Loss of staminodes in Aquilegia jonesii reveals a fading stamen-staminode boundary.

The modification of fertile stamens into sterile staminodes has occurred independently many times in the flowering plant lineage. In the genus Aquilegia (columbine) and its closest relatives, the two stamen whorls closest to the carpels have been converted to staminodes. In Aquilegia, the only genetic analyses of staminode development have been reverse genetic approaches revealing that B-class floral identity genes are involved. A. jonesii, the only species of columbine where staminodes have reverted to fertile stamens, allows us to explore the genetic architecture of staminode development using a forward genetic approach. We performed QTL analysis using an outcrossed F2 population between A. jonesii and a horticultural variety that makes fully developed staminodes, A. coerulea 'Origami'. Our results reveal a polygenic basis for staminode loss where the two staminode whorls are under some level of independent control. We also discovered that staminode loss in A. jonesii is not complete, in which staminode-like traits sometimes occur in the inner fertile stamens, potentially representing a fading boundary of gene expression. The QTLs identified in this study provide a map to guide future reverse genetic and functional studies examining the genetic basis and evolutionary significance of this trait.

Genetic architecture underlying variation in floral meristem termination in Aquilegia.

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.

Non-pollinator selection for a floral homeotic mutant conferring loss of nectar reward in Aquilegia coerulea.

Here, we describe a polymorphic population of Aquilegia coerulea with a naturally occurring floral homeotic mutant, A. coerulea var. daileyae, where the characteristic petals with nectar spurs are replaced with a second set of sepals. Although it would be expected that this loss of pollinator reward would be disadvantageous to the mutant, we find that it has reached relatively high frequency (∼25%) and is under strong, positive selection across multiple seasons (s = 0.17-0.3) primarily due to reduced floral herbivory. We identify the underlying locus (APETALA3-3) and multiple causal loss-of-function mutations indicating an ongoing soft sweep. Elevated linkage disequilibrium around the two most common causal alleles indicates that positive selection has been occurring for many generations. Lastly, genotypic frequencies at AqAP3-3 indicate a degree of positive assortative mating by morphology. Together, these data provide both a compelling example that large-scale discontinuous morphological changes differentiating taxa can occur due to single mutations and a particularly clear example of linking genotype, phenotype, and fitness.

Genetic architecture of floral traits in bee- and hummingbird-pollinated sister species of Aquilegia (columbine).

Interactions with animal pollinators have helped shape the stunning diversity of flower morphologies across the angiosperms. A common evolutionary consequence of these interactions is that some flowers have converged on suites of traits, or pollination syndromes, that attract and reward specific pollinator groups. Determining the genetic basis of these floral pollination syndromes can help us understand the processes that contributed to the diversification of the angiosperms. Here, we characterize the genetic architecture of a bee-to-hummingbird pollination shift in Aquilegia (columbine) using QTL mapping of 17 floral traits encompassing color, nectar composition, and organ morphology. In this system, we find that the genetic architectures underlying differences in floral color are quite complex, and we identify several likely candidate genes involved in anthocyanin and carotenoid floral pigmentation. Most morphological and nectar traits also have complex genetic underpinnings; however, one of the key floral morphological phenotypes, nectar spur curvature, is shaped by a single locus of large effect.

POPOVICH, encoding a C2H2 zinc-finger transcription factor, plays a central role in the development of a key innovation, floral nectar spurs, in Aquilegia.

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.

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

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.

The Aquilegia genome provides insight into adaptive radiation and reveals an extraordinarily polymorphic chromosome with a unique history.

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.

Genetic variation among mainland and island populations of a native perennial grass used in restoration.

Genetic marker studies can assist restoration practice through selection of seed sources that conserve historical levels of gene diversity and population genetic differentiation. We examined genetic variation and structure within and among mainland and island populations of Elymus glaucus, a perennial bunchgrass species native to western North American grasslands that is targeted for grassland restoration. Island populations of E. glaucus represent sensitive sites and potentially distinctive seed sources for reintroduction, and little is known of their genetic composition. Genetic diversity and structure were estimated using amplified fragment length polymorphism markers for 21 populations and 416 individuals distributed across two coastal California mainland locations and three California Channel Islands. Eight primer combinations resulted in 166 markers, of which 165 (99.4 %) were polymorphic. The number of polymorphic bands was significantly greater among mainland populations relative to island sites, and locally common alleles were present for each sampled island and mainland location. Population structure was high (62.9 %), with most variation (55.8 %) distributed among populations, 7.1 % between mainland and island locations, and the remainder (37.1 %) within populations. Isolation by distance was only apparent among islands. Using marker data to recommend appropriate seed sources for restoration, E. glaucus seeds are best derived within islands with collections representing a large number of individuals from matching environments. Given the limited gene flow and prior evidence of adaptive divergence among populations of this species, regional collections are recommended in all cases to maintain diversity and to avoid long-distance introductions of highly differentiated plant material.

Understanding the development and evolution of novel floral form in Aquilegia.

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.

Disruption of the petal identity gene APETALA3-3 is highly correlated with loss of petals within the buttercup family (Ranunculaceae).

Absence of petals, or being apetalous, is usually one of the most important features that characterizes a group of flowering plants at high taxonomic ranks (i.e., family and above). The apetalous condition, however, appears to be the result of parallel or convergent evolution with unknown genetic causes. Here we show that within the buttercup family (Ranunculaceae), apetalous genera in at least seven different lineages were all derived from petalous ancestors, indicative of parallel petal losses. We also show that independent petal losses within this family were strongly associated with decreased or eliminated expression of a single floral organ identity gene, APETALA3-3 (AP3-3), apparently owing to species-specific molecular lesions. In an apetalous mutant of Nigella, insertion of a transposable element into the second intron has led to silencing of the gene and transformation of petals into sepals. In several naturally occurring apetalous genera, such as Thalictrum, Beesia, and Enemion, the gene has either been lost altogether or disrupted by deletions in coding or regulatory regions. In Clematis, a large genus in which petalous species evolved secondarily from apetalous ones, the gene exhibits hallmarks of a pseudogene. These results suggest that, as a petal identity gene, AP3-3 has been silenced or down-regulated by different mechanisms in different evolutionary lineages. This also suggests that petal identity did not evolve many times independently across the Ranunculaceae but was lost in numerous instances. The genetic mechanisms underlying the independent petal losses, however, may be complex, with disruption of AP3-3 being either cause or effect.

Spatiotemporal reconstruction of the Aquilegia rapid radiation through next-generation sequencing of rapidly evolving cpDNA regions.

Aquilegia is a well-known model system in the field of evolutionary biology, but obtaining a resolved and well-supported phylogenetic reconstruction for the genus has been hindered by its recent and rapid diversification. Here, we applied 454 next-generation sequencing to PCR amplicons of 21 of the most rapidly evolving regions of the plastome to generate c. 24 kb of sequences from each of 84 individuals from throughout the genus. The resulting phylogeny has well-supported resolution of the main lineages of the genus, although recent diversification such as in the European taxa remains unresolved. By producing a chronogram of the whole Ranunculaceae family based on published data, we inferred calibration points for dating the Aquilegia radiation. The genus originated in the upper Miocene c. 6.9 million yr ago (Ma) in Eastern Asia, and diversification occurred c. 4.8 Ma with the split of two main clades, one colonizing North America, and the other Western Eurasia through the mountains of Central Asia. This was followed by a back-to-Asia migration, originating from the European stock using a North Asian route. These results provide the first backbone phylogeny and spatiotemporal reconstruction of the Aquilegia radiation, and constitute a robust framework to address the adaptative nature of speciation within the group.

Evolution of spur-length diversity in Aquilegia petals is achieved solely through cell-shape anisotropy.

The role of petal spurs and specialized pollinator interactions has been studied since Darwin. Aquilegia petal spurs exhibit striking size and shape diversity, correlated with specialized pollinators ranging from bees to hawkmoths in a textbook example of adaptive radiation. Despite the evolutionary significance of spur length, remarkably little is known about Aquilegia spur morphogenesis and its evolution. Using experimental measurements, both at tissue and cellular levels, combined with numerical modelling, we have investigated the relative roles of cell divisions and cell shape in determining the morphology of the Aquilegia petal spur. Contrary to decades-old hypotheses implicating a discrete meristematic zone as the driver of spur growth, we find that Aquilegia petal spurs develop via anisotropic cell expansion. Furthermore, changes in cell anisotropy account for 99 per cent of the spur-length variation in the genus, suggesting that the true evolutionary innovation underlying the rapid radiation of Aquilegia was the mechanism of tuning cell shape.

Genomic tools development for Aquilegia: construction of a BAC-based physical map.

BACKGROUND: The genus Aquilegia, consisting of approximately 70 taxa, is a member of the basal eudicot lineage, Ranuculales, which is evolutionarily intermediate between monocots and core eudicots, and represents a relatively unstudied clade in the angiosperm phylogenetic tree that bridges the gap between these two major plant groups. Aquilegia species are closely related and their distribution covers highly diverse habitats. These provide rich resources to better understand the genetic basis of adaptation to different pollinators and habitats that in turn leads to rapid speciation. To gain insights into the genome structure and facilitate gene identification, comparative genomics and whole-genome shotgun sequencing assembly, BAC-based genomics resources are of crucial importance. RESULTS: BAC-based genomic resources, including two BAC libraries, a physical map with anchored markers and BAC end sequences, were established from A. formosa. The physical map was composed of a total of 50,155 BAC clones in 832 contigs and 3939 singletons, covering 21X genome equivalents. These contigs spanned a physical length of 689.8 Mb (~2.3X of the genome) suggesting the complex heterozygosity of the genome. A set of 197 markers was developed from ESTs induced by drought-stress, or involved in anthocyanin biosynthesis or floral development, and was integrated into the physical map. Among these were 87 genetically mapped markers that anchored 54 contigs, spanning 76.4 Mb (25.5%) across the genome. Analysis of a selection of 12,086 BAC end sequences (BESs) from the minimal tiling path (MTP) allowed a preview of the Aquilegia genome organization, including identification of transposable elements, simple sequence repeats and gene content. Common repetitive elements previously reported in both monocots and core eudicots were identified in Aquilegia suggesting the value of this genome in connecting the two major plant clades. Comparison with sequenced plant genomes indicated a higher similarity to grapevine (Vitis vinifera) than to rice and Arabidopsis in the transcriptomes. CONCLUSIONS: The A. formosa BAC-based genomic resources provide valuable tools to study Aquilegia genome. Further integration of other existing genomics resources, such as ESTs, into the physical map should enable better understanding of the molecular mechanisms underlying adaptive radiation and elaboration of floral morphology.

Within and between whorls: comparative transcriptional profiling of Aquilegia and Arabidopsis.

BACKGROUND: The genus Aquilegia is an emerging model system in plant evolutionary biology predominantly because of its wide variation in floral traits and associated floral ecology. The anatomy of the Aquilegia flower is also very distinct. There are two whorls of petaloid organs, the outer whorl of sepals and the second whorl of petals that form nectar spurs, as well as a recently evolved fifth whorl of staminodia inserted between stamens and carpels. METHODOLOGY/PRINCIPAL FINDINGS: We designed an oligonucleotide microarray based on EST sequences from a mixed tissue, normalized cDNA library of an A. formosa x A. pubescens F2 population representing 17,246 unigenes. We then used this array to analyze floral gene expression in late pre-anthesis stage floral organs from a natural A. formosa population. In particular, we tested for gene expression patterns specific to each floral whorl and to combinations of whorls that correspond to traditional and modified ABC model groupings. Similar analyses were performed on gene expression data of Arabidopsis thaliana whorls previously obtained using the Ath1 gene chips (data available through The Arabidopsis Information Resource). CONCLUSIONS/SIGNIFICANCE: Our comparative gene expression analyses suggest that 1) petaloid sepals and petals of A. formosa share gene expression patterns more than either have organ-specific patterns, 2) petals of A. formosa and A. thaliana may be independently derived, 3) staminodia express B and C genes similar to stamens but the staminodium genetic program has also converged on aspects of the carpel program and 4) staminodia have unique up-regulation of regulatory genes and genes that have been implicated with defense against microbial infection and herbivory. Our study also highlights the value of comparative gene expression profiling and the Aquilegia microarray in particular for the study of floral evolution and ecology.

Genetic variation at nuclear loci fails to distinguish two morphologically distinct species of Aquilegia.

Aquilegia formosa and pubescens are two closely related species belonging to the columbine genus. Despite their morphological and ecological differences, previous studies have revealed a large degree of intercompatibility, as well as little sequence divergence between these two taxa. We compared the inter- and intraspecific patterns of variation for 9 nuclear loci, and found that the two species were practically indistinguishable at the level of DNA sequence polymorphism, indicating either very recent speciation or continued gene flow. As a comparison, we also analyzed variation at two loci across 30 other Aquilegia taxa; this revealed slightly more differentiation among taxa, which seemed best explained by geographic distance. By contrast, we found no evidence for isolation by distance on a more local geographic scale. We conclude that the extremely low levels of genetic differentiation between A. formosa and A. pubescens at neutral loci will facilitate future genome-wide scans for speciation genes.

Aquilegia as a model system for the evolution and ecology of petals.

The ranunculid genus Aquilegia holds extraordinary promise as a model system for investigating a wide range of questions relating to the evolution and ecology of petals. New genetic and genomic resources, including an extensive EST database, BAC libraries and physical maps, as well as virus-induced gene silencing are facilitating this research on multiple fronts. At the developmental genetic level, Aquilegia has been important for elucidating the developmental programme for specifying petals and petaloid characteristics. Data suggest that duplication events among the petal and stamen identity genes have resulted in sub- and neofunctionalization. This expansion of gene function does not include the petaloidy of Aquilegia sepals, however, which does not depend on the same loci that control identity of the second whorl petals. Of special interest is the elaboration of the petal into a nectar spur, a major innovation for the genus. Intra- and interspecific variation in the shape and colour of petals, especially the spurs, has been shown to be adaptative for different pollinators. Thus, understanding the genetic basis of these traits will help us connect the ecological interactions driving speciation with the genetic changes responsible for remodelling morphology. Progress in this area has focused on the multiple, parallel transitions in flower colour and nectar spur length across the genus. For flower colour, upstream transcription factors appear to be primarily targets of natural selection. Thus research in Aquilegia spans the initial evolution of petals and petaloidy to the diversification of petal morphology to the ecological basis of petal form, thereby providing a comprehensive picture of the evolutionary biology of this critical angiosperm feature.

Adaptive radiations: from field to genomic studies.

Adaptive radiations were central to Darwin's formation of his theory of natural selection, and today they are still the centerpiece for many studies of adaptation and speciation. Here, we review the advantages of adaptive radiations, especially recent ones, for detecting evolutionary trends and the genetic dissection of adaptive traits. We focus on Aquilegia as a primary example of these advantages and highlight progress in understanding the genetic basis of flower color. Phylogenetic analysis of Aquilegia indicates that flower color transitions proceed by changes in the types of anthocyanin pigments produced or their complete loss. Biochemical, crossing, and gene expression studies have provided a wealth of information about the genetic basis of these transitions in Aquilegia. To obtain both enzymatic and regulatory candidate genes for the entire flavonoid pathway, which produces anthocyanins, we used a combination of sequence searches of the Aquilegia Gene Index, phylogenetic analyses, and the isolation of novel sequences by using degenerate PCR and RACE. In total we identified 34 genes that are likely involved in the flavonoid pathway. A number of these genes appear to be single copy in Aquilegia and thus variation in their expression may have been key for floral color evolution. Future studies will be able to use these sequences along with next-generation sequencing technologies to follow expression and sequence variation at the population level. The genetic dissection of other adaptive traits in Aquilegia should also be possible soon as genomic resources such as whole-genome sequencing become available.

Are we there yet? Tracking the development of new model systems.

It is increasingly clear that additional 'model' systems are needed to elucidate the genetic and developmental basis of organismal diversity. Whereas model system development previously required enormous investment, recent advances including the decreasing cost of DNA sequencing and the power of reverse genetics to study gene function are greatly facilitating the process. In this review, we consider two aspects of the development of new genetic model systems: first, the types of questions being advanced using these new models; and second, the essential characteristics and molecular tools for new models, depending on the research focus. We hope that researchers will be inspired to explore this array of emerging models and even consider developing new molecular tools for their own favorite organism.

Pollinator shifts drive increasingly long nectar spurs in columbine flowers.

Directional evolutionary trends have long garnered interest because they suggest that evolution can be predictable. However, the identification of the trends themselves and the underlying processes that may produce them have often been controversial. In 1862, in explaining the exceptionally long nectar spur of Angraecum sesquipedale, Darwin proposed that a coevolutionary 'race' had driven the directional increase in length of a plant's spur and its pollinator's tongue. Thus he predicted the existence of an exceptionally long-tongued moth. Though the discovery of Xanthopan morgani ssp. praedicta in 1903 with a tongue length of 22 cm validated Darwin's prediction, his 'race' model for the evolution of long-spurred flowers remains contentious. Spurs may also evolve to exceptional lengths by way of pollinator shifts as plants adapt to a series of unrelated pollinators, each with a greater tongue length. Here, using a species-level phylogeny of the columbine genus, Aquilegia, we show a significant evolutionary trend for increasing spur length during directional shifts to pollinators with longer tongues. In addition, we find evidence for 'punctuated' change in spur length during speciation events, suggesting that Aquilegia nectar spurs rapidly evolve to fit adaptive peaks predefined by pollinator morphology. These findings show that evolution may proceed in predictable pathways without reversals and that change may be concentrated during speciation.