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.

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.

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.

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.

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: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 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.

*For correspondence: scott.hodges@lifesci.ucsb.edu (SH); magnus.nordborg@gmi.oeaw.ac.at (MN) 1Gregor Mendel Institute, Austrian Academy of Sciences, Vienna BioCenter (VBC), Vienna, 11 Austria; 2Department of Ecology, Evolution, and Marine Biology, University of California 12 Santa Barbara, Santa Barbara, California, USA; 3Central-European Institute of Technology 13 (CEITEC), Masaryk University, Brno, Czech Republic; 4Vienna Graduate School of 14 Population Genetics, Vienna, Austria; 5Department of Energy Joint Genome Institute, 15 Walnut Creek, California, USA; 6HudsonAlpha Institute of Biotechnology, Huntsville, 16 Alabama, USA; 7Institute of Experimental Botany, Centre of the Region Haná for 17 Biotechnological and Agricultural Research, Olomouc, Czech Republic; 8Department of 18 Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts, USA 19

The Louisiana Irises serve as an important system for the study of the evolutionary processes of speciation, including reproductive isolation, hybridization, and adaptation. Sequencing methods today allow for the generation of resources key to elucidating the genetic basis of these phenomena. Here we describe the transcriptomes of floral and young leaf tissue from Iris fulva generated by massively parallel pyrosequencing. In order to identify potential candidates for the study of reproductive isolation and adaptation in the Louisiana Irises we phylogenetically analyzed the type II MIKC(C) MADS-box and R2R3 MYB transcription factors expressed in these tissues. A total of 25 Iris MIKC(C) MADS-box genes in 9 clades and 42 Iris R2R3 MYB genes in 19 clades were identified. Through the identification of eudicot and monocot specific clades, these analyses contribute to our understanding of the evolution of these prominent transcription factor families in the angiosperms.

The formation of hybrids among closely related species has been observed in numerous plant taxa. Selection by pollinators on floral traits can act as an early reproductive isolating barrier and may be especially important when there is overlap in distribution and flowering time. In this study, we use Quantitative Trait Locus (QTL) mapping based on 293 codominant SNP markers in an F2 population (n = 328) to assess the size, magnitude, and location of the genetic regions controlling floral traits known to be important for pollinator attraction in 2 species of Lousiana Irises, Iris fulva and Iris hexagona. We also evaluate correlations among F2 traits and identify transgression in the hybrid population. Overall, we observe that differences in most floral traits between I. fulva and I. hexagona are controlled by multiple QTLs and are distributed across several linkage groups. We also find evidence of transgression at several QTL, suggesting that hybridization can contribute to generating phenotypic variation, which may be adaptive in rapidly changing environments.

Natural hybridization is a widespread phenomenon, detected among clades of fungi, plants, and animals. The potential outcomes of genetic exchange include adaptive trait evolution, speciation, and even the radiation of entire species complexes. Not only does natural hybridization have the potential to affect evolutionary processes, it often does so very rapidly. Indeed, adaptations can be transferred in only a handful of hybrid generations. Likewise, entire species assemblages may arise from a hybrid swarm over the space of a few thousand years. This chapter tests these conclusions with examples from a diverse array of fungal, botanical, and zoological examples. Each example illustrates the potential for natural hybridization to provide the genetic and phenotypic variation necessary for rapid, adaptive, diversifying evolution.

Background: Genomic analyses are being applied to 'non-model' species. These complexes are now 'models' for applying genomic approaches to test evolutionary hypotheses. These approaches have revealed the genetic underpinnings of natural selection and adaptation.Aims: We wish to illustrate that the newest techniques in genomics would not be possible without older technological and methodological advances. We also point to the necessity for detailed phenotypic information to reveal the genomics of natural selection and adaptation.Methods: We review studies starting with Watson and Crick through the definition of the polymerase chain reaction to illustrate how newer technologies rest on the platform of older discoveries. We review studies from two plant clades that illustrate various genomic approaches taken to test for natural selection and adaptive evolution.Results: We find that older and newer genomic methodological approaches are necessary to dissect the genetics of natural selection and adaptation.Conclusions: The basis for the newest genomic technologies continues to rest on much older methodological advances. It is not possible to understand the genomic components affecting natural selection and adaptation without phenotypic and genomic data. When such data are combined, they reflect a powerful approach for understanding the mode and tempo of selection and adaptation.

Estimates of hybrid fitness have been used as either a platform for testing the potential role of natural hybridization in the evolution of species and species complexes or, alternatively, as a rationale for dismissing hybridization events as being of any evolutionary significance. From the time of Darwin's publication of The Origin, through the neo-Darwinian synthesis, to the present day, the observation of variability in hybrid fitness has remained a challenge for some models of speciation. Yet, Darwin and others have reported the elevated fitness of hybrid genotypes under certain environmental conditions. In modern scientific terminology, this observation reflects the fact that hybrid genotypes can demonstrate genotype × environment interactions. In the current review, we illustrate the development of one plant species complex, namely the Louisiana Irises, into a 'model system' for investigating hybrid fitness and the role of genetic exchange in adaptive evolution and diversification. In particular, we will argue that a multitude of approaches, involving both experimental and natural environments, and incorporating both manipulative analyses and surveys of natural populations, are necessary to adequately test for the evolutionary significance of introgressive hybridization. An appreciation of the variability of hybrid fitness leads to the conclusion that certain genetic signatures reflect adaptive evolution. Furthermore, tests of the frequency of allopatric versus sympatric/parapatric divergence (that is, divergence with ongoing gene flow) support hybrid genotypes as a mechanism of evolutionary diversification in numerous species complexes.

Background Hybridization among Louisiana Irises has been well established and the genetic architecture of reproductive isolation is known to affect the potential for and the directionality of introgression between taxa. Here we use co-dominant markers to identify regions where QTL are located both within and between backcross maps to compare the genetic architecture of reproductive isolation and fitness traits across treatments and years. Results QTL mapping was used to elucidate the genetic architecture of reproductive isolation between Iris fulva and Iris brevicaulis . Homologous co-dominant EST-SSR markers scored in two backcross populations between I. fulva and I. brevicaulis were used to generate genetic linkage maps. These were used as the framework for mapping QTL associated with variation in 11 phenotypic traits likely responsible for reproductive isolation and fitness. QTL were dispersed throughout the genome, with the exception of one region of a single linkage group (LG) where QTL for flowering time, sterility, and fruit production clustered. In most cases, homologous QTL were not identified in both backcross populations, however, homologous QTL for flowering time, number of growth points per rhizome, number of nodes per inflorescence, and number of flowers per node were identified on several linkage groups. Conclusions Two different traits affecting reproductive isolation, flowering time and sterility, exhibit different genetic architectures, with numerous QTL across the Iris genome controlling flowering time and fewer, less distributed QTL affecting sterility. QTL for traits affecting fitness are largely distributed across the genome with occasional overlap, especially on LG 4, where several QTL increasing fitness and decreasing sterility cluster. Given the distribution and effect direction of QTL affecting reproductive isolation and fitness, we have predicted genomic regions where introgression may be more likely to occur (those regions associated with an increase in fitness and unlinked to loci controlling reproductive isolation) and those that are less likely to exhibit introgression (those regions linked to traits decreasing fitness and reproductive isolation).

In order to maximize reproductive success, plants have evolved different strategies to control the critical developmental shift marked by the transition to flowering. As plants have adapted to diverse environments across the globe, these strategies have evolved to recognize and respond to local seasonal cues through the induction of specific downstream genetic pathways, thereby ensuring that the floral transition occurs in favorable conditions. Determining the genetic factors involved in controlling the floral transition in many species is key to understanding how this trait has evolved. Striking genetic discoveries in Arabidopsis thaliana (Arabidopsis) and Oryza sativa (rice) revealed that similar genes in both species control flowering in response to photoperiod, suggesting that this genetic module could be conserved between distantly related angiosperms. However, as we have gained a better understanding of the complex evolution of these genes and their functions in other species, another possibility must be considered: that the genetic module controlling flowering in response to photoperiod is the result of convergence rather than conservation. In this review, we show that while data clearly support a central role of FLOWERING LOCUS T (FT) homologs in floral promotion across a diverse group of angiosperms, there is little evidence for a conserved role of CONSTANS (CO) homologs in the regulation of these loci. In addition, although there is an element of conserved function for FT homologs, even this component has surprising complexity in its regulation and evolution.

Background Flowering is a critical transition in plant development, the timing of which can have considerable fitness consequences. Until recently, research into the genetic control of flowering time and its associated developmental changes was focused on core eudicots (for example, Arabidopsis) or monocots (for example, Oryza ). Here we examine the flowering response of Aquilegia formosa , a member of the eudicot order Ranunculales that is emerging as an important model for the investigation of plant ecology and evolution. Results We have determined that A. formosa has a strong vernalization requirement but little or no photoperiod response, making it a day neutral (DN) plant. Consistent with this, the Aquilegia homolog of FLOWERING LOCUS T ( AqFT ) is expressed in both long and short days but surprisingly, the locus is expressed before the transition to flowering. In situ hybridizations with homologs of several Arabidopsis Floral Pathway Integrators (FPIs) do not suggest conserved functions relative to Arabidopsis, the potential exceptions being AqLFY and AqAGL24.2 . Conclusions In Aquilegia , vernalization is critical to flowering but this signal is not strictly required for the transcriptional activation of AqFT . The expression patterns of AqLFY and AqAGL24.2 suggest a hypothesis for the development of Aquilegia 's determinate inflorescence whereby their differential expression controls the progression of each meristem from inflorescence to floral identity. Interestingly, none of the Aquilegia expression patterns are consistent with a function in floral repression which, combined with the lack of a FLC homolog, means that new candidate genes must be identified for the control of vernalization response in Aquilegia .