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An endemic Aquilegia not seen on Mt Parnassos for more than one and a half centuries is rediscovered. It also occurs on Mt Giona in south central Greece. Its taxonomic status was reassessed leading to a new combination at subspecies rank. [ABSTRACT FROM AUTHOR]

Floral organs are produced by floral meristems (FMs), which harbor stem cells in their centers. Since each flower only has a finite number of organs, the stem cell activity of an FM will always terminate at a specific time point, a process termed floral meristem termination (FMT). Variation in the timing of FMT can give rise to floral morphological diversity, but how this process is fine-tuned at a developmental and evolutionary level is poorly understood. Flowers from the genus Aquilegia share identical floral organ arrangement except for stamen whorl number (SWN), making Aquilegia a well-suited system for investigation of this process: differences in SWN between species represent differences in the timing of FMT. By crossing A. canadensis and A. brevistyla, quantitative trait locus (QTL) mapping has revealed a complex genetic architecture with seven QTL. We explored potential candidate genes under each QTL and characterized novel expression patterns of select loci of interest using in situ hybridization. To our knowledge, this is the first attempt to dissect the genetic basis of how natural variation in the timing of FMT is regulated, and our results provide insight into how floral morphological diversity can be generated at the meristematic level.

Abstract Background Aquilegia 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. Results The 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. Conclusions In 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.

Background: Aquilegia 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. Results: The 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. Conclusions: In 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. [ABSTRACT FROM AUTHOR]

Knowing the determinants of biodiversity is crucial to understanding the differentiation of living organisms. Several gene-flow limiting processes can drive divergence, including adaptive ecological differentiation or geographical isolation both constituting environmental-dependent reproductive barriers. Furthermore, the strength of these processes may be associated to the degree to which ecological niches are conserved and shared by phylogenetically related species. Here, we aim to disentangle these two alternatives reproductive barriers by evaluating the degree of ecological niche similarity among phylogenetically related species. We performed ecological niche modelling (ENM) on four species belonging to a recently diversified genus (Aquilegia, Ranunculaceae) occurring in montane environments of the Alpine and Apennine chains (southern Europe). Results show that niche differentiation among species is related to phylogenetic distinctiveness. Nevertheless, niche similarity tests indicated that the conditions selected by the species are more similar than expected from the null models, even though they are sparsely available in the respective ecological spaces, suggesting that the observed pattern may result from phylogenetic niche conservatism. These results, highlight that paleogeographic events in south-European mountains may have driven divergence by isolation in Aquilegia while partially retaining the possibly ancestral characters of the niches. [ABSTRACT FROM AUTHOR]

The evolution of novel features, such as eyes or wings, that allow organisms to exploit their environment in new ways can lead to increased diversification rates. Therefore, understanding the genetic and developmental mechanisms involved in the origin of these key innovations has long been of interest to evolutionary biologists. In flowering plants, floral nectar spurs are a prime example of a key innovation, with the independent evolution of spurs associated with increased diversification rates in multiple angiosperm lineages due to their ability to promote reproductive isolation via pollinator specialization. As none of the traditional plant model taxa have nectar spurs, little is known about the genetic and developmental basis of this trait. Nectar spurs are a defining feature of the columbine genus Aquilegia (Ranunculaceae), a lineage that has experienced a relatively recent and rapid radiation. We use a combination of genetic mapping, gene expression analyses, and functional assays to identify a gene crucial for nectar spur development, POPOVICH (POP), which encodes a C2H2 zinc-finger transcription factor. POP plays a central role in regulating cell proliferation in the Aquilegia petal during the early phase (phase I) of spur development and also appears to be necessary for the subsequent development of nectaries. The identification of POP opens up numerous avenues for continued scientific exploration, including further elucidating of the genetic pathway of which it is a part, determining its role in the initial evolution of the Aquilegia nectar spur, and examining its potential role in the subsequent evolution of diverse spur morphologies across the genus.

Background and aimsPendulous flowers (due to a flexible pedicel) are a common, convergent trait of hummingbird-pollinated flowers. However, the role of flexible pedicels remains uncertain despite several functional hypotheses. Here we present and test the 'lever action hypothesis': flexible pedicels allow pendulous flowers to move upwards from all sides, pushing the stigma and anthers against the underside of the feeding hummingbird regardless of which nectary is being visited.MethodsTo test whether this lever action increased pollination success, we wired emasculated flowers of serpentine columbine, Aquilegia eximia, to prevent levering and compared pollination success of immobilized flowers with emasculated unwired and wire controls.Key resultsSeed set was significantly lower in wire-immobilized flowers than unwired control and wire control flowers. Video analysis of visits to wire-immobilized and unwired flowers demonstrated that birds contacted the stigmas and anthers of immobilized flowers less often than those of flowers with flexible pedicels.ConclusionsWe conclude that flexible pedicels permit the levering of reproductive structures onto a hovering bird. Hummingbirds, as uniquely large, hovering pollinators, differ from flies or bees which are too small to cause levering of flowers while hovering. Thus, flexible pedicels may be an adaptation to hummingbird pollination, in particular due to hummingbird size. We further speculate that this mechanism is effective only in radially symmetric flowers; in contrast, zygomorphic hummingbird-pollinated flowers are usually more or less horizontally oriented rather than having pendulous flowers and flexible pedicels.

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.

Summary: Floral meristem termination (FMT) represents one of the defining features of a floral meristem relative to a vegetative meristem. Timing of FMT is a major determinant of the total number of organs in a flower, and canalization toward relatively rapid FMT is considered to have been a major force in shaping angiosperm evolution. For decades, investigation of FMT has been focused on model systems that only produce four whorls of organs in a flower, while little is known about the molecular basis that underlies nature variation in the timing of FMT. Here, we hypothesize on how known pathways could have been modified to generate variation in FMT and explain how developing new model systems will help to deepen our understanding of the genetic control and evolution of FMT. [ABSTRACT FROM AUTHOR]

The columbine genus Aquilegia is a classic example of an adaptive radiation, involving a wide variety of pollinators and habitats. Here we present the genome assembly of A. coerulea 'Goldsmith', complemented by high-coverage sequencing data from 10 wild species covering the world-wide distribution. Our analyses reveal extensive allele sharing among species and demonstrate that introgression and selection played a role in the Aquilegia radiation. We also present the remarkable discovery that the evolutionary history of an entire chromosome differs from that of the rest of the genome - a phenomenon that we do not fully understand, but which highlights the need to consider chromosomes in an evolutionary context.

BACKGROUND:Pathogen perception by plants is mediated by plasma membrane-localized immune receptors that have varied extracellular domains. Lectin receptor kinases (LecRKs) are among these receptors and are subdivided into 3 classes, C-type LecRKs (C-LecRKs), L-type LecRKs (L-LecRKs) and G-type LecRKs (G-LecRKs). While C-LecRKs are represented by one or two members in all plant species investigated and have unknown functions, L-LecRKs have been characterized in a few plant species and have been shown to play roles in plant defense against pathogens. Whereas Arabidopsis G-LecRKs have been characterized, this family of LecRKs has not been studied in tomato. RESULTS:This investigation updates the current characterization of Arabidopsis G-LecRKs and characterizes the tomato G-LecRKs, using LecRKs from the monocot rice and the basal eudicot columbine to establish a basis for comparisons between the two core eudicots. Additionally, revisiting parameters established for Arabidopsis nomenclature for LecRKs is suggested for both Arabidopsis and tomato. Moreover, using phylogenetic analysis, we show the relationship among and between members of G-LecRKs from all three eudicot plant species. Furthermore, investigating presence of motifs in G-LecRKs we identified conserved motifs among members of G-LecRKs in tomato and Arabidopsis, with five present in at least 30 of the 38 Arabidopsis members and in at least 45 of the 73 tomato members. CONCLUSIONS:This work characterized tomato G-LecRKs and added members to the currently characterized Arabidopsis G-LecRKs. Additionally, protein sequence analysis showed an expansion of this family in tomato as compared to Arabidopsis, and the existence of conserved common motifs in the two plant species as well as conserved species-specific motifs.

Traits 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. [ABSTRACT FROM AUTHOR]

Premise: 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. Methods: 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. Results: 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. Conclusions: 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In-depth investigation of any developmental process in plants requires knowledge of both the underpinning molecular networks and how they directly determine patterns of cell division and expansion over time. Floral meristems (FMs) produce floral organs, after which they undergo floral meristem termination (FMT); precise control of organ initiation and FMT is crucial to the reproductive success of any flowering plant. Using live confocal imaging, we characterized developmental dynamics during floral organ primordia initiation and FMT in Aquilegia coerulea (Ranunculaceae). Our results uncover distinct patterns of primordium initiation between stamens and staminodes compared with carpels, and provide insight into the process of FMT, which is discernable based on cell division dynamics that precede carpel initiation. To our knowledge, this is the first quantitative live imaging of meristem development in a system with numerous whorls of floral organs, as well as an apocarpous gynoecium. This study provides crucial information for our understanding of how the spatial-temporal regulation of floral meristem behavior is achieved in both evolutionary and developmental contexts. [ABSTRACT FROM AUTHOR]

Background and Aims Aquilegia produce elongated, three-dimensional petal spurs that fill with nectar to attract pollinators. Previous studies have shown that the diversity of spur length across the Aquilegia genus is a key innovation that is tightly linked with its recent and rapid diversification into new ranges, and that evolution of increased spur lengths is achieved via anisotropic cell elongation. Previous work identified a brassinosteroid response transcription factor as being enriched in the early developing spur cup. Brassinosteroids are known to be important for cell elongation, suggesting that brassinosteroid-mediated response may be an important regulator of spur elongation and potentially a driver of spur length diversity in Aquilegia. In this study, we investigated the role of brassinosteroids in the development of the Aquilegia coerulea petal spur. Methods We exogenously applied the biologically active brassinosteroid brassinolide to developing petal spurs to investigate spur growth under high hormone conditions. We used virus-induced gene silencing and gene expression experiments to understand the function of brassinosteroid-related transcription factors in A. coerulea petal spurs. Key Results We identified a total of three Aquilegia homologues of the BES1/BZR1 protein family and found that these genes are ubiquitously expressed in all floral tissues during development, yet, consistent with the previous RNAseq study, we found that two of these paralogues are enriched in early developing petals. Exogenously applied brassinosteroid increased petal spur length due to increased anisotropic cell elongation as well as cell division. We found that targeting of the AqBEH genes with virus-induced gene silencing resulted in shortened petals, a phenotype caused in part by a loss of cell anisotropy. Conclusions Collectively, our results support a role for brassinosteroids in anisotropic cell expansion in Aquilegia petal spurs and highlight the brassinosteroid pathway as a potential player in the diversification of petal spur length in Aquilegia. [ABSTRACT FROM AUTHOR]

Speciation can be described as a reduction, and the eventual cessation, in the ability to interbreed. Thus, determining how gene flow differs within and between nascent species can illuminate the relative stage the taxa have attained in the speciation process. Aquilegia formosa and A. pubescens are fully intercompatible, yet occur in different habitats and have flowers specialized for pollination by hummingbirds and hawkmoths, respectively. Using 79 SNP loci, we genotyped nearly 1000 individuals from populations of both species in close proximity to each other and from putative hybrid zones. The species shared all but one SNP polymorphism, and on average, allele frequencies differed by only 0.14. However, the species were clearly differentiated using Structure, and admixed individuals were primarily identified at putative hybrid zones. PopGraph identified a highly integrated network among all populations, but populations of each species and hybrid zones occupied distinct regions in the network. Using either conditional graph distance (cGD) or Fst/(1-Fst), we found significant isolation by distance (IBD) among populations. Within species, IBD was strong, indicating high historic gene flow. IBD extended approximately 100 km in A. pubescens and 30 km in A. formosa. However, IBD between the species was very weak and extended only a few km beyond hybrid zones, suggesting little recent gene flow. The extensive sharing of SNP polymorphisms between these species suggests that they are very early in the speciation process while the low signal of IBD suggests that they have largely ceased gene exchange.

Flowers of the lower eudicot Aquilegia (columbine) possess morphological innovations, namely elaborate petal spurs and a fifth distinct organ identity, the staminodium, that are well suited to the investigation of key questions in developmental evolution. The recent evolution of these characteristics combined with a growing set of genetic and genomic resources has provided insight into how the traits arose and diversified. The petal spur appears to represent a key innovation that diversified largely via modification of specific aspects of cell expansion. In the case of the staminodium, gene duplication has played a role in allowing a novel organ identity to be carved out of the traditional ABC program.

Abstract Background Petal 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. Results Petal 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. Conclusions The 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.

Summary: The petal spur of the basal eudicot Aquilegia is a key innovation associated with the adaptive radiation of the genus. Previous studies have shown that diversification of Aquilegia spur length can be predominantly attributed to variation in cell elongation. However, the genetic pathways that control the development of petal spurs are still being investigated.Here, we focus on a pair of closely related homologs of the AUXIN RESPONSE FACTOR family, AqARF6 and AqARF8, to explore their roles in Aquileiga coerulea petal spur development.Expression analyses of the two genes show that they are broadly expressed in vegetative and floral organs, but have relatively higher expression in petal spurs, particularly at later stages. Knockdown of the two AqARF6 and AqARF8 transcripts using virus‐induced gene silencing resulted in largely petal‐specific defects, including a significant reduction in spur length due to a decrease in cell elongation. These spurs also exhibited an absence of nectar production, which was correlated with downregulation of STYLISH homologs that have previously been shown to control nectary development.This study provides the first evidence of ARF6/8 homolog‐mediated petal development outside the core eudicots. The genes appear to be specifically required for cell elongation and nectary maturation in the Aquilegia petal spur. [ABSTRACT FROM AUTHOR]

Summary: Identification and comparison of the conserved and variable downstream genes of floral organ identity regulators are critical to understanding the mechanisms underlying the commonalities and peculiarities of floral organs. Yet, because of the lack of studies in nonmodel species, a general picture of the regulatory evolution between floral organ identity genes and their targets is still lacking.Here, by conducting extensive chromatin immunoprecipitation followed by high‐throughput sequencing (ChIP-seq), electrophoretic mobility shift assay and bioinformatic analyses, we identify and predict the target genes of a petal identity gene, AqAPETALA3‐3 (AqAP3‐3), in Aquilegia coerulea (Ranunculaceae) and compare them with those of its counterpart in Arabidopsis thaliana, AP3.In total, 7049 direct target genes are identified for AqAP3‐3, of which 2394 are highly confident and 1085 are shared with AP3. Gene Ontology enrichment analyses further indicate that conserved targets are largely involved in the formation of identity‐related features, whereas nonconserved targets are mostly required for the formation of species‐specific features.These results not only help understand the molecular bases of the conserved and nonconserved features of petals, but also pave the way to studying the regulatory evolution between floral organ identity genes and their targets. [ABSTRACT FROM AUTHOR]

Background and Aims 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. Methods 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'. Key Results 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. Conclusions 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. [ABSTRACT FROM AUTHOR]

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Abstract The model Aquilegia coerulea x “Origami” possesses several interesting floral features, including petaloid sepals that are morphologically distinct from the true petals and a broad domain containing many whorls of stamens. We undertook the current study in an effort to understand the former trait, but additionally uncovered data that inform on the latter. The Aquilegia B gene homolog AqPI is shown to contribute to the production of anthocyanin in the first whorl sepals, although it has no major role in their morphology. Surprisingly, knockdown of AqPI in Aquilegia coerulea x “Origami” also reveals a role for the B class genes in maintaining the expression of the C gene homolog AqAG1 in the outer whorls of stamens. These findings suggest that the transference of pollinator function to the first whorl sepals included a non-homeotic recruitment of the B class genes to promote aspects of petaloidy. They also confirm results in several other Ranunculales that have revealed an unexpected regulatory connection between the B and C class genes.

Homologs of the transcription factor LEAFY (LFY) and the F-box family member UNUSUAL FLORAL ORGANS (UFO) have been found to promote floral meristem identity across diverse dicot model systems. The lower eudicot model Aquilegia produces cymose inflorescences that are independently evolved from the well-studied cymose models Petunia and tomato. We have previously characterized the expression pattern of the Aquilegia homolog AqLFY but in the current study, we add expression data on the two UFO homologs, AqUFO1 and 2, and conduct virus-induced gene silencing of all the loci. Down-regulation of AqLFY or AqUFO1 and 2 does not eliminate floral meristem identity but, instead, causes the transition from inflorescence to floral identity to become gradual rather than discrete. Inflorescences in down-regulated plants generate several nodes of bract/sepal chimeras and, once floral development does commence, flowers initiate several whorls of sepals before finally producing the wildtype floral whorls. In addition, silencing of AqUFO1/2 appears to specifically impact petal identity and/or the initiation of petal and stamen whorls. In general, however, there is no evidence for an essential role of AqLFY or AqUFO1/2 in transcriptional activation of the B or C gene homologs. These findings highlight differences between deeply divergent dicot lineages in the functional conservation of the floral meristem identity program.

Homologs of the transcription factor LEAFY (LFY) and the F-box family member UNUSUAL FLORAL ORGANS (UFO) have been found to promote floral meristem identity across diverse dicot model systems. The lower eudicot model Aquilegia produces cymose inflorescences that are independently evolved from the well-studied cymose models Petunia and tomato. We have previously characterized the expression pattern of the Aquilegia homolog AqLFY but in the current study, we add expression data on the two UFO homologs, AqUFO1 and 2 , and conduct virus-induced gene silencing of all the loci. Down-regulation of AqLFY or AqUFO1 and 2 does not eliminate floral meristem identity but, instead, causes the transition from inflorescence to floral identity to become gradual rather than discrete. Inflorescences in down-regulated plants generate several nodes of bract/sepal chimeras and, once floral development does commence, flowers initiate several whorls of sepals before finally producing the wildtype floral whorls. In addition, silencing of AqUFO1/2 appears to specifically impact petal identity and/or the initiation of petal and stamen whorls. In general, however, there is no evidence for an essential role of AqLFY or AqUFO1/2 in transcriptional activation of the B or C gene homologs. These findings highlight differences between deeply divergent dicot lineages in the functional conservation of the floral meristem identity program. [ABSTRACT FROM AUTHOR]

For diagnostics and control of emerging plant diseases, accurate species determination of the causal pathogens is a prerequisite. Downy mildew disease, caused by an unknown species of Peronospora, has speedily spread throughout numerous gardens and nurseries of the ornamental plant Aquilegia in the UK, but so far does not seem to have reached continental Europe. Apart from cultivated Aquilegia, downy mildew from wild columbines has only been reported from East Asia, where natural populations of Semiaquilegia are affected by downy mildew. To resolve the phylogenetic relationships of the causal pathogens on Aquilegia and Semiaquilegia, a phylogeny based on nine loci was performed. In addition, detailed morphological comparisons were carried out to determine if the downy mildew agents on Aquilegia and Semiaquilegia are conspecific and can be discriminated from related downy mildew species. Strong evidence was found that the downy mildew pathogens on Aquilegia and Semiaquilegia are conspecific, but distinct from other species of Peronospora affecting Ranunculaceae, Papaveraceae, and Saxifragaceae. Thus, a new species, Peronospora aquilegiicola, is introduced. The quick spread of the pathogen throughout the UK and the current absence from continental Europe highlights the importance to consider quarantine measures to restrict the further spread of this pathogen. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: 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 Aquilegia coerulea.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. See also the Commentary on this article by Moyroud, 221: 614–617. [ABSTRACT FROM AUTHOR]

Aquaporins (AQPs) play a pivotal role in the cellular transport of water and many other small solutes, influencing many physiological and developmental processes in plants. In the present study, extensive bioinformatics analysis of AQPs was performed in Aquilegia coerulea L., a model species belonging to basal eudicots, with a particular focus on understanding the AQPs role in the developing petal nectar spur. A total of 29 AQPs were identified in Aquilegia, and their phylogenetic analysis performed with previously reported AQPs from rice, poplar and Arabidopsis depicted five distinct subfamilies of AQPs. Interestingly, comparative analysis revealed the loss of an uncharacterized intrinsic protein II (XIP-II) group in Aquilegia. The absence of the entire XIP subfamily has been reported in several previous studies, however, the loss of a single clade within the XIP family has not been characterized. Furthermore, protein structure analysis of AQPs was performed to understand pore diversity, which is helpful for the prediction of solute specificity. Similarly, an AQP AqcNIP2-1 was identified in Aquilegia, predicted as a silicon influx transporter based on the presence of features such as the G-S-G-R aromatic arginine selectivity filter, the spacing between asparagine-proline-alanine (NPA) motifs and pore morphology. RNA-seq analysis showed a high expression of tonoplast intrinsic proteins (TIPs) and plasma membrane intrinsic proteins (PIPs) in the developing petal spur. The results presented here will be helpful in understanding the AQP evolution in Aquilegia and their expression regulation, particularly during floral development.

The columbine genus Aquilegia is a classic example of an adaptive radiation, involving a wide variety of pollinators and habitats. Here we present the genome assembly of A. coerulea ‘Goldsmith’, complemented by high-coverage sequencing data from 10 wild species covering the world-wide distribution. Our analyses reveal extensive allele sharing among species and demonstrate that introgression and selection played a role in the Aquilegia radiation. We also present the remarkable discovery that the evolutionary history of an entire chromosome differs from that of the rest of the genome – a phenomenon that we do not fully understand, but which highlights the need to consider chromosomes in an evolutionary context.

Molecular study of the nucleotide sequence of the rpl32-trnL spacer and the ITS1-ITS2 fragment of rRNA genes in eight species of the genus Aquilegia sampled in Northern Asia has shown that populations of A. borodinii and A. jucunda from various localities are genetically diverse. The results confi rm that A. viridifl ora and A. atropurpurea are genetically distant from other species studied. The relationship degrees among A. aradanica, A. sibirica, and A. synakensis have been determined. The hybrid origin of A. jucunda is proven.

The model Aquilegia coerulea x "Origami" possesses several interesting floral features, including petaloid sepals that are morphologically distinct from the true petals and a broad domain containing many whorls of stamens. We undertook the current study in an effort to understand the former trait, but additionally uncovered data that inform on the latter. The Aquilegia B gene homolog AqPI is shown to contribute to the production of anthocyanin in the first whorl sepals, although it has no major role in their morphology. Surprisingly, knockdown of AqPI in Aquilegia coerulea x "Origami" also reveals a role for the B class genes in maintaining the expression of the C gene homolog AqAG1 in the outer whorls of stamens. These findings suggest that the transference of pollinator function to the first whorl sepals included a non-homeotic recruitment of the B class genes to promote aspects of petaloidy. They also confirm results in several other Ranunculales that have revealed an unexpected regulatory connection between the B and C class genes. [ABSTRACT FROM AUTHOR]

Southern European columbines (genus Aquilegia) are involved in active processes of diversification, and the Iberian Peninsula offers a privileged observatory to witness the process. Studies on Iberian columbines have provided significant advances on species diversification, but we still lack a complete perspective of the genetic diversification in the Iberian scenario. This work explores how genetic diversity of the genus Aquilegia is geographically structured across the Iberian Peninsula. We used Bayesian clustering methods, principal coordinates analyses, and NJ phenograms to assess the genetic relationships among 285 individuals from 62 locations and detect the main lineages. Genetic diversity of Iberian columbines consists of five geographically structured lineages, corresponding to different Iberian taxa. Differentiation among lineages shows particularly complex admixture patterns at Northeast and highly homogeneous toward Northwest and Southeast. This geographic genetic structure suggests the existence of incomplete lineage sorting and interspecific hybridization as could be expected in recent processes of diversification under the influence of quaternary postglacial migrations. This scenario is consistent with what is proposed by the most recent studies on European and Iberian columbines, which point to geographic isolation and divergent selection by habitat specialization as the main diversification drivers of the Iberian Aquilegia complex. [ABSTRACT FROM AUTHOR]