Aquilegia Species Research Articles

Characterizing complete mitochondrial genome of Aquilegia amurensis and its evolutionary implications

BackgroundAquilegia is a model system for studying the evolution of adaptive radiation. However, very few studies have been conducted on the Aquilegia mitochondrial genome. Since mitochondria play a key role in plant adaptation to abiotic stress, analyzing the mitochondrial genome may provide a new perspective for understanding adaptive evolution.ResultsThe Aquilegia amurensis mitochondrial genome was characterized by a circular chromosome and two linear chromosomes, with a total length of 538,736 bp; the genes included 33 protein-coding genes, 24 transfer RNA (tRNA) genes and 3 ribosomal RNA (rRNA) genes. We subsequently conducted a phylogenetic analysis based on single nucleotide polymorphisms (SNPs) in the mitochondrial genomes of 18 Aquilegia species, which were roughly divided into two clades: the European-Asian clade and the North American clade. Moreover, the genes mttB and rpl5 were shown to be positively selected in European-Asian species, and they may help European and Asian species adapt to environmental changes.ConclusionsIn this study, we assembled and annotated the first mitochondrial genome of the adaptive evolution model plant Aquilegia. The subsequent analysis provided us with a basis for further molecular studies on Aquilegia mitochondrial genomes and valuable information on adaptive evolution in Aquilegia.

The past, present, and future of ecogeographic isolation between closely related Aquilegia plants.

Quantifying the strength of the ecogeographic barrier is an important aspect of plant speciation research, and serves as a practical step to understanding the evolutionary trajectory of plants under climate change. Here, we quantified the extent of ecogeographic isolation in four closely related Aquilegia species that radiated in the Mountains of SW China and adjacent regions, often lacking intrinsic barriers. We used environmental niche models to predict past, present, and future species potential distributions and compared them to determine the degree of overlap and ecogeographic isolation. Our investigation found significant ecological differentiation in all studied species pairs except A. kansuensis and A. ecalacarata. The current strengths of ecogeographic isolation are above 0.5 in most cases. Compared with current climates, most species had an expanding range in the Last Glacial Maximum, the Mid Holocene, and under four future climate scenarios. Our results suggested that ecogeographic isolation contributes to the diversification and maintenance of Aquilegia species in the Mountains of northern and SW China and would act as an essential reproductive barrier in the future.

Characterization and expression analysis of bHLH transcription factors reveal their putative regulatory effects on nectar spur development in Aquilegia species

Nectar spur is a hollow extension of certain flower parts and shows strikingly diverse size and shape in Aquilegia. Nectar spur development is involved in cell division and expansion processes. The basic helix-loop-helix (bHLH) transcription factors (TFs) control a diversity of organ morphogenesis, including cell division and cell expansion processes. However, the role of bHLH genes in nectar spur development in Aquilegia is mainly unknown. We conducted a genome-wide identification of the bHLH gene family in Aquilegia to determine structural characteristics and phylogenetic relationships, and to analyze expression profiles of these genes during the development of nectar spur in spurless and spurred species. A total of 120 AqbHLH genes were identified from the Aquilegia coerulea genome. The phylogenetic tree showed that AqbHLH proteins were divided into 15 subfamilies, among which S7 and S8 subfamilies occurred marked expansion. The AqbHLH genes in the same clade had similar motif composition and gene structure characteristics. Conserved residue analysis indicated nineteen residues with conservation of more than 50% were found in the four conserved regions. In the upstream sequence of AqbHLH genes, the light-responsive element was the most abundant cis-acting element. Eighteen AqbHLH genes showed syntenic relationships, and eight genes from four syntenic pairs underwent tandem duplications. According to the expression profiling analysis by public RNA-Seq data and qRT-PCR results, five AqbHLH genes, including AqbHLH027, AqbHLH046, AqbHLH082, AqbHLH083 and AqbHLH092, were differentially expressed between different tissues in A. coerulea at early developmental stages, as well as between spurless and spurred Aquilegia species. Of them, AqbHLH046 was not only highly expressed in spur compared with blade, but also showed higher expression levels in spurred species than spurless specie, suggesting it plays an essential role in the development of spur by regulating cell division. This study lays a foundation to investigate the function of AqbHLH genes family in nectar spur development, and has potential implications for speciation and genetic breeding in the genus Aquilegia.

Physio-Biochemical Responses of Three Aquilegia Species Seedlings to Salt Stress

Road deicing salts are widely used during winter in northern China, which makes it essential to choose proper salt-tolerant plant species in urban landscapes. Columbine (Aquilegia) is a herb with high ornamental and commercial values. This study evaluated three Aquilegia species (A. oxysepala, A. parviflora, and A. viridiflora) for salt tolerance by monitoring their germination state under different electrical conductivity (EC) of 0.1 dS·m−1 (distilled water), 1.0 dS·m−1, 2.0 dS·m−1, 3.0 dS·m−1, 4.0 dS·m−1, 5.0 dS·m−1, 6.0 dS·m−1, physio-biochemical responses to different EC of 0.3 dS·m−1 (tap water), 5.0 dS·m−1, and 10.0 dS·m−1. The germination and growth parameters, visual scores, dry weight, leaf stomatal conductance, photosynthetic rate, and chlorophyll contents of three species decreased under salt stress, which was opposite to the changes of electrolyte leakage, malondialdehyde, proline, and soluble sugar contents. Superoxide dismutase and peroxidase activity trend differently among species. These results showed that the germination threshold of three species was 6.0 dS·m−1. A. oxysepala was the most salt-tolerant species, with a tolerance threshold of soil conductivity in 2.83 dS·m−1, followed by A. viridiflora and A. parviflora. Therefore, A. oxysepala is suitable for planting as a ground cover in urban areas where deicing salt is applied.

Complex developmental and transcriptional dynamics underlie pollinator-driven evolutionary transitions in nectar spur morphology in Aquilegia (columbine).

Determining the developmental programs underlying morphological variation is key to elucidating the evolutionary processes that generated the stunning biodiversity of the angiosperms. Here, we characterized the developmental and transcriptional dynamics of the elaborate petal nectar spur of Aquilegia (columbine) in species with contrasting pollination syndromes and spur morphologies. We collected petal epidermal cell number and length data across four Aquilegia species, two with short, curved nectar spurs of the bee-pollination syndrome and two with long, straight spurs of the hummingbird-pollination syndrome. We also performed RNA-seq on A. brevistyla (bee) and A. canadensis (hummingbird) distal and proximal spur compartments at multiple developmental stages. Finally, we intersected these data sets with a previous QTL mapping study on spur length and shape to identify new candidate loci. The differential growth between the proximal and distal surfaces of curved spurs is primarily driven by differential cell division. However, independent transitions to straight spurs in the hummingbird syndrome have evolved by increasing differential cell elongation between spur surfaces. The RNA-seq data reveal these tissues to be transcriptionally distinct and point to auxin signaling as being involved with the differential cell elongation responsible for the evolution of straight spurs. We identify several promising candidate genes for future study. Our study, taken together with previous work in Aquilegia, reveals the complexity of the developmental mechanisms underlying trait variation in this system. The framework we established here will lead to exciting future work examining candidate genes and processes involved in the rapid radiation of the genus.

Genetic and Epigenetic Signatures Associated with the Divergence of Aquilegia Species

Widely grown in the Northern Hemisphere, the genus Aquilegia (columbine) is a model system in adaptive radiation research. While morphological variations between species have been associated with environmental factors, such as pollinators, how genetic and epigenetic factors are involved in the rapid divergence in this genus remains under investigated. In this study, we surveyed the genomes and DNA methylomes of ten Aquilegia species, representative of the Asian, European and North American lineages. Our analyses of the phylogeny and population structure revealed high genetic and DNA methylomic divergence across these three lineages. By multi-level genome-wide scanning, we identified candidate genes exhibiting lineage-specific genetic or epigenetic variation patterns that were signatures of inter-specific divergence. We demonstrated that these species-specific genetic variations and epigenetic variabilities are partially independent and are both functionally related to various biological processes vital to adaptation, including stress tolerance, cell reproduction and DNA repair. Our study provides an exploratory overview of how genetic and epigenetic signatures are associated with the diversification of the Aquilegia species.

Intraspecific independent evolution of floral spur length in response to local flower visitor size in Japanese Aquilegia in different mountain regions.

Geographic differences in floral traits may reflect geographic differences in effective pollinator assemblages. Independent local adaptation to pollinator assemblages in multiple regions would be expected to cause parallel floral trait evolution, although sufficient evidence for this is still lacking. Knowing the intraspecific evolutionary history of floral traits will reveal events that occur in the early stages of trait diversification. In this study, we investigated the relationship between flower spur length and pollinator size in 16 populations of Aquilegia buergeriana var. buergeriana distributed in four mountain regions in the Japanese Alps. We also examined the genetic relationship between yellow‐ and red‐flowered individuals, to see if color differences caused genetic differentiation by pollinator isolation. Genetic relationships among 16 populations were analyzed based on genome‐wide single‐nucleotide polymorphisms. Even among populations within the same mountain region, pollinator size varied widely, and the average spur length of A. buergeriana var. buergeriana in each population was strongly related to the average visitor size of that population. Genetic relatedness between populations was not related to the similarity of spur length between populations; rather, it was related to the geographic proximity of populations in each mountain region. Our results indicate that spur length in each population evolved independently of the population genetic structure but in parallel in response to local flower visitor size in different mountain regions. Further, yellow‐ and red‐flowered individuals of A. buergeriana var. buergeriana were not genetically differentiated. Unlike other Aquilegia species in Europe and America visited by hummingbirds and hawkmoths, the Japanese Aquilegia species is consistently visited by bumblebees. As a result, genetic isolation by flower color may not have occurred.

Characterization of Terpene synthase variation in flowers of wild aquilegia species from Northeastern Asia.

There are several causes for the great diversity in floral terpenes. The terpene products are determined by the catalytic fidelity, efficiency and plasticity of the active sites of terpene synthases (TPSs). However, the molecular mechanism of TPS in catalyzing terpene biosynthesis and its evolutionary fate in wild plant species remain largely unknown. In this study, the functionality of terpene synthases and their natural variants were assessed in two Northeastern Asia endemic columbine species and their natural hybrid. Synoptically, TPS7, TPS8, and TPS9 were highly expressed in these Aquilegia species from the Zuojia population. The in vitro and in vivo enzymatic assays revealed that TPS7 and TPS8 mainly produced (+)-limonene and β-sesquiphellandrene, respectively, whereas TPS9 produced pinene, similar to the major components released from Aquilegia flowers. Multiple sequence alignment of Aquilegia TPS7 and TPS8 in the Zuojia population revealed amino acid polymorphisms. Domain swapping and amino acid substitution assays demonstrated that 413A, 503I and 529D had impacts on TPS7 catalytic activity, whereas 420G, 538F and 545L affected the ratio of β-sesquiphellandrene to β-bisabolene in TPS8. Moreover, these key polymorphic amino acid residues were found in Aquilegia species from the Changbai Mountain population. Interestingly, amino acid polymorphisms in TPSs were present in individuals with low expression levels, and nonsynonymous mutations could impact the catalytic activity or product specificity of these genes. The results of this study will shed new light on the function and evolution of TPS genes in wild plant species and are beneficial to the modification of plant fragrances.

Petal ontogeny, structure, and pollination system of four Aquilegia species in Midwest China

The diversification of spur length in Aquilegia is often thought to be driven by pollinators. In this study, the morphology and pollination biology of four sympatric Aquilegia species distributed in China with different petal spur lengths were investigated through scanning electron microscopy, light microscopy, and field observations and measurements. The morphology and spur lengths of the four Aquilegia species differed: Aquilegia kansuensis has long incurved spurs (14.82 ± 0.18 mm), A. rockii has long straight spurs (16.34 ± 0.18 mm), A. semicalcarata has short slender spurs (5.16 ± 0.17 mm), and A. ecalcarata is effectively spurless (1.20 ± 0.03 mm). Early petal development was similar among the species; petals undergo initiation and delayed development, resulting in mature petals with different spur lengths and morphologies via developmental remodeling. The long spurs of A. kansuensis and A. rockii were derived from an increased cell number and anisotropic cell expansion, with well-developed secretory tissue forming at the spur bottom. No secretory tissue formed in the spurs of A. semicalcarata and A. ecalcarata, even though their cell number increased. Although their spur morphology and nectary structure show considerable differences, the pollination systems of the four species are similar. Rhingia campestris is the dominant pollinator of A. kansuensis, A. semicalcarata, and A. ecalcarata, whereas Pipiza familiaris is the dominant pollinator of A. rockii. Both R. campestris and P. familiaris forage for pollen when visiting flowers. Given that the four species studied differ in spur length but are all pollinated by short proboscid pollinators, there is no obvious one-to-one correspondence between spur length and pollinator mouthpart length in A. rockii, A. kansuensis, A. semicalcarata and A. ecalcarata, which is similar to the pattern in the European Aquilegia alpina, and abiotic factors may be an important explanation for spur lengths in our study.

Loss of innovative traits underlies multiple origins of Aquilegia ecalcarata

AbstractTraits are basic attributes of organisms that form the basis for speciation and diversity. The floral nectar spur is a classic example of a key innovative trait. Differences in nectar spur morphology can lead to pollinator specialization and thereby promote reproductive isolation between species. Despite its importance, the nectar spur has been lost in some members of the columbine genus (Aquilegia), resulting in a new spurless trait, and the evolutionary influence of this trait has become a topic of scientific interest. Aquilegia ecalcarata is an important representative columbine species that lacks spurs. Here, we resequenced the genomes of 324 individuals from A. ecalcarata and four related species. We found that A. ecalcarata was divided into three groups based on the phylogenetic relationships and population genetic structures. Topology weighting analysis revealed that A. ecalcarata has multiple origins, and Patterson′s D statistic showed that the spurless trait may have one origin. Floral morphological analysis revealed significant differences between A. ecalcarata and its spurred sister groups, and the floral phenotypes of the three A. ecalcarata groups have identical or similar floral phenotypes. Our results confirmed that the spurless trait not only produced the phenotype of A. ecalcarata but also contributed to the emergence of the A. rockii phenotype. Moreover, the spurless trait promoted the divergence between A. ecalcarata and its close, spurred relatives. Our research shows that the loss of key innovative traits can play a very important role in speciation and species diversity.

<p><strong>Nomenclatural notes and typification of three <em>Aquilegia</em> (Ranunculaceae) names described from the Himalayas</strong></p>

As part of the ongoing revision of the Himalayas species of Aquilegia (Ranunculaceae), three names applicable to three accepted species are typified, and some nomenclatural and taxonomic remarks are provided. Lectotypes are designated for A. glauca (a taxonomic synonym of A. fragrans) and A. vulgaris subsp. nivalis (a nomenclatural synonym of A. nivalis). Epitypes for A. fragrans and A. glauca were also designated in order to avoid any ambiguity in the taxonomic interpretation of the lectotypes and hence the application of the names.

Comparative chloroplast genomes and phylogenetic analysis of Aquilegia.

Premise Aquilegia is an ideal taxon for studying the evolution of adaptive radiation. Current phylogenies of Aquilegia based on different molecular markers are inconsistent, and therefore a clear and accurate phylogeny remains uncertain. Analyzing the chloroplast genome, with its simple structure and low recombination rate, may help solve this problem.MethodsNext‐generation sequencing data were generated or downloaded for Aquilegia species, enabling their chloroplast genomes to be assembled. The assemblies were used to estimate the genome characteristics and infer the phylogeny of Aquilegia.ResultsIn this study, chloroplast genome sequences were assembled for Aquilegia species distributed across Asia, North America, and Europe. Three of the genes analyzed (petG, rpl36, and atpB) were shown to be under positive selection and may be related to adaptation. The phylogenetic tree of Aquilegia showed that its member species formed two clades with high support, North American and European species, with the Asian species being paraphyletic; A. parviflora and A. amurensis clustered with the North American species, while the remaining Asian species were found in the European clade. In addition, A. oxysepala var. kansuensis should be considered as a separate species rather than a variety.DiscussionThe complete chloroplast genomes of these Aquilegia species provide new insights into the reconstruction of the phylogeny of related species and contribute to the further study of this genus.

Erythroid induction activity of Aquilegia fragrans and Aquilegia pubiflora and identification of compounds using liquid chromatography-tandem mass spectrometry

Aquilegia fragrans (AF) and Aquilegia pubiflora (AP) are the two medicinally important species of genus Aquilegia used for the treatment of various diseases and infections. This paper describes the potential of fetal hemoglobin induction activity of the methanolic extracts of AF and AP in K562 cell line. AF and AP have shown 27.147 ± 1.376 and 32.786 ± 1.048 percent erythroid induction, respectively at 15.625 (µg/mL) concentration which suggested that both plants can be the source of potential fetal hemoglobin inducers and may be used for the treatment of β-thalassemia. Phytochemical analyses of both species were also evaluated by using high-resolution LC-ESI-QTOF-MS/MS techniques. A Total of thirty compounds were identified using positive and negative ionization modes. The identification was based on the matching of high-resolution masses, isotopic pattern, and MS/MS fragmentation. Several statistical analyses were performed to evaluate the distribution of compounds in both species. Identified compounds belong to various classes including flavonoids, steroids, lignans, terpenoids, benzofuran and coumarins. The established chemical fingerprints will be helpful in standardization and quality control of plant extracts.

<p><strong>Two new taxa and one new record of <em>Aquilegia</em> (Ranunculaceae) from India and Pakistan</strong></p>

A new species Aquilegia bashahrica and a new nothospecies Aquilegia × emodi from North-western Himalayas, are described and illustrated. In addition A. lactiflora is recorded for India and Pakistan for the first time and Aquilegia kareliniana is excluded from the flora of India. An identification key to the species of Aquilegia from the North-western Himalayas is provided and diagnostic characters are discussed.

Developmental and molecular characterization of novel staminodes in Aquilegia.

The ranunculid model system Aquilegia is notable for the presence of a fifth type of floral organ, the staminode, which appears to be the result of sterilization and modification of the two innermost whorls of stamens. Previous studies have found that the genetic basis for the identity of this new organ is the result of sub- and neofunctionalization of floral organ identity gene paralogues; however, we do not know the extent of developmental and molecular divergence between stamens and staminodes. We used histological techniques to describe the development of the Aquilegia coerulea 'Origami' staminode relative to the stamen filament. These results have been compared with four other Aquilegia species and the closely related genera Urophysa and Semiaquilegia. As a complement, RNA sequencing has been conducted at two developmental stages to investigate the molecular divergence of the stamen filaments and staminodes in A. coerulea 'Origami'. Our developmental study has revealed novel features of staminode development, most notably a physical interaction along the lateral margin of adjacent organs that appears to mediate their adhesion. In addition, patterns of abaxial/adaxial differentiation are observed in staminodes but not stamen filaments, including asymmetric lignification of the adaxial epidermis in the staminodes. The comparative transcriptomics are consistent with the observed lignification of staminodes and indicate that stamen filaments are radialized due to overexpression of adaxial identity, while the staminodes are expanded due to the balanced presence of abaxial identity. These findings suggest a model in which the novel staminode identity programme interacts with the abaxial/adaxial identity pathways to produce two whorls of laterally expanded organs that are highly differentiated along their abaxial/adaxial axis. While the ecological function of Aquilegia staminodes remains to be determined, these data are consistent with a role in protecting the early carpels from herbivory and/or pathogens.

Comparative transcriptomics of early petal development across four diverse species of Aquilegia reveal few genes consistently associated with nectar spur development

BackgroundPetal nectar spurs, which facilitate pollination through animal attraction and pollen placement, represent a key innovation promoting diversification in the genus Aquilegia (Ranunculaceae). Identifying the genetic components that contribute to the development of these three-dimensional structures will inform our understanding of the number and types of genetic changes that are involved in the evolution of novel traits. In a prior study, gene expression between two regions of developing petals, the laminar blade and the spur cup, was compared at two developmental stages in the horticultural variety A. coerulea ‘Origami’. Several hundred genes were differentially expressed (DE) between the blade and spur at both developmental stages. In order to narrow in on a set of genes crucial to early spur formation, the current study uses RNA sequencing (RNAseq) to conduct comparative expression analyses of petals from five developmental stages between four Aquilegia species, three with morphologically variable nectar spurs, A. sibirica, A. formosa, and A. chrysantha, and one that lacks nectar spurs, A. ecalcarata.ResultsPetal morphology differed increasingly between taxa across the developmental stages assessed, with petals from all four taxa being indistinguishable pre-spur formation at developmental stage 1 (DS1) and highly differentiated by developmental stage 5 (DS5). In all four taxa, genes involved in mitosis were down-regulated over the course of the assessed developmental stages, however, many genes involved in mitotic processes remained expressed at higher levels later in development in the spurred taxa. A total of 690 genes were identified that were consistently DE between the spurred taxa and A. ecalcarata at all five developmental stages. By comparing these genes with those identified as DE between spur and blade tissue in A. coerulea ‘Origami’, a set of only 35 genes was identified that shows consistent DE between petal samples containing spur tissue versus those without spur tissue.ConclusionsThe results of this study suggest that expression differences in very few loci are associated with the presence and absence of spurs. In general, it appears that the spurless petals of A. ecalcarata cease cell divisions and enter the cell differentiation phase at an earlier developmental time point than those that produce spurs. This much more tractable list of 35 candidates genes will greatly facilitate targeted functional studies to assess the genetic control and evolution of petal spurs in Aquilegia.

Rapid Divergence Followed by Adaptation to Contrasting Ecological Niches of Two Closely Related Columbine Species Aquilegia japonica and A. oxysepala.

Elucidating the mechanisms underlying the genetic divergence between closely related species is crucial to understanding the origin and evolution of biodiversity. The genus Aquilegia L. has undergone rapid adaptive radiation, generating about 70 well-recognized species that are specialized to distinct habitats and pollinators. In this study, to address the underlying evolutionary mechanisms that drive the genetic divergence, we analyzed the whole genomes of two ecologically isolated Aquilegia species, A. oxysepala and A. japonica as well as their putative hybrid. Our comparative genomic analyses reveal that while the two species diverged only recently and experienced recurrent gene flow, a high level of genetic divergence is observed in their nuclear genomes. In particular, candidate genomic regions that show signature of selection differ dramatically between the two species. Given that the splitting time of the two species is broadly matched with the decrease in effective population sizes, we propose that allopatric isolation together with natural selection have preceded the interspecific gene flow in the process of speciation. The observed high genetic divergence is likely an outcome of combined effects of natural selection, genetic drift and divergent sorting of ancestral polymorphisms. Our study provides a genome-wide view of how genetic divergence has evolved between closely related species.

Homologs of the STYLISH gene family control nectary development in Aquilegia.

Floral nectaries are an interesting example of a convergent trait in flowering plants, and are associated with the diversification of numerous angiosperm lineages, including the adaptive radiation of the New World Aquilegia species. However, we know very little as to what genes contribute to nectary development and evolution, particularly in noncore eudicot taxa. We analyzed expression patterns and used RNAi-based methods to investigate the functions of homologs from the STYLISH (STY) family in nectar spur development in Aquilegiacoerulea. We found that AqSTY1 exhibits concentrated expression in the presumptive nectary of the growing spur tip, and triple gene silencing of the three STY-like genes revealed that they function in style and nectary development. Strong expression of STY homologs was also detected in the nectary-bearing petals of Delphinium and Epimedium. Our results suggest that the novel recruitment of STY homologs to control nectary development is likely to have occurred before the diversification of the Ranunculaceae and Berberidaceae. To date, the STY homologs of the Ranunculales are the only alternative loci for the control of nectary development in flowering plants, providing a critical data point in understanding the evolutionary origin and developmental basis of nectaries.

Genetic diversity and evolutionary history of four closely related Aquilegia species revealed by 10 nuclear gene fragments

AbstractThe genus Aquilegia is emerging as the new model system for plant development, ecology, and evolution studies. Previous research showed that pollinator shift might drive the diversification of North American Aquilegia species, and natural selection on the length of petal nectar spur might play a crucial role. In this genus, A. ecalcarata Maxim. is the only taxon that has lost nectar spurs. Previous phylogenetic results indicated that A. ecalcarata, A. yabeana Kitag., A. oxysepala var. kansuensis Bruhl., and A. rockii Munz comprised a monophyletic group. However, their pattern of genetic diversity remains unknown. In addition, little is known about the evolutionary relationship among the four species on the population level. We carried out a population genetics study with 21 representative populations based on 10 single‐copy nuclear gene fragments and found that: (i) A. yabeana conserved the highest genetic diversity (both πsil and θsil) and A. oxysepala var. kansuensis had the lowest level; (ii) A. ecalcarata split into two groups, with one population clustered with A. rockii and the other five populations clustered with A. oxysepala var. kansuensis; and (iii) the allele frequency spectrum showed an excess of low frequency alleles in all four species, implying that they may undergo the mutation‐drift equilibrium. Our findings provide the first investigation of genetic diversity and evolutionary relationships in A. yabeana, A. oxysepala var. kansuensis, A. rockii, and A. ecalcarata. They lay the foundation for future evolutionary studies, such as speciation mediated by pollinators.

Characterisation and risk assessment of the emerging Peronospora disease on Aquilegia

Aquilegia is a popular garden plant in the northern hemisphere as well as a native plant in the UK and continental Europe. In 2000, Semiaquilegia adoxoides was found infected by downy mildew in Korea, and since 2013 there have been several confirmed records of a Peronospora sp. affecting Aquilegia in the UK, with symptomatic plants being observed several years prior. Symptoms include yellow patches delineated by the veins on the leaves of affected plants, but systemically infected plants were also recorded, which are generally chlorotic and show curled leaf margins. A greyish down of conidiophores and conidia was observed on the lower side of infected leaves. Preliminary molecular phylogenetic analyses could not identify the causal agent at the species level, but revealed its affinities to other Peronospora species parasitic on the Ranunculales and Saxifragaceae. To our knowledge, this is the first occurrence of a downy mildew affecting a species of Aquilegia. Already, about 1 year after its confirmed first occurrence in the UK and 3 years after reported symptoms, a huge impact on infested gardens and nurseries has occurred. As oospore production has been observed and the pathogen can grow systemically, rendering seed transmission likely, this pathogen should be classified as a high risk pathogen for Aquilegia. Appropriate quarantine measures should be taken to restrict the pathogen from spreading.