very characteristic, the incorporation into mitogenome could be explained rather by intracellular transfer than mitochondrial HGT. The mitogenome contains almost a complete set of genes known from other vascular plants with exception of rps10 and sdh3, the latter being present but pseudogenized. Analysis of long ORFs enabled the identification of genes which are rarely present in plant mitogenomes, including RNA and DNA polymerases, albeit their presence even at species level is variable. Mitochondrial transcripts of P. patens were edited with a high frequency, which exceeded the level known in other analyzed angiosperms, despite the strict qualification criteria of counting the editing events and taking into analysis generally less frequently edited leaf transcriptome. The total number of edited sites was 902 and nad4 was identified as the most heavily edited gene with 65 C to U changes. Non-canonical, reverse U to C editing was not detected. Comparative analysis of mitochondrial genes of three Pulsatilla species revealed a level of variation comparable to chloroplast CDS dataset and much higher infrageneric differentiation than in other known angiosperm genera. The variation found in CDS of mitochondrial genes is comparable to values found among Pulsatilla plastomes. Despite the complicated mitogenome structure, 14 single copy regions of 329 kbp, not splitted by repeats or plastid-derived sequences (MTPT), revealed the potential for phylogenetic, phylogeographic and population genetics studies by revealing intra- and interspecific collinearity. CONCLUSIONS: This study provides valuable new information about mitochondrial genome of early divergent eudicots, Pulsatilla patens, revealed multi-chromosomal structure and shed new light on mitogenomics of early eudicots.

Evolutionary history of the Pasque-flowers (Pulsatilla, Ranunculaceae): Molecular phylogenetics, systematics and rDNA evolution.

Pulsatilla (Anemoneae, Ranunculaceae) is sister to Anemone s.s. and contains ca 40 perennial species of considerable horticultural and medical importance. We sequenced 31 of those species, plus nine subspecies, two cultivars and six outgroups, for two nuclear regions (high-copy nrITS and low-copy MLH1) and three plastid regions (rbcL, accD-psaI, trnL intron) in order to generate the first comprehensive species-level phylogeny of the genus. Phylogenetic trees were constructed using both concatenation-based (maximum likelihood and Bayesian inference) and coalescence methods. The better supported among the internal nodes were subjected to molecular clock dating and ancestral area reconstruction, and karyotypic characters identified by us using Fluorescence In Situ Hybridization were mapped across the tree. The preferred species tree from the coalescence analysis formed the basis of a new infrageneric classification based on monophyly plus degree of divergence. The earliest divergent of the three subgenera, Kostyczewianae, is represented by only a single species that is morphologically intermediate between Anemone s.s. and 'core' Pulsatilla. Subgenus Pulsatilla is considerably richer in species than its sister subgenus Preonanthus and contains three monophyletic sections. Species possessing nodding flowers and pectinately dissected leaves are phylogenetically derived compared with groups possessing erect flowers and palmately lobed leaves. Pulsatilla separated from Anemone s.s. at ca 25 Ma. Our results indicate a central Asian mountain origin of the genus and an initial diversification correlated with late Tertiary global cooling plus regional mountain uplift, aridification and consequent expansion of grasslands. The more rapid and extensive diversification within subgenus Pulsatilla began at ca 3 Ma and continued throughout the Quaternary, driven not only by major perturbations in global climate but also by well-documented polyploidy.

Conservation of the Threatened Species, Pulsatilla vulgaris Mill. (Pasqueflower), is Aided by Reproductive System and Polyploidy.

Population loss due to habitat disturbance is a major concern in biodiversity conservation. Here we investigate the genetic causes of the demographic decline observed in English populations of Pulsatilla vulgaris and the consequences for conservation. Using 10 nuclear microsatellite markers, we compare genetic variation in wild populations with restored and seed-regenerated populations (674 samples). Emergence of genetic structure and loss of allelic variation in natural populations are not as evident as expected from demographic trends. Restored populations show genetic variation comparable to their source populations and, in general, to the wild ones. Genetic homogeneity is observed in regeneration trials, although some alleles not captured in source populations are detected. We infer that polyploidy, longevity, and clonal reproduction have provided P. vulgaris with the standing genetic variation necessary to make the species resilient to the effects of demographic decline, suggesting that the use of multiple sources for reintroduction may be beneficial to mimic natural gene flow and the availability of multiple allele copies typical of polyploid species.

Genomic Resources of Three Pulsatilla Species Reveal Evolutionary Hotspots, Species-Specific Sites and Variable Plastid Structure in the Family Ranunculaceae.

BACKGROUND: The European continent is presently colonized by nine species of the genus Pulsatilla, five of which are encountered only in mountainous regions of southwest and south-central Europe. The remaining four species inhabit lowlands in the north-central and eastern parts of the continent. Most plants of the genus Pulsatilla are rare and endangered, which is why most research efforts focused on their biology, ecology and hybridization. The objective of this study was to develop genomic resources, including complete plastid genomes and nuclear rRNA clusters, for three sympatric Pulsatilla species that are most commonly found in Central Europe. The results will supply valuable information about genetic variation, which can be used in the process of designing primers for population studies and conservation genetics research. The complete plastid genomes together with the nuclear rRNA cluster can serve as a useful tool in hybridization studies. METHODOLOGY/PRINCIPAL FINDINGS: Six complete plastid genomes and nuclear rRNA clusters were sequenced from three species of Pulsatilla using the Illumina sequencing technology. Four junctions between single copy regions and inverted repeats and junctions between the identified locally-collinear blocks (LCB) were confirmed by Sanger sequencing. Pulsatilla genomes of 120 unique genes had a total length of approximately 161-162 kb, and 21 were duplicated in the inverted repeats (IR) region. Comparative plastid genomes of newly-sequenced Pulsatilla and the previously-identified plastomes of Aconitum and Ranunculus species belonging to the family Ranunculaceae revealed several variations in the structure of the genome, but the gene content remained constant. The nuclear rRNA cluster (18S-ITS1-5.8S-ITS2-26S) of studied Pulsatilla species is 5795 bp long. Among five analyzed regions of the rRNA cluster, only Internal Transcribed Spacer 2 (ITS2) enabled the molecular delimitation of closely-related Pulsatilla patens and Pulsatilla vernalis. CONCLUSIONS/SIGNIFICANCE: The determination of complete plastid genome and nuclear rRNA cluster sequences in three species of the genus Pulsatilla is an important contribution to our knowledge of the evolution and phylogeography of those endangered taxa. The resulting data can be used to identify regions that are particularly useful for barcoding, phylogenetic and phylogeographic studies. The investigated taxa can be identified at each stage of development based on their species-specific SNPs. The nuclear and plastid genomic resources enable advanced studies on hybridization, including identification of parent species, including their roles in that process. The identified nonsynonymous mutations could play an important role in adaptations to changing environments. The results of the study will also provide valuable information about the evolution of the plastome structure in the family Ranunculaceae.

Are the Organellar Genomes Useful for Fine Scale Population Structure Analysis of Endangered Plants?-A Case Study of Pulsatilla patens (L.) Mill.

Pulsatilla patens is a rare and endangered species in Europe and its population resources have significantly decreased over the past decades. Previous genetic studies of this species made it possible to estimate the genetic diversity of the European population and to describe the structure of chloroplast and mitochondrial genomes. The main aim of these studies was to characterize the variability of chloroplast and mitochondrial genomes in more detail at the intra-population and inter-population levels. Our study presents new organelle genome reference sequences that allow the design of novel markers that can be the starting point for testing hypotheses, past and modern biogeography of rare and endangered species P. patens, and adaptive responses of this species to changing environments. The study included sixteen individuals from five populations located in Northeastern Poland. Comparative analysis of 16 P. patens plastomes from 5 populations enabled us to identify 160 point mutations, including 64 substitutions and 96 InDels. The most numerous detected SNPs and Indels (75%) were accumulated in three intergenic spacers: ndhD-ccsA, rps4-rps16, and trnL(UAG)-ndhF. The mitogenome dataset, which was more than twice as large as the plastome (331 kbp vs. 151 kbp), revealed eight times fewer SNPs (8 vs. 64) and six times fewer InDels (16 vs. 96). Both chloroplast and mitochondrial genome identified the same number of haplotypes-11 out of 16 individuals, but both organellar genomes slightly differ in haplotype clustering. Despite the much lower variation, mitogenomic data provide additional resolution in the haplotype detection of P. patens, enabling molecular identification of individuals, which were unrecognizable based on the plastome dataset.

Re-introduction of an extinct population of Pulsatilla patens using different propagation techniques.

The study focuses on the propagation of a rare and endangered plant species (Pulsatilla patens) to re-introduce an extinct population from calamine area in Southern Poland. The plants were propagated from seeds, rhizome cuttings, or regenerated in vitro from shoot tips, hypocotyls with roots or cotyledons of seedlings on Murashige & Skoog (MS) medium supplemented with 0.25 or 0.50 mg L-1 BAP (Benzylaminopurine) via direct and indirect organogenesis or somatic embryogenesis (SE). The most efficient micropropagation method was with shoot tips as an explant on MS + 0.25 mg L-1 BAP where 97% of the explants produced multiple shoots, mass SE was observed after transfer on ½ MS with 2% saccharose; 267 (35%) shoots rooted on ½ MS + 2% saccharose were acclimatized to ex vitro conditions. Flow cytometry revealed genome size stability of propagated plantlets. Low genetic differentiation between micropropagated plantlets and initial material was indicated by ISSR (Inter Simple Sequence Repeat) markers. Totally, 132 vigorous plantlets obtained on various pathways were introduced to the field plots in 2020; 30.33% survived the winter, and several reached the generative stage and flowered in the spring 2021. In next season (March/April 2022) the number of introduced plants decreased to 25% while the number of flowering and fruiting shoots in different clumps increased in some plots. This is the first report of successful re-introduction of the endangered P. patens based on micropropagation, rhizome cuttings, and seed germination.

Chloroplast genomes elucidate diversity, phylogeny, and taxonomy of Pulsatilla (Ranunculaceae).

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.

Rapidly discriminate commercial medicinal Pulsatilla chinensis (Bge.) Regel from its adulterants using ITS2 barcoding and specific PCR-RFLP assay.

Pulsatillae radix is a conventional traditional Chinese medicine (TCM) with common name Baitouweng, and has notable effects on inflammation and dysentery. Pulsatilla chinensis (Bge.) Regel is the only source plant of Baitouweng recorded in Chinese Pharmacopoeia, but its adulteration often occurs in the market that possibly affects medicinal efficacy and safety. We have established an internal transcribed spacer 2 (ITS2) barcode library based on 105 plant samples from 12 Pulsatilla species and 10 common adulterants. Results indicate that ITS2 barcoding can accurately distinguish Pulsatilla species from their adulterants. Pulsatilla chinensis can be discriminated from 11 congeneric species by two stable single nucleotide polymorphisms (SNPs) in the ITS2 region. Additionally, a quick specific PCR-RFLP identification assay based on the ITS2 barcode was developed. Using specific primers ITS2/PR1 combined with restriction enzyme Bgl I, Pu. chinensis can rapidly be differentiated from other species via simple and low-cost test procedures. Furthermore, 30 commercial Baitouweng products were tested and only two products were derived from authentic Pu. chinensis. Thus, these two molecular approaches provide practical tools for quick identification of commercial Baitouweng products and can help ensure the safe use of this TCM product.

Genetic Diversity and Population Structure of the Rare and Endangered Plant Species Pulsatilla patens (L.) Mill in East Central Europe.

Pulsatilla patens s.s. is a one of the most endangered plant species in Europe. The present range of this species in Europe is highly fragmented and the size of the populations has been dramatically reduced in the past 50 years. The rapid disappearance of P. patens localities in Europe has prompted the European Commission to initiate active protection of this critically endangered species. The aim of this study was to estimate the degree and distribution of genetic diversity within European populations of this endangered species. We screened 29 populations of P. patens using a set of six microsatellite primers. The results of our study indicate that the analyzed populations are characterized by low levels of genetic diversity (Ho = 0.005) and very high levels of inbreeding (FIS = 0.90). These results suggest that genetic erosion could be partially responsible for the lower fitness in smaller populations of this species. Private allelic richness was very low, being as low as 0.00 for most populations. Average genetic diversity over loci and mean number of alleles in P. patens populations were significantly correlated with population size, suggesting severe genetic drift. The results of AMOVA point to higher levels of variation within populations than between populations.The results of Structure and PCoA analyses suggest that the genetic structure of the studied P. patens populations fall into three clusters corresponding to geographical regions. The most isolated populations (mostly from Romania) formed a separate group with a homogeneous gene pool located at the southern, steppic part of the distribution range. Baltic, mostly Polish, populations fall into two genetic groups which were not fully compatible with their geographic distribution.Our results indicate the serious genetic depauperation of P. patens in the western part of its range, even hinting at an ongoing extinction vortex. Therefore, special conservation attention is required to maintain the populations of this highly endangered species of European Community interest.

The use of AFLP markers in conservation genetics--a case study on Pulsatilla vernalis in the Polish lowlands.

Pulsatilla vernalis is a rare species in the Polish lowlands, strongly threatened by anthropogenic disturbance of its habitats. A grave decrease in its populations has been observed during the past 60-80 years (analogous populations in Eastern Austria and the Czech Republic are almost or completely extinct). An analysis of the genetic diversity of populations in the Polish lowlands was performed to estimate its level and distribution. The AFLP method was used for the study of seven populations. An analysis using five pairs of selective primers revealed 446 scorable fragments; 62.1% of them were polymorphic. The average gene diversity indices was 0.17 (the mean value for all the populations), ranging from 0.139 to 0.204. A weak relationship between diversity and population size was revealed. Most of the genetic diversity was contributed to by the within-population level (AMOVA) and only a weak geographical structure was shown by UPGMA clustering. Four populations formed population-specific clusters while three others (from one region) were intermixed. These preliminary results show a moderate genetic diversity of the studied populations, which was still rather high when compared with their size. This result, together with the low between-population differentiation in the region, suggests that these populations are the remnants of larger populations that, only a few decades ago, were much less isolated.