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.

Developmental and Molecular Changes Underlying the Vernalization-Induced Transition to Flowering in Aquilegia coerulea (James).

Reproductive success in plants is dependent on many factors but the precise timing of flowering is certainly among the most crucial. Perennial plants often have a vernalization or over-wintering requirement in order to successfully flower in the spring. The shoot apical meristem undergoes drastic developmental and molecular changes as it transitions into inflorescence meristem (IM) identity, which then gives rise to floral meristems (FMs). In this study, we have examined the developmental and gene expression changes underlying the transition from the vegetative to reproductive phases in the basal eudicot Aquilegia coerulea, which has evolved a vernalization response independently relative to other established model systems. Results from both our histology and scanning electron studies demonstrate that developmental changes in the meristem occur gradually during the third and fourth weeks of vernalization. Based on RNAseq data and cluster analysis, several known flowering time loci, including AqFT and AqFL1, exhibit dramatic changes in expression during the fourth week. Further consideration of candidate gene homologs as well as unexpected loci of interest creates a framework in which we can begin to explore the genetic basis of the flowering time transition in Aquilegia.

Sub- and neo-functionalization of APETALA3 paralogs have contributed to the evolution of novel floral organ identity in Aquilegia (columbine, Ranunculaceae).

Previous studies of the lower eudicot model Aquilegia have revealed differential expression patterns of two APETALA3 (AP3) paralogs that appear to coincide with the development of a distinct fifth floral organ type, the staminodium. The AqAP3-1 locus quickly becomes limited to the staminodia while AqAP3-2 becomes stamen-specific. We used transient RNAi-based methods to silence each of these loci individually and in combination, followed by detailed studies of the resultant morphologies and the effects on gene expression patterns. Silencing of AqAP3-1 had a strong effect on the staminodia, causing transformation into carpeloid organs, while silencing of AqAP3-2 only affected the stamens, resulting in sterility, stunting or weak transformation towards carpel identity. Much more dramatic phenotypes were obtained in the doubly silenced flowers, where all stamens and staminodia were transformed into carpels. Quantitative reverse-transcription polymerase chain reaction analyses of B gene homolog expression in these flowers are consistent with complex patterns of regulatory feedback among the loci. These findings suggest that the presence of ancient AP3 paralogs in the Ranunculaceae has facilitated the recent evolution of a novel organ identity program in Aquilegia. Specifically, it appears that downregulation of AqAP3-2 in the innermost whorl of stamens was a critical step in the evolution of elaborated sterile organs in this position.

Glandular trichomes as an inflorescence defence mechanism against insect herbivores in Iberian columbines.

Glandular trichomes play a defensive role against herbivores in the leaves of many plant species. However, their functional role in inflorescences has not been studied, even though theory suggests that tissues with a higher fitness value, such as inflorescences, should be better defended. Using manipulative experiments, we analysed the defensive role of glandular trichomes against herbivorous insects in the inflorescence of Iberian columbines (genus Aquilegia), and its inter-population and inter-taxa variation in relation to herbivore abundance and potential selective pressure. The experiments were conducted in eight populations belonging to four subspecies of two columbines (Aquilegia vulgaris and Aquilegia pyrenaica). For each population, we estimated the density of glandular trichomes in the inflorescences, the abundance of insects stuck in the inflorescences, the abundance of small herbivorous insects, the incidence of damage on flowers and fruits, and the fruit set. The density of glandular trichomes on the inflorescence of A. vulgaris and A. pyrenaica was higher in regions of higher herbivore abundance. We also found that when the plants lose the protection of glandular trichomes, small insects have better access to flowers and fruits, causing more damage and reducing plant fitness. This study concludes that glandular trichomes are part of an adaptive response against phytophagous insect herbivory. The observed variation in herbivore pressure between taxa, likely caused by habitat differentiation, might have played a role in trait differentiation through divergent selection. This result adds evidence to the differentiation of the Iberian columbines through habitat specialization.

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.

Petal-specific subfunctionalization of an APETALA3 paralog in the Ranunculales and its implications for petal evolution.

• The petals of the lower eudicot family Ranunculaceae are thought to have been derived many times independently from stamens. However, investigation of the genetic basis of their identity has suggested an alternative hypothesis: that they share a commonly inherited petal identity program. This theory is based on the fact that an ancient paralogous lineage of APETALA3 (AP3) in the Ranunculaceae appears to have a conserved, petal-specific expression pattern. • Here, we have used a combination of approaches, including RNAi, comparative gene expression and molecular evolutionary studies, to understand the function of this petal-specific AP3 lineage. • Functional analysis of the Aquilegia locus AqAP3-3 has demonstrated that the paralog is required for petal identity with little contribution to the identity of the other floral organs. Expanded expression studies and analyses of molecular evolutionary patterns provide further evidence that orthologs of AqAP3-3 are primarily expressed in petals and are under higher purifying selection across the family than the other AP3 paralogs. • Taken together, these findings suggest that the AqAP3-3 lineage underwent progressive subfunctionalization within the order Ranunculales, ultimately yielding a specific role in petal identity that has probably been conserved, in stark contrast with the multiple independent origins predicted by botanical theories.

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.

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.

Elaboration of B gene function to include the identity of novel floral organs in the lower eudicot Aquilegia.

The basal eudicot Aquilegia (columbine) has an unusual floral structure that includes two morphologically distinct whorls of petaloid organs and a clearly differentiated fifth organ type, the staminodium. In this study, we have sought to determine how Aquilegia homologs of the B class genes APETALA3 (AP3) and PISTILLATA (PI) contribute to these novel forms of organ identity. Detailed expression analyses of the three AP3 paralogs and one PI homolog in wild-type and floral homeotic mutant lines reveal complex patterns that suggest that canonical B class function has been elaborated in Aquilegia. Yeast two-hybrid studies demonstrate that the protein products of Aquilegia's AP3 and PI homologs can form heterodimers, much like what has been observed for their core eudicot homologs. Downregulation of AqvPI using virus-induced gene silencing indicates that in addition to petal and stamen identity, this locus is essential to staminodial identity but may not control the identity of the petaloid sepals. Our findings show that preexisting floral organ identity programs can be partitioned and modified to produce additional organ types. In addition, they indicate that some types of petaloid organs are not entirely dependent on AP3/PI homologs for their identity.

Impact of insect pollinator group and floral display size on outcrossing rate.

Despite the strong influence of pollination ecology on the evolution of selfing, we have little information on how distinct groups of insect pollinators influence outcrossing rate. However, differences in behavior between pollinator groups could easily influence how each group affects outcrossing rate. We examined the influence of distinct insect pollinator groups on outcrossing rate in the rocky mountain columbine, Aquilegia coerulea. The impact of population size, plant density, size of floral display, and herkogamy (spatial separation between anthers and stigmas) on outcrossing rate was also considered as these variables were previously found to affect outcrossing rate in some plant species. We quantified correlations between all independent variables and used simple and two-factor regressions to determine direct and indirect impact of each independent variable on outcrossing rate. Outcrossing rate increased significantly with hawkmoth abundance but not with the abundance of any of the other groups of floral visitors, which included bumblebees, solitary bees, syrphid flies, and muscidae. Outcrossing rate was also significantly affected by floral display size and together, hawkmoth abundance and floral display size explained 87% of the variation in outcrossing rate. None of the other independent variables directly affected the outcrossing rate. This is the first report of a significant impact of pollinator type on outcrossing rate. Hawkmoths did not visit fewer flowers per plant relative to other pollinator groups but preferred visiting female-phase flowers first on a plant. Both the behavior of pollinators and floral display size affected outcrossing rate via their impact on the level of geitonogamous (among flower) selfing. Given that geitonogamous selfing is never advantageous, the variation in outcrossing rate and maintenance of mixed mating systems in populations of A. coerulea may not require an adaptive explanation.

Flexible stigma presentation assists context-dependent pollination in a wild columbine.

Stigma presentation has received much less attention than pollen presentation. Investigation of the various forms of stigma presentation may reveal previously undescribed reproductive mechanisms in plants. Here we investigated stigma presentation in the spring-flowering herb Aquilegia yabeana, whose linear stigma gradually develops over the lifetime of the flower. The degree of stigma development and recurvature are influenced by the timing of pollen deposition, apparently to favor cross-pollination. In this species, autogamous self-pollination was found to occur in the middle of the receptive period of the stigma if prior cross-pollination did not occur. However, because later-arriving cross-pollen was deposited closer to the ovary, it reached the base of the style before self-pollen which had been deposited earlier. Flexible stigma presentation helps the flower to maximize pollen resources under unreliable pollination conditions, taking advantage of both selfing and outcrossing. This is the first description of stigma development regulated by pollen deposition in a wild plant.

Detecting the historical signature of key innovations using stochastic models of character evolution and cladogenesis.

Phylogenetic evidence for biological traits that increase the net diversification rate of lineages (key innovations) is most commonly drawn from comparisons of clade size. This can work well for ancient, unreversed traits and for correlating multiple trait origins with higher diversification rates, but it is less suitable for unique events, recently evolved innovations, and traits that exhibit homoplasy. Here I present a new method for detecting the phylogenetic signature of key innovations that tests whether the evolutionary history of the candidate trait is associated with shorter waiting times between cladogenesis events. The method employs stochastic models of character evolution and cladogenesis and integrates well into a Bayesian framework in which uncertainty in historical inferences (such as phylogenetic relationships) is allowed. Applied to a well-known example in plants, nectar spurs in columbines, the method gives much stronger support to the key innovation hypothesis than previous tests.

Limitations on reproductive success in endemic Aquilegia viscosa (Ranunculaceae) relative to its widespread congener Aquilegia vulgaris: the interplay of herbivory and pollination.

Plant reproduction can be strongly affected by herbivory and different features of pollination ecology, such as pollinator visitation rates and capacity for self-pollination. The purpose of this study is to compare the relative impact of herbivory and pollination on maternal reproductive success in endemic Aquilegia viscosa and its widespread congener Aquilegia vulgaris. We conducted herbivore exclusion experiments in two populations of each species in 2 different years and showed that the maternal fertility of A. viscosa was significantly more limited by floral predation and pre-dispersal seed predation than its widespread congener. In the absence of herbivory, A. viscosa retained significantly lower maternal fertility than A. vulgaris. Experimental pollinations in an insect-free glasshouse showed that the two species have an equal seed/ovule ratio both in the absence of pollinators and in the presence of non-limiting outcross pollination. Pollinator visitation rates were significantly higher in populations of A. vulgaris than in populations of A. viscosa. In addition, path analyses showed that spur length, an important trait for pollinator attraction in Aquilegia, and, indirectly sepal and petal width, contribute positively to the seed/ovule ratio in A. vulgaris, but not in A. viscosa. These results indicate that maternal fertility of endemic A. viscosa is strongly reduced by flower and seed predation despite low rates of pollinator visitation, and that pollen or resource limitation in the wild may further reduce maternal fertility. Finally, floral trait variation appears to be decoupled from fertility variation in endemic A. viscosa, which possibly constrains the evolution of reproductive traits in this species.