Bee-pollination promotes rapid divergent evolution in plants growing in different soils.

Divergent evolution leads to variation among populations and thus promotes diversification. In plants, adaptation to different soils, pollinator guilds, and herbivores is thought to be a key ecological driver of adaptive divergence, but few studies have investigated this process experimentally. Here we use experimental evolution with fast cycling Brassica rapa plants to study the impact of soil, pollination, herbivory, and their interactions on divergent evolution in various traits during eight generations of selection. We found significant evolutionary changes in plant phenotypes caused by all three factors and their interactions. In the richer soil type, plants showed higher evolutionary rates, especially with bumblebee-pollination, which led to the evolution of increased attractiveness of plants to bumblebees. Plants that had experienced aphid-herbivory showed lower attractiveness. We found the strongest evolutionary divergence when plants evolved in different soils with bee-pollination rather than hand-pollination, irrespective of herbivory. This "soil-pollinator effect" impacted divergence in diverse suites of traits, for example leaf size, flowering time, flower petal length, some floral volatiles and leaf glucosinolates. We conclude that the interaction between soil and biotic pollination may be an important cause for divergent evolution of plants growing on different soil types, even without a shift in pollinator guilds.

Rapid genomic evolution in Brassica rapa with bumblebee selection in experimental evolution.

BACKGROUND: Insect pollinators shape rapid phenotypic evolution of traits related to floral attractiveness and plant reproductive success. However, the underlying genomic changes remain largely unknown despite their importance in predicting adaptive responses to natural or to artificial selection. Based on a nine-generation experimental evolution study with fast cycling Brassica rapa plants adapting to bumblebees, we investigate the genomic evolution associated with the previously observed parallel phenotypic evolution. In this current evolve and resequencing (E&R) study, we conduct a genomic scan of the allele frequency changes along the genome in bumblebee-pollinated and hand-pollinated plants and perform a genomic principal component analysis (PCA). RESULTS: We highlight rapid genomic evolution associated with the observed phenotypic evolution mediated by bumblebees. Controlling for genetic drift, we observe significant changes in allelic frequencies at multiple loci. However, this pattern differs according to the replicate of bumblebee-pollinated plants, suggesting putative non-parallel genomic evolution. Finally, our study underlines an increase in genomic variance implying the putative involvement of multiple loci in short-term pollinator adaptation. CONCLUSIONS: Overall, our study enhances our understanding of the complex interactions between pollinator and plants, providing a stepping stone towards unravelling the genetic basis of plant genomic adaptation to biotic factors in the environment.

Putative Signals of Generalist Plant Species Adaptation to Local Pollinator Communities and Abiotic Factors.

The reproductive success of flowering plants with generalized pollination systems is influenced by interactions with a diverse pollinator community and abiotic factors. However, knowledge about the adaptative potential of plants to complex ecological networks and the underlying genetic mechanisms is still limited. Based on a pool-sequencing approach of 21 natural populations of Brassica incana in Southern Italy, we combined a genome-environmental association analysis with a genome scan for signals of population genomic differentiation to discover genetic variants associated with the ecological variation. We identified genomic regions putatively involved in the adaptation of B. incana to the identity of local pollinator functional categories and pollinator community composition. Interestingly, we observed several shared candidate genes associated with long-tongue bees, soil texture, and temperature variation. We established a genomic map of potential generalist flowering plant local adaptation to complex biotic interactions, and the importance of considering multiple environmental factors to describe the adaptive landscape of plant populations.

Plant phenotypic plasticity changes pollinator-mediated selection.

Many organisms change their phenotype in response to the environment, a phenomenon called phenotypic plasticity. Although plasticity can dramatically change the phenotype of an organism, we hardly understand how this can affect biotic interactions and the resulting phenotypic selection. Here, we use fast cycling Brassica rapa plants in an experiment in the greenhouse to study the link between plasticity and selection. We detected strong plasticity in morphology, nectar, and floral scent in response to different soil types and aphid herbivory. We found positive selection on nectar and morphological traits in hand- and bumblebee-pollinated plants. Bumblebee-mediated selection on a principal component representing plant height, flower number, and flowering time (mPC3) differed depending on soil type and herbivory. For plants growing in richer soil, selection was stronger in the absence of herbivores, whereas for plants growing in poorer soil selection was stronger with herbivory. We showed that bumblebees visited tall plants with many flowers overproportionally in plants in poor soil with herbivory (i.e., when tall plants were rare), thus causing stronger positive selection on this trait combination. We suggest that with strong plasticity under most stressful conditions, pollinator-mediated selection may promote adaptation to local environmental factors given sufficient heritability of the traits under selection.

Hyperbolic odorant mixtures as a basis for more efficient signaling between flowering plants and bees.

Animals use odors in many natural contexts, for example, for finding mates or food, or signaling danger. Most analyses of natural odors search for either the most meaningful components of a natural odor mixture, or they use linear metrics to analyze the mixture compositions. However, we have recently shown that the physical space for complex mixtures is 'hyperbolic', meaning that there are certain combinations of variables that have a disproportionately large impact on perception and that these variables have specific interpretations in terms of metabolic processes taking place inside the flower and fruit that produce the odors. Here we show that the statistics of odorants and odorant mixtures produced by inflorescences (Brassica rapa) are also better described with a hyperbolic rather than a linear metric, and that combinations of odorants in the hyperbolic space are better predictors of the nectar and pollen resources sought by bee pollinators than the standard Euclidian combinations. We also show that honey bee and bumble bee antennae can detect most components of the B. rapa odor space that we tested, and the strength of responses correlates with positions of odorants in the hyperbolic space. In sum, a hyperbolic representation can be used to guide investigation of how information is represented at different levels of processing in the CNS.

Do floral and ecogeographic isolation allow the co-occurrence of two ecotypes of Anacamptis papilionacea (Orchidaceae)?

Ecotypes are relatively frequent in flowering plants and considered central in ecological speciation as local adaptation can promote the insurgence of reproductive isolation. Without geographic isolation, gene flow usually homogenizes the allopatrically generated phenotypic and ecological divergences, unless other forms of reproductive isolation keep them separated. Here, we investigated two orchid ecotypes with marked phenotypic floral divergence that coexist in contact zones. We found that the two ecotypes show different ecological habitat preferences with one being more climatically restricted than the other. The ecotypes remain clearly morphologically differentiated both in allopatry and in sympatry and differed in diverse floral traits. Despite only slightly different flowering times, the two ecotypes achieved floral isolation thanks to different pollination strategies. We found that both ecotypes attract a wide range of insects, but the ratio of male/female attracted by the two ecotypes was significantly different, with one ecotype mainly attracts male pollinators, while the other mainly attracts female pollinators. As a potential consequence, the two ecotypes show different pollen transfer efficiency. Experimental plots with pollen staining showed a higher proportion of intra- than interecotype movements confirming floral isolation between ecotypes in sympatry while crossing experiments excluded evident postmating barriers. Even if not completely halting the interecotypes pollen flow in sympatry, such incipient switch in pollination strategy between ecotypes may represent a first step on the path toward evolution of sexual mimicry in Orchidinae.

Combining biotechnology and evolution for understanding the mechanisms of pollinator attraction.

Many flowering plants rely on pollinators for their reproductive success. Plant-pollinator interactions usually depend on a complex combination of traits based on a fine-tuned biosynthetic machinery, with many structural and regulatory genes involved. Yet, the physiological mechanisms in plants are the product of evolutionary processes. While evolution has been modifying flowers through millions of years, it is also a rapid process that can change plant traits within few generations. Here we discuss both mechanistic and evolutionary aspects of pollinator attraction. We also propose how latest advances in biotechnology and evolutionary studies, and their combination, will improve the elucidation of molecular mechanisms and evolutionary dynamics of pollinator attraction in changing environments.

Herbivory and pollination impact on the evolution of herbivore-induced plasticity in defense and floral traits.

Theory predicts that herbivory should primarily determine the evolution of herbivore-induced plasticity in plant defenses, but little is known about the influence of other interactions such as pollination. Pollinators may exert negative selection on the herbivore-induced plasticity of chemical defenses when floral signals and rewards are indirectly affected, provoking deterrent effects on these mutualists. We investigated the influence of constant herbivory and pollination on the evolved patterns and degree of herbivore-induced plasticity in chemical plant defenses and floral morphometry and volatiles in fast-cycling Brassica rapa plants. To do this, we used plants from an evolution experiment that had evolved under bee/hand pollination and herbivory manipulated in a 2 × 2 factorial design during six generations, producing four selection treatments. We grew sibling plant pairs from each of the four selection treatments of the last generation and infested one group with herbivores and left the other uninfested. Herbivore-induced plasticity was analyzed within- and between-selection treatments. We found support for the hypothesis that constant herbivory favors the evolution of higher constitutive yet lower herbivore-induced plasticity in defenses. However, this only occurred in plants that evolved under hand pollination and constant herbivory. Bee pollination had a strong influence on the evolution of herbivore-induced plasticity of all traits studied. Plants that evolved under bee pollination, with and without constant herbivory, showed remarkably similar patterns of herbivore-induced plasticity in their defense- and floral traits and had a higher number of plastic responses compared to plants with hand pollination. Such patterns support the hypothesis that bee pollination influenced the evolution of herbivore-induced plasticity, most likely via indirect effects, such as links between defense- and floral traits. We conclude that interactions other than herbivory, such as pollination, may impact herbivore-induced plasticity, through indirect effects and metabolic trade-offs, when it contributes to trait evolution in plants.

Floral signals evolve in a predictable way under artificial and pollinator selection in Brassica rapa.

BACKGROUND: Angiosperms employ an astonishing variety of visual and olfactory floral signals that are generally thought to evolve under natural selection. Those morphological and chemical traits can form highly correlated sets of traits. It is not always clear which of these are used by pollinators as primary targets of selection and which would be indirectly selected by being linked to those primary targets. Quantitative genetics tools for predicting multiple traits response to selection have been developed since long and have advanced our understanding of evolution of genetically correlated traits in various biological systems. We use these tools to predict the evolutionary trajectories of floral traits and understand the selection pressures acting on them. RESULTS: We used data from an artificial selection and a pollinator (bumblebee, hoverfly) evolution experiment with fast cycling Brassica rapa plants to predict evolutionary changes of 12 floral volatiles and 4 morphological floral traits in response to selection. Using the observed selection gradients and the genetic variance-covariance matrix (G-matrix) of the traits, we showed that the observed responses of most floral traits including volatiles were predicted in the right direction in both artificial- and bumblebee-selection experiment. Genetic covariance had a mix of constraining and facilitating effects on evolutionary responses. We further revealed that G-matrices also evolved in the selection processes. CONCLUSIONS: Overall, our integrative study shows that floral signals, especially volatiles, evolve under selection in a mostly predictable way, at least during short term evolution. Evolutionary constraints stemming from genetic covariance affected traits evolutionary trajectories and thus it is important to include genetic covariance for predicting the evolutionary changes of a comprehensive suite of traits. Other processes such as resource limitation and selfing also need to be considered for a better understanding of floral trait evolution.

The role of volatiles in plant communication.

Volatiles mediate the interaction of plants with pollinators, herbivores and their natural enemies, other plants and micro-organisms. With increasing knowledge about these interactions the underlying mechanisms turn out to be increasingly complex. The mechanisms of biosynthesis and perception of volatiles are slowly being uncovered. The increasing scientific knowledge can be used to design and apply volatile-based agricultural strategies.

Rapid plant evolution driven by the interaction of pollination and herbivory.

Pollination and herbivory are both key drivers of plant diversity but are traditionally studied in isolation from each other. We investigated real-time evolutionary changes in plant traits over six generations by using fast-cycling Brassica rapa plants and manipulating the presence and absence of bumble bee pollinators and leaf herbivores. We found that plants under selection by bee pollinators evolved increased floral attractiveness, but this process was compromised by the presence of herbivores. Plants under selection from both bee pollinators and herbivores evolved higher degrees of self-compatibility and autonomous selfing, as well as reduced spatial separation of sexual organs (herkogamy). Overall, the evolution of most traits was affected by the interaction of bee pollination and herbivory, emphasizing the importance of the cross-talk between both types of interactions for plant evolution.

Emergence of a floral colour polymorphism by pollinator-mediated overdominance.

Maintenance of polymorphism by overdominance (heterozygote advantage) is a fundamental concept in evolutionary biology. In most examples known in nature, overdominance is a result of homozygotes suffering from deleterious effects. Here we show that overdominance maintains a non-deleterious polymorphism with black, red and white floral morphs in the Alpine orchid Gymnadenia rhellicani. Phenotypic, metabolomic and transcriptomic analyses reveal that the morphs differ solely in cyanidin pigments, which are linked to differential expression of an anthocyanidin synthase (ANS) gene. This expression difference is caused by a premature stop codon in an ANS-regulating R2R3-MYB transcription factor, which is heterozygous in the red colour morph. Furthermore, field observations show that bee and fly pollinators have opposite colour preferences; this results in higher fitness (seed set) of the heterozygous morph without deleterious effects in either homozygous morph. Together, these findings demonstrate that genuine overdominance exists in nature.

Real-time evolution supports a unique trajectory for generalized pollination.

Whereas specialized pollination is well recognized to cause floral adaptation, we know little about the evolutionary impact of generalized pollination. For example, it is largely unknown whether such pollination can lead to adaptive floral divergence and to what degree pollinators with different effectiveness determine evolutionary trajectories. Here, we investigated the evolutionary consequences of combined bumblebee- and hoverfly-pollination ("generalized" pollination) in comparison with those of each individual pollinator species (specialized pollination), using fast-cycling Brassica rapa plants during seven generations of experimental evolution. Bumblebees were twice as efficient as hoverflies in pollinating B. rapa flowers, but phenotypic selection and evolutionary change in plants with generalized pollination was different from both bumblebee- and hoverfly-pollinated plants for several traits. After seven generations evolution, plants with generalized pollination resembled bumblebee-pollinated plants in having little spontaneous selfing and tall size, but were more similar to hoverfly-pollinated plants in having low floral scent emission. This unique trait combination supports the idea of a generalized-pollination ecotype, coined neither by the most efficient pollinator, nor by an evolutionary average between the changes caused by each individual pollinator. For a better understanding of such "nonadditive evolution," future research should target interactions of pollinators and their effect on phenotypic selection.

Crab spiders impact floral-signal evolution indirectly through removal of florivores.

The puzzling diversity of flowers is primarily shaped by selection and evolutionary change caused by the plant's interaction with animals. The contribution of individual animal species to net selection, however, may vary depending on the network of interacting organisms. Here we document that in the buckler mustard, Biscutella laevigata, the crab spider Thomisus onustus reduces bee visits to flowers but also benefits plants by feeding on florivores. Uninfested plants experience a trade-off between pollinator and spider attraction as both bees and crab spiders are attracted by the floral volatile ß-ocimene. This trade-off is reduced by the induced emission of ß-ocimene after florivore infestation, which is stronger in plant populations where crab spiders are present than where they are absent, suggesting that plants are locally adapted to the presence of crab spiders. Our study demonstrates the context-dependence of selection and shows how crab spiders impact on floral evolution.

Trans-generational inheritance of herbivory-induced phenotypic changes in Brassica rapa.

Biotic stress can induce plastic changes in fitness-relevant plant traits. Recently, it has been shown that such changes can be transmitted to subsequent generations. However, the occurrence and extent of transmission across different types of traits is still unexplored. Here, we assessed the emergence and transmission of herbivory-induced changes in Brassica rapa and their impact on interactions with insects. We analysed changes in morphology and reproductive traits as well as in flower and leaf volatile emission during two generations with leaf herbivory by Mamestra brassicae and Pieris brassicae and two subsequent generations without herbivory. Herbivory induced changes in all trait types, increasing attractiveness of the plants to the parasitoid wasp Cotesia glomerata and decreasing visitation by the pollinator Bombus terrestris, a potential trade-off. While changes in floral and leaf volatiles disappeared in the first generation after herbivory, some changes in morphology and reproductive traits were still measurable two generations after herbivory. However, neither parasitoids nor pollinators further discriminated between groups with different past treatments. Our results suggest that transmission of herbivore-induced changes occurs preferentially in resource-limited traits connected to plant growth and reproduction. The lack of alterations in plant-insect interactions was likely due to the transient nature of volatile changes.

Covariation and phenotypic integration in chemical communication displays: biosynthetic constraints and eco-evolutionary implications.

Chemical communication is ubiquitous. The identification of conserved structural elements in visual and acoustic communication is well established, but comparable information on chemical communication displays (CCDs) is lacking. We assessed the phenotypic integration of CCDs in a meta-analysis to characterize patterns of covariation in CCDs and identified functional or biosynthetically constrained modules. Poorly integrated plant CCDs (i.e. low covariation between scent compounds) support the notion that plants often utilize one or few key compounds to repel antagonists or to attract pollinators and enemies of herbivores. Animal CCDs (mostly insect pheromones) were usually more integrated than those of plants (i.e. stronger covariation), suggesting that animals communicate via fixed proportions among compounds. Both plant and animal CCDs were composed of modules, which are groups of strongly covarying compounds. Biosynthetic similarity of compounds revealed biosynthetic constraints in the covariation patterns of plant CCDs. We provide a novel perspective on chemical communication and a basis for future investigations on structural properties of CCDs. This will facilitate identifying modules and biosynthetic constraints that may affect the outcome of selection and thus provide a predictive framework for evolutionary trajectories of CCDs in plants and animals.

Floral scent and species divergence in a pair of sexually deceptive orchids.

Speciation is typically accompanied by the formation of isolation barriers between lineages. Commonly, reproductive barriers are separated into pre- and post-zygotic mechanisms that can evolve with different speed. In this study, we measured the strength of different reproductive barriers in two closely related, sympatric orchids of the Ophrys insectifera group, namely Ophrys insectifera and Ophrys aymoninii to infer possible mechanisms of speciation. We quantified pre- and post-pollination barriers through observation of pollen flow, by performing artificial inter- and intraspecific crosses and analyzing scent bouquets. Additionally, we investigated differences in mycorrhizal fungi as a potential extrinsic factor of post-zygotic isolation. Our results show that floral isolation mediated by the attraction of different pollinators acts apparently as the sole reproductive barrier between the two orchid species, with later-acting intrinsic barriers seemingly absent. Also, the two orchids share most of their fungal mycorrhizal partners in sympatry, suggesting little or no importance of mycorrhizal symbiosis in reproductive isolation. Key traits underlying floral isolation were two alkenes and wax ester, present predominantly in the floral scent of O. aymoninii. These compounds, when applied to flowers of O. insectifera, triggered attraction and a copulation attempt of the bee pollinator of O. aymoninii and thus led to the (partial) breakdown of floral isolation. Based on our results, we suggest that adaptation to different pollinators, mediated by floral scent, underlies species isolation in this plant group. Pollinator switches may be promoted by low pollination success of individuals in dense patches of plants, an assumption that we also confirmed in our study.

Real-time divergent evolution in plants driven by pollinators.

Pollinator-driven diversification is thought to be a major source of floral variation in plants. Our knowledge of this process is, however, limited to indirect assessments of evolutionary changes. Here, we employ experimental evolution with fast cycling Brassica rapa plants to demonstrate adaptive evolution driven by different pollinators. Our study shows pollinator-driven divergent selection as well as divergent evolution in plant traits. Plants pollinated by bumblebees evolved taller size and more fragrant flowers with increased ultraviolet reflection. Bumblebees preferred bumblebee-pollinated plants over hoverfly-pollinated plants at the end of the experiment, showing that plants had adapted to the bumblebees' preferences. Plants with hoverfly pollination became shorter, had reduced emission of some floral volatiles, but increased fitness through augmented autonomous self-pollination. Our study demonstrates that changes in pollinator communities can have rapid consequences on the evolution of plant traits and mating system.