Chloroplast Markers Research Articles

DNA barcoding of the genus Verbascum (Scrophulariaceae) in the Arabian Peninsula

AbstractVerbascum and Rhabdotosperma are members of the family Scrophulariaceae. The first genus comprises approximately 360 species from almost all parts of the world, while the second contains a total of 8 species from tropical Africa and the Arabian Peninsula. Since 1977, the relationships between Verbascum and Rhabdotosperma continue to be contested. The present study aims to present the phylogenetic relationships and status among Verbascum species in the Arabian Peninsula. For phylogenetic analyses, maximum parsimony and Bayesian inference were performed. In total, 236 DNA sequences from 59 specimens of Arabian Verbascum were analysed. The phylogenetic analysis of one nuclear (ITS) and three chloroplastic (rbcL, matK, trnL) markers confirmed the monophyly of Verbascum, including the genus Rhabdotosperma. In addition to presenting novel phylogenetic relationships among the different Verbascum species in the Arabian Peninsula, our study reduced the species count of Arabian Verbascum to 16. Moreover, the phylogenetic analysis strongly supports the reinstatement of the genus Rhabdotosperma into Verbascum based on the Bayesian and maximum parsimony analyses.

Population Structure and Genetic Diversity of the Toona ciliata (Meliaceae) Complex Assayed with Chloroplast DNA Markers.

Toona ciliata is a deciduous or semi-deciduous tree species and belongs to the Toona genus of the Meliaceae family. Owing to low natural regeneration and over-exploitation, the species is listed as an endangered species at level II in China and its conservation has received increasing concern. Here, we sampled 447 individuals from 29 populations across the range-wide distribution of the T. ciliata complex in China and assessed their genetic variation using two chloroplast DNA markers. The results showed that the overall haplotype diversity and nucleotide diversity per site were high at h = 0.9767 and π = 0.0303 for the psbA-trnH fragment and h= 0.8999 and π = 0.0189 for the trnL-trnL fragment. Phylogenetic analysis supported the division of the natural distribution of T. ciliata complex into western and eastern regions. The genetic diversity was higher in the western region than in the eastern region, showing significant phylogeographic structure. Genetic differentiation among populations was moderate (Φst=42.87%), and the effects of isolation by distance (IBD) were significant. A neutrality test and mismatch distribution analysis indicated that the distribution of the T. ciliata complex generally did not expand, although a few local populations could likely expand after bottleneck effects. The overall results were complementary to and consolidated previous studies using mitochondrial and nuclear DNA markers. We finally discussed strategies for the genetic conservation of the T. ciliata complex.

Complete chloroplast genomes of eight Artemisia species: Comparative analysis, molecular identification, and phylogenetic analysis.

Artemisia L. is the largest genus in the Asteraceae, and well known for its high medicinal value. The morphological features of Artemisia species are similar, making taxonomic identification and evolutionary research difficult. We sequenced chloroplast genomes of eight Artemisia species, all of which are common adulterants of A. argyi. We used novel genetic data and compared these data to the published A. argyi chloroplast genome in to develop molecular markers for species identification and reconstructing phylogenetic relationships between Artemisia species. The eight chloroplast sequences were highly similar in gene order, content, and structure, encoding a total of 114 genes (82 protein-coding genes, 28 tRNAs, and four rRNAs). All species harboured similar repeat sequences and simple sequence repeats (SSRs), ranging from 47 to 49 and 38 to 40 repeats, respectively. In addition, we identified five hypervariable regions (rpl32-trnL, rps16-trnQ, petN-psbM, trnE-rpoB, and atpA-trnR) and ten variable coding genes (ycf1, psbG, rpl36, psaC, psaI, accD, psbT, ndhD, ndhE, and psbH), which can be used to develop chloroplast molecular markers. Finally, phylogenetic reconstructions based on six datasets produced similar topologies, revealing A. argyi is closely related to species often found as adulterants, as expected. Our research provides valuable new information on the evolution and phylogenetic relationships between Artemisia chloroplast genomes and identifies valuable molecular makers to distinguish it from closely related species.

Hybrids as mirrors of the past: genomic footprints reveal spatio-temporal dynamics and extinction risk of alpine extremophytes in the mountains of Central Asia.

Hybridization is one of the key processes shaping lineage diversification, particularly in regions that experienced strong climate oscillations. The alpine biome with its rich history of glacial-interglacial cycles and complex patterns of species distribution shifts offers an excellent system to investigate the impact of gene flow on population dynamics and speciation, important issues for evolutionary biology and biodiversity conservation. In this study, we combined genomic data (DArTseq), chloroplast markers, and morphology to examine phylogenetic relationships and the permeability of species boundaries and their evolutionary outcomes among the alpine extremophilic species of Puccinellia (Poaceae) in the Pamir Mountains, a part of the Mountains of Central Asia biodiversity hotspot. We determined the occurrence of interspecific hybrids between P. himalaica and P. pamirica, which demonstrated almost symmetric ancestry from their parental species and did not show signals of introgression. According to our integrative revision, the natural hybrids between P. himalaica and P. pamirica should be classified as Puccinellia ×vachanica (pro species). Using approximate Bayesian computation for population history inference, we uncovered that P. himalaica hybridized with P. pamirica independently in multiple localities over the Holocene. Hybrids inherited the fine-scale genetic structure from their parental species, which developed these patterns earlier, during the Late Pleistocene. Hybridization had different consequences for the involved parental lineages, likely playing an important role in a continuing decline of P. himalaica in the Pamir Mountains over the Holocene. Our results show that P. himalaica should be considered a critically endangered species in the Pamir Mountains and could also be retreating across its entire range of distribution in High Mountain Asia. Using a comparative phylogeographic framework, we revealed the risk of extinction of a cold-adapted alpine species in a global biodiversity hotspot. This study highlights that genomics could unravel diversity trends under climate change and provides valuable evidence for conservation management.

Population Structure and Genetic Diversity Analyses Provide New Insight into the Endemic Species Aster spathulifolius Maxim. and Its Evolutionary History.

Aster spathulifolius, an ecologically significant plant species native to the coastal regions of Korea and Japan, remains understudied in terms of its genetic structure and evolutionary history. In this study, we employed four chloroplast markers and the nuclear ITS region from 15 populations of A. spathulifolius from both Korea and Japan, including their islands, to unravel the spatial genetic structure, differentiation, gene flow, phylogenetic, and biogeographical relationships. Analysis based on multiple methods identified a low level of genetic diversity, genetic differentiation and gene flow among A. spathulifolius populations. Network analysis and principal coordinates analysis showed that 15 populations could be divided into two groups: mainland and island. Furthermore, UPGMA, neighbor-net, maximum-likelihood and Bayesian inference-based phylogenetic tree confirmed that these populations formed two distinct clades. Therefore, the island populations might be treated as A. spathulifolius populations rather than A. oharai populations. Divergence time analysis estimated the divergence of A. spathulifolius lineages approximately 23.09 million years ago, while ancestral area reconstruction analysis suggested Korea as the potential origin, conflicting with alternative scenarios. These findings contribute to a comprehensive understanding of the evolutionary history, genetic structure, and adaptive strategies of A. spathulifolius in coastal environments. Our study challenges previous assumptions and underscores the necessity for further population studies to elucidate the intricate dynamics of this distinctive plant species.

Morphological and molecular evidence for natural hybridization between Sorbus pohuashanensis and S. discolor (Rosaceae)

In overlapping distribution areas of Sorbus pohuashanensis and S. discolor in North China (Mount Tuoliang, Mount Xiling and Mount Baihua), Sorbus individuals were found with pink fruit, which have never been recorded for the flora of China. Fourteen morphological characters combined with four chloroplast DNA markers and internal transcribed spacer (ITS) were used to analyze the origin of the Sorbus individuals with pink fruits and their relationship to S. pohuashanensis and S. discolor. PCA, SDA and one-way (taxon) ANOVA of morphological characters provided convincing evidence of the hybrid origin of Sorbus individuals with pink fruits based on a novel morphological character and many intermediate characters. Haplotype analysis based on four cpDNA markers showed that either S. pohuashanensis or S. discolor were maternal parents of Sorbus individuals with pink fruits. Incongruence of the position of Sorbus individuals with pink fruits between cpDNA and ITS in cluster trees supported by DNA sequence comparative analysis, implying former hybridization events between S. pohuashanensis and S. discolor. Multiple hybridization events between S. pohuashanensis and S. discolor might have contributed to the generation of Sorbus individuals with pink fruits. This study has provided insights into hybridization between species of the same genus in sympatric areas, which is of great significance for the study of interspecific hybridization.

Genetic relatedness of rice landraces in North East India with wild relatives using chloroplast markers

AbstractThe evolutionary relationship between domesticated Oryza species and their wild relatives in North East India is not well understood. To improve the understanding of the evolutionary relationship, this study investigates the genetic diversity of 68 indigenous rice landraces from North East India, ten O. rufipogon genotypes, and nine O. nivara genotypes using chloroplast variable repeat markers which, due to non-recombination and uniparental inheritance, enable better phylogenetic inference. Reference genotypes IR64 (indica) and Nipponbare (japonica) were included to characterize various phylogenetic clusters. Using distance-based hierarchical clustering, model-based structuring and principal component analysis, selected landraces from the three North Eastern Indian states of Assam, Manipur and Arunachal Pradesh were grouped into two phylogenetically different clusters that represented the IR64 and Nipponbare groupings. Interestingly, despite the fact that a cluster analysis combining North East landraces and wild relatives likewise produced two separate clusters (cluster I: Nipponbare, cluster II: IR64), the majority of the wild relatives were only clustered in the IR64 cluster. This suggests that the two distinct evolutionary histories of the rice landraces in North East India are distinguished by their genetic affinity for wild relatives and their variation in the indica and japonica pools. These results highlight chloroplast divergence influencing the genetic diversity of North East landraces with wild relatives. Further, these findings will enable in-depth studies on the functional significance of chloroplast diversity on trait adaptation in rice landraces.

Nuclear and chloroplast DNA phylogeography reveals high genetic diversity and postglacial range expansion in Quercus mexicana.

Phylogeographical studies are fundamental for understanding factors that influence the spatial distribution of genetic lineages within species. Population expansions and contractions, distribution shifts, and climate changes are among the most important factors shaping the genetic compositions of populations. We investigated the phylogeography of an endemic oak, Quercus mexicana (Fagaceae), which has a restricted distribution in northeastern Mexico along the Sierra Madre Oriental and adjacent areas. Nuclear and chloroplast DNA microsatellite markers were used to describe the genetic diversity and structure of 39 populations of Q. mexicana along its entire distribution area. We tested whether population expansion or contraction events influenced the genetic diversity and structure of the species. We also modeled the historical distributional range of Q. mexicana (for the Mid Holocene, the Last Glacial Maximum, and the Last Interglacial) to estimate the extent to which climate fluctuations have impacted the distribution of this oak species. Our results revealed high genetic diversity and low genetic structure in Q. mexicana populations. Ecological niche models suggested historical fluctuations in the distributional range of Q. mexicana. Historical range changes, gene flow, and physical barriers seem to have played an important role in shaping the phylogeographic structure of Q. mexicana. Our study indicates that the genetic structure of Q. mexicana may have been the result of responses of oak trees not only to heterogeneous environments present in the Sierra Madre Oriental and adjacent areas, but also to elevational and latitudinal shifts in response to climate changes in the past.

Biochemical and molecular identification of ten plum (Prunus salicina L.) cultivars in Egypt based on their physiological traits, chloroplast and mitochondrial DNA markers

Biochemical and molecular identification of ten plum (Prunus salicina L.) cultivars in Egypt based on their physiological traits, chloroplast and mitochondrial DNA markers

Exploring the genetic diversity and population structure of Ailanthus altissima using chloroplast and nuclear microsatellite DNA markers across its native range

Exploring the genetic diversity and population structure of Ailanthus altissima using chloroplast and nuclear microsatellite DNA markers across its native range

Haplotype diversity patterns in Quercus suber (Fagaceae) inferred from cpDNA sequence data

Chloroplast genome diversity in cork oak (Quercus suber) is characterised by the occurrence of haplotypes that are akin to those found in other Mediterranean oak species, particularly in Q. ilex and Q. rotundifolia, suggesting the possible presence of an introgressed chloroplast lineage. To further investigate this pattern, we reconstructed chloroplast haplotypes by sequencing four chloroplast markers (cpDNA), sampled across 181 individuals and 10 taxa. Our analyses resulted in the identification of two diversified chloroplast haplogroups in Q. suber, corresponding to a geographically widespread lineage and an Afro-Iberian lineage. Time-calibrated phylogenetic analyses of cpDNA point to a Miocene origin of the two haplogroups in Q. suber, suggesting that the Afro-Iberian lineage was present in cork oak before the onset of glaciation periods. The persistence of the two haplogroups in the western part of the species distribution range may be a consequence of either ancient introgression events or chloroplast lineage sorting, combined with different fixation in refugia through glaciation periods. Our results provide a comprehensive insight on the origins of chloroplast diversity in these ecologically and economically important Mediterranean oaks.

Genetic diversity and occupation strategy of Egeria najas (Hydrocharitaceae) in different habitats along the Upper Paraná River corridor

Egeria najas is a submerged aquatic macrophyte native to South America, with high propagation in reservoirs and natural lakes, whose reproductive strategy is little known. Understanding the genetic diversity of macrophyte populations can provide important information about this species' dispersion and colonization strategies, and support management actions. We aimed to genetically characterize populations of E. najas that colonize reservoirs and natural aquatic habitats (in a floodplain) in the Upper Paraná River basin, using the molecular markers ITS and trnL-trnF. The results showed the absence of genetic variation for the nuclear marker ITS and 13 distinct haplotypes for trnL-trnF. One of these haplotypes occurred in all habitats and 11 are unique haplotypes, of which 5 occurred in the Itaipu Reservoir and 6 in the floodplain. The null genetic diversity for the nuclear marker and the genetic homogeneity of the studied populations indicates that the reproduction of E. najas is mostly vegetative. The source of chloroplast marker haplotype variability may be somatic mutations. The connectivity among aquatic environments associated with river flow favors the transport of aquatic macrophyte propagules to different habitats. In the case of E. najas, whose vegetative propagules regenerate easily, the frequency of migrations supports the low genetic variability observed in populations of the Upper Paraná. In addition, the ability to occupy new habitats and recolonize disturbed ones strongly indicates that E. najas populations follow the metapopulation dynamics.

Relict stands of Central European oaks: Unravelling autochthony and genetic structure based on a multi-population study

Central European white oaks expanded rapidly after the last glacial period and reached their current distribution range during the early Holocene. They have been an important resource of timber, fuelwood and animal feed for humans, who actively promoted their presence in forests and other landscape types at least since the early historical times. Besides stands with intensive management, putatively relict populations of three native oak species can be found on unproductive sites with restricted accessibility. Here, we apply chloroplast and nuclear microsatellite markers in order to address the autochthony of relict and managed stands and compare the spatial distribution of genetic variation between them. Based on data from more than 150 populations, we demonstrate that oak autochthony was preserved throughout historical times which is likely the result of traditional silvicultural treatment. This is supported by the fact that the spatial pattern of chloroplast haplotype distribution still reflects the post-glacial recolonization in both relict and old managed stands. We observed significant admixture of haplotypes only in stands established after the Second World War, which is attributable to the transfer of reproductive material used for afforestation. In terms of nuclear genetic variation, we observed marked differences among species. Quercus pubescens exhibited a pronounced genetic structure. Genetic drift and limited gene flow among its small and isolated populations in our study area might have contributed to this pattern. Varying extent of genetic introgression with other sympatric oak species could offer an additional explanation. On the contrary, the gene pools of Q. petraea and Q. robur are highly homogenous, displaying only weak isolation-by-distance. We found no significant differences of genetic diversity and differentiation between relict and managed stands. This suggests that seed transfer mostly occurred within our study area, even in those stands established in post-war times, verifying previous findings which point out limited human interference. We recommend consideration of population genetic structure for gene conservation, with a finer resolution of gene conservation units needed for Q. pubescens due to its spatial genetic structure. Both relict and old managed stands, species-pure or mixed, are suitable for conservation, as they host autochthonous gene pools. Coppice-with-standard management could contribute to preservation of autochthony. In the face of climate change, it is also important to maintain the evolutionary potential of the stands, by facilitating generative reproduction and allowing for hybridization in mixed stands.

Genetic polymorphism of populations and phylogeny of the Central Siberian Polygonum (Polygonaceae) section using ISSR and chloroplast markers

This article is devoted to the study of genetic polymorphism of populations and phylogeny of Polygonum section using ISSR and chloroplast markers. Section Polygonum is a taxonomically complex group. Identification of individuals is complicated because of the small size of plants, the processes of interspecific hybridization, heterocarpy, phenological and ecological plasticity. In this work, the genetic variability of six populations of five knotweed species growing in Krasnoyarsk was studied. ISSR analysis revealed that the level of genetic diversity in the populations varies from 4.17 % to 35.83 % and has maximum values for genetically close species P. sabulosum (P = 35.83 %) and P. arenastrum (P = 22.5–30 %). Minimum values of genetic diversity were observed for populations of P. aviculare (P = 4.17 %) and P. calcatum (P = 5 %). The studied populations are characterized by a very high level of genetic differentiation (Gst = 0.78) and reliably belong to different species. On the similarity dendrogram (UPGMA), individuals of these species form clearly distinct clades. The genetic structure of populations using STRUCTURE also shows a reliable division of individuals into 5 clusters corresponding to the studied species. Phylogenetic analysis using the trnL-trnF chloroplast region shows the presence of a 5-nucleotide deletion and two transversions that makes it possible to divide the studied group of Polygonum into two morphologically different clusters. The first group is formed by P. arenastrum, P. calcatum, P. caspicum, P. sabulosum and characterized by the absence of a main shoot, a gradual reduction of leaves to the top of shoots and the perianth dissection by 1/2 of its length. The second group includes P. aviculare, P. boreale, P. neglectum, P rectum that have a main shoot, heterophilly, perianth dissection by 2/3–3/4 of its length. Two substitutions have been identified in the matK sequences, however it is less informative for solving taxonomic issues of the studied group.

Characterization and evaluation of nine Cannabis sativa chloroplast SNP markers for crop type determination and biogeographical origin on European samples

Cannabis sativa can be classified in two main types, according to psychotropic cannabinoid ∆9-tetrahydrocannabinol (∆9-THC) content: the drug-type and the fiber-type. According to the European Monitoring Center for Drugs and Drug Addiction, most of the European Union countries consider the possession of cannabis, for personal use, a minor offense with possibility of incarceration. Despite of the model of legal supply (i.e., Spanish cannabis clubs, Netherlands coffee shops) or medical use (i.e., Italy), cannabis remains the most used and trafficked illicit plant in the European Union. Differentiating cannabis crops or tracing the biogeographical origin is crucial for law enforcement purposes. Chloroplast DNA (cpDNA) markers may assist to determine biogeographic origin and to differentiate hemp from marijuana. This research aims: to identify and to evaluate nine C. sativa cpDNA polymorphic SNP sites to differentiate crop type and to provide information about its biogeographical origin. Five SNaPshot™ assays for nine chloroplast markers were developed and conducted in marijuana samples seized in Chile, the USA-Mexico border and Spain, and hemp samples grown in Spain and in Italy. The SNapShot™ assays were tested on 122 cannabis samples, which included 16 blind samples, and were able to differentiate marijuana crop type from hemp crop type in all samples. Using phylogenetic analysis, genetic differences were observed between marijuana and hemp samples. Moreover, principal component analysis (PCA) supported the relationship among hemp samples, as well as for USA-Mexico border, Spanish, and Chilean marijuana samples. Genetic differences between groups based on the biogeographical origin and their crop type were observed. Increasing the number of genetic markers, including the most recently studied ones, and expanding the sample database will provide more accurate information about crop differentiation and biogeographical origin.

First insights into genetic structure and diversity of Corylus colurna L. for conservation of its genetic resources and possible assisted migration

Climate change is already affecting all forest ecosystems and threatening species therein. By establishing mixed forests incorporating other tree species (e. g. scattered broadleaves) adaptation of forest stands to changing environmental conditions can be improved and the risks minimized. Turkish hazel (Corylus colurna L.) has been currently discussed as a valuable so-called alternative tree species in some European regions because it is well adapted to difficult environmental conditions. However, in its natural distribution area Turkish hazel has been overexploited because of its valuable wood and can only be found in small and isolated populations. Therefore, we examined the genetic structure and variation of Turkish hazel covering most of its natural distribution range in Asia Minor, the Caucasus, and the Balkan Peninsula. Altogether, 689 Turkish hazel trees from 25 populations were analysed at 15 nuclear and five chloroplast microsatellite markers. Both data sets revealed distinct genetic clusters of Turkish hazel in its natural distribution range, suggesting separate refugia and migration pathways during the Holocene. Medium to high level genetic variation was found, with somewhat lower values in Georgia and Turkey compared to the Balkan populations. Overall, our results highlight the need to delineate provenance regions, identify gene conservation units (GCUs) and seed stands for Turkish hazel which are essential for species conservation, provenance research and possible assisted migration attempts. Based on our results each country is able now to start conservation programs and select GCUs for Turkish hazel.

Molecular phylogenetic study of Utricularia section Oligocista in Australia and a new Cape York endemic species

Utricularia section Oligocista A.DC. (Lentibulariaceae) is distributed across the old and new world tropics. Wepresent a molecular phylogeny using a matrix of the nuclear ITS and two chloroplast markers that includes 19of the 37 recognised species sampled across the range of each species. The phylogenetic study also includesmembers of the closely allied sections Enskide (Raf.) P.Taylor (including section Minutae Lowrie, Cowie &Conran) and Stomoisia (Raf.) Kuntze from across each of their distributions. The phylogeny shows that Australiacontains species from the two major clades of section Oligocista, with each involving ancestors with yellow andpurple/mauve corolla colours. This study provides morphological and molecular evidence for recognition of anew species, Utricularia irwinica R.W.Jobson & Baleeiro, from Cape York Peninsula, Queensland. A lectotypeis designated for the name Utricularia cyanea var. alba Benth. We also recommend changes to the taxonomyof sections Enskide and Oligocista based on the phylogenetic results and morphology.

Genealogical tracing of Olea europaea species and pedigree relationships of var. europaea using chloroplast and nuclear markers

BackgroundOlive is one of the most cultivated species in the Mediterranean Basin and beyond. Despite being extensively studied for its commercial relevance, the origin of cultivated olive and the history of its domestication remain open questions. Here, we present a genealogical and kinship relationships analysis by mean of chloroplast and nuclear markers of different genera, subgenus, species, subspecies, ecotypes, cultivated, ancient and wild types, which constitutes one of the most inclusive research to date on the diversity within Olea europaea species. A complete survey of the variability across the nuclear and plastid genomes of different genotypes was studied through single nucleotide polymorphisms, indels (insertions and deletions), and length variation.ResultsFifty-six different chlorotypes were identified among the Oleaceae family including Olea europaea, other species and genera. The chloroplast genome evolution, within Olea europaea subspecies, probably started from subsp. cuspidata, which likely represents the ancestor of all the other subspecies and therefore of wild types and cultivars. Our study allows us to hypothesize that, inside the subspecies europaea containing cultivars and the wild types, the ancestral selection from var. sylvestris occurred both in the eastern side of the Mediterranean and in the central-western part of Basin. Moreover, it was elucidated the origin of several cultivars, which depends on the introduction of eastern cultivars, belonging to the lineage E1, followed by crossing and replacement of the autochthonous olive germplasm of central-western Mediterranean Basin. In fact, our study highlighted that two main ‘founders’ gave the origin to more than 60% of analyzed olive cultivars. Other secondary founders, which strongly contributed to give origin to the actual olive cultivar diversity, were already detected.ConclusionsThe application of comparative genomics not only paves the way for a better understanding of the phylogenetic relationships within the Olea europaea species but also provides original insights into other elusive evolutionary processes, such as chloroplast inheritance and parentage inside olive cultivars, opening new scenarios for further research such as the association studies and breeding programs.

Phylogeny and morphological evolution of the Neotropical genus Cuspidaria (Bignonieae, Bignoniaceae): Combining high‐throughput sequencing and targeted loci data

AbstractThe combined use of high‐throughput sequencing (HTS) with targeted loci data offers an excellent opportunity to reconstruct robust and comprehensive phylogenies at fine taxonomic scales using a hybrid approach. In this study, we infer the phylogeny of Cuspidaria (Bignonieae, Bignoniaceae), a diverse clade of lianas and shrubs centered in South America's wet and dry forests. We used HTS to obtain complete or nearly complete plastome sequences of seven individuals of Cuspidaria selected to represent the main clades in the genus. This sampling strategy aimed to reconstruct relationships at deeper nodes (i.e., backbone). We also used targeted loci data obtained through Sanger sequencing to obtain sequences of two chloroplast markers (ndhF, rpl32‐trnL) and one nuclear marker (PepC) for multiple individuals of 18 out of the 21 species of Cuspidaria recognized in the most recent treatment of the genus; only C. emmonsii, C. lachnaea, and C. simplicifolia were not sampled. This broad sampling strategy aimed to test the monophyly of individual species and reconstruct fine‐scale interspecific relationships within the genus. Both datasets were analyzed separately and combined using Bayesian inference and maximum likelihood approaches. The combined dataset includes 65 individuals representing 18 previously recognized species of Cuspidaria plus outgroup taxa. The analysis of the combined dataset recovered a monophyletic Cuspidaria, excluding C. bracteata, which was nested within the outgroup and is best treated elsewhere. Ancestral character state reconstructions provided novel insights into the evolution of morphology and identified multiple putative morphological synapomorphies for key lineages of Cuspidaria. Namely, anthers curved forward was reconstructed as a potential synapomorphy for the Cuspidaria clade, whereas interpetiolar gland fields in stems and inflorescence, subulate or inconspicuous prophylls of the axillary buds, biternate leaves, actinodromous venation, calyx cupulate or spathaceous, calyx apices dentate or irregularly lobed, fruits with winged valves, and fruit midrib limited by two longitudinal ridges were reconstructed as potential synapomorphies of different clades within the genus. Other traits such as habit, tertiary venation, inflorescence type, corolla color, number of ovule series, and pollen unit were highly labile. As circumscribed here, Cuspidaria is composed of eight main clades supported by molecular data and morphological synapomorphies: Sideropogon, Tetrastichella, Cinerea, Paracarpaea, Cremastus, Blepharitheca, Saldanhaea, and Cuspidaria s.str. clades. All species are monophyletic, except for C. pulchra and C. sceptrum, which form a species complex and are best treated as a single taxon. The necessary taxonomic changes are proposed, leading to the recognition of 19 species in Cuspidaria.

No wonder, it is a hybrid. Natural hybridization between Jacobaea vulgaris and J. erucifolia revealed by molecular marker systems and its potential ecological impact.

Progressive changes in the environment are related to modifications of the habitat. Introducing exotic species, and interbreeding between species can lead to processes that in the case of rare species or small populations threatens their integrity. Given the declining trends of many populations due to increased hybridization, early recognition of hybrids becomes important in conservation management. Natural hybridization is prevalent in Jacobaea. There are many naturally occurring interspecific hybrids in this genus, including those between Jacobaea vulgaris and its relatives. Although Jacobaea erucifolia and J. vulgaris often co-occur and are considered closely related, apart from the few reports of German botanists on the existence of such hybrids, there is no information on research confirming hybridization between them. Morphologically intermediate individuals, found in the sympatric distributions of J. vulgaris and J. erucifolia, were hypothesized to be their hybrids. Two molecular marker systems (nuclear and chloroplast DNA markers) were employed to test this hypothesis and characterize putative hybrids. Nuclear and chloroplast DNA sequencing results and taxon-specific amplified fragment length polymorphism (AFLP) fragment distribution analysis confirmed the hybrid nature of all 25 putative hybrids. The AFLP patterns of most hybrids demonstrated a closer relationship to J. erucifolia, suggesting frequent backcrossing. Moreover, they showed that several individuals previously described as pure were probably also of hybrid origin, backcrosses to J. erucifolia and J. vulgaris. This study provides the first molecular confirmation that natural hybrids between J. vulgaris and J. erucifolia occur in Poland. Hybridization appeared to be bidirectional but asymmetrical with J. vulgaris as the usual maternal parent.