The function of the floral corona in the pollination of a Mediterranean style dimorphic daffodil.

Narcissus papyraceus is a style dimorphic species with two floral forms, with anthers at similar height and stigmas above (long-styled L) and below (short-styled S) the anther level. The species is self-incompatible, but intra- and inter-morph compatible. Populations are either dimorphic (including both morphs) in the region of the Strait of Gibraltar, or L-monomorphic (with only L plants) in the inland of the Iberian Peninsula. This variation correlates with the most common floral visitors, being primarily long-tongued and short-tongued pollinators, respectively, a rare condition in Mediterranean plants. The maintenance of S-flowers relies on long-tongued insects, as only those deliver pollen to short-styled stigmas. Narcissus flowers present a long and narrow tube, at the bottom of which nectar accumulates, and a floral corona, which has been proposed as an important trait for the attraction of pollinators. Here we tested the importance of the corona on pollination of L and S flowers. We described UV reflectance patterns of the corona and tepals, and characterised VOCs in intact flowers and flowers with trimmed coronas. We also conducted a field experiment in the dimorphic and monomorphic region to estimate the importance of corona removal on seed production in stands with solitary plants and in groups to control for compatible pollen limitation. Reflectance was higher in the tepals than the corona, although both traits presented a reflectance peak around 450 nm wavelength. L- and S-flowers produced similar volatiles, regardless of the manipulation of the corona. Across dimorphic and monomorphic regions, S-flowers with the corona removed suffered a reduction in seed production of ca. 50%, while seed production remained similar in L flowers both with the corona intact and removed. Plants in solitary stands suffered a strong reduction in seed production, which was more pronounced in the monomorphic region. Our results suggest that the corona in Narcissus is more important for the pollination of S-flowers, which generally have lower seed production compared to L-flowers. Taken together, these results suggest that the floral corona indirectly plays an important role for maintenance of the polymorphism.

Pollen transfer efficiency and its effect on inflorescence size in deceptive pollination strategies.

Pollination systems differ in pollen transfer efficiency, a variable that may influence the evolution of flower number. Here we apply a comparative approach to examine the link between pollen transfer efficiency and the evolution of inflorescence size in food and sexually deceptive orchids. We examined pollination performance in nine food-deceptive, and eight sexually deceptive orchids by recording pollen removal and deposition in the field. We calculated correlations between reproductive success and flower number (as a proxy for resources allocated during reproductive process), and directional selection differentials were estimated on flower number for four species. Results indicate that sexually deceptive species experience decreased pollen loss compared to food-deceptive species. Despite producing fewer flowers, sexually deceptive species attained levels of overall pollination success (through male and female function) similar to food-deceptive species. Furthermore, a positive correlation between flower number and pollination success was observed in food-deceptive species, but this correlation was not detected in sexually deceptive species. Directional selection differentials for flower number were significantly higher in food compared to sexually deceptive species. We suggest that pollination systems with more efficient pollen transfer and no correlation between pollination success and number of flowers produced, such as sexual deception, may allow the production of inflorescences with fewer flowers that permit the plant to allocate fewer resources to floral displays and, at the same time, limit transpiration. This strategy can be particularly important for ecological success in Mediterranean water-deprived habitats, and might explain the high frequency of sexually deceptive species in these specialised ecosystems.

Floral isolation is the major reproductive barrier between a pair of rewarding orchid sister species.

The crucial role of reproductive isolation in speciation has long been recognized; however, a limited number of studies quantify different isolation barriers and embed reproductive isolation in a phylogenetic context. In this study, we investigate reproductive isolation between the often sympatrically occurring orchid species, Gymnadenia conopsea and G. odoratissima. We examine the phylogenetic relationship between the two species and analyse floral isolation, fruit set and seed viability from interspecies crosses, as well as the ploidy level. Additionally, we quantify interspecies differences in floral signals and morphology. The results suggest that the two species have a sister-species relationship. In terms of reproductive isolation, we found complete floral isolation between the two species, but little to no post-pollination isolation; the species also mostly had the same ploidy level in the studied populations. We also show clear distinctions in floral signals, as well as in floral size and spur length. We propose that respective adaptation to short- vs. long-tongued pollinators was the driver of speciation in the here studied Gymnadenia species. Our study supports the key role of floral isolation in orchid speciation and shows that floral isolation is not restricted to highly specialized pollination systems, but can also occur between species with less specialized pollination.

Bees use honest floral signals as indicators of reward when visiting flowers.

Pollinators visit flowers for rewards and should therefore have a preference for floral signals that indicate reward status, so called 'honest signals'. We investigated honest signalling in Brassica rapa L. and its relevance for the attraction of a generalised pollinator, the bumble bee Bombus terrestris (L.). We found a positive association between reward amount (nectar sugar and pollen) and the floral scent compound phenylacetaldehyde. Bumble bees developed a preference for phenylacetaldehyde over other scent compounds after foraging on B. rapa. When foraging on artificial flowers scented with synthetic volatiles, bumble bees developed a preference for those specific compounds that honestly indicated reward status. These results show that the honesty of floral signals can play a key role in their attractiveness to pollinators. In plants, a genetic constraint, resource limitation in reward and signal production, and sanctions against cheaters may contribute to the evolution and maintenance of honest signalling.

Pollinator shifts between Ophrys sphegodes populations: might adaptation to different pollinators drive population divergence?

Local adaptation to different pollinators is considered one of the possible initial stages of ecological speciation as reproductive isolation is a by-product of the divergence in pollination systems. However, pollinator-mediated divergent selection will not necessarily result in complete reproductive isolation, because incipient speciation is often overcome by gene flow. We investigated the potential of pollinator shift in the sexually deceptive orchids Ophrys sphegodes and Ophrys exaltata and compared the levels of floral isolation vs. genetic distance among populations with contrasting predominant pollinators. We analysed floral hydrocarbons as a proxy for floral divergence between populations. Floral adoption of pollinators and their fidelity was tested using pollinator choice experiments. Interpopulation gene flow and population differentiation levels were estimated using AFLP markers. The Tyrrhenian O. sphegodes population preferentially attracted the pollinator bee Andrena bimaculata, whereas the Adriatic O. sphegodes population exclusively attracted A. nigroaenea. Significant differences in scent component proportions were identified in O. sphegodes populations that attracted different preferred pollinators. High interpopulation gene flow was detected, but populations were genetically structured at species level. The high interpopulation gene flow levels independent of preferred pollinators suggest that local adaptation to different pollinators has not (yet) generated detectable genome-wide separation. Alternatively, despite extensive gene flow, few genes underlying floral isolation remain differentiated as a consequence of divergent selection. Different pollination ecotypes in O. sphegodes might represent a local selective response imposed by temporal variation in a geographical mosaic of pollinators as a consequence of the frequent disturbance regimes typical of Ophrys habitats.

Production of plant growth modulating volatiles is widespread among rhizosphere bacteria and strongly depends on culture conditions.

Recent studies have suggested that bacterial volatiles play an important role in bacterial-plant interactions. However, few reports of bacterial species that produce plant growth modulating volatiles have been published, raising the question whether this is just an anecdotal phenomenon. To address this question, we performed a large screen of strains originating from the soil for volatile-mediated effects on Arabidopsis thaliana. All of the 42 strains tested showed significant volatile-mediated plant growth modulation, with effects ranging from plant death to a sixfold increase in plant biomass. The effects of bacterial volatiles were highly dependent on the cultivation medium and the inoculum quantity. GC-MS analysis of the tested strains revealed over 130 bacterial volatile compounds. Indole, 1-hexanol and pentadecane were selected for further studies because they appeared to promote plant growth. None of these compounds triggered a typical defence response, using production of ethylene and of reactive oxygen species (ROS) as read-outs. However, when plants were challenged with the flg-22 epitope of bacterial flagellin, a prototypical elicitor of defence responses, additional exposure to the volatiles reduced the flg-22-induced production of ethylene and ROS in a dose-dependent manner, suggesting that bacterial volatiles may act as effectors to inhibit the plant's defence response.

The discovery of 2,5-dialkylcyclohexan-1,3-diones as a new class of natural products.

Orchids employing sexual deceit attract males of their pollinator species through specific volatile signals that mimic female-released sex pheromones. One of these signals proved to be 2-ethyl-5-propylcyclohexan-1,3-dione (chiloglottone1), a new natural product that was shown to be most important in the relations between orchids of the genus Chiloglottis, native to Australia, and corresponding pollinator species. Systematic investigations on the mass spectrometric fragmentation pattern of 2,5-dialkylcyclohexan-1,3-diones identified key ions providing information about the structures of the substituents at positions 2 and 5. Results enabled us to identify 2-ethyl-5-pentylcyclohexan-1,3-dione (chiloglottone2) and 2-butyl-5-methylcyclohexan-1,3-dione (chiloglottone3) as new natural products that play a decisive role in the pollination syndrome of some Chiloglottis species. During field bioassays, pure synthetic samples of chiloglottone1-3 or mixtures thereof proved to be attractive to the corresponding orchid pollinators. Because of their likely biogenesis from ubiquitous fatty acid precursors, 2,5-dialkylcyclohexan-1,3-diones may represent a hitherto overlooked, widespread class of natural products.

Pollinator convergence and the nature of species' boundaries in sympatric Sardinian Ophrys (Orchidaceae).

BACKGROUND AND AIMS: In the sexually deceptive Ophrys genus, species isolation is generally considered ethological and occurs via different, specific pollinators, but there are cases in which Ophrys species can share a common pollinator and differ in pollen placement on the body of the insect. In that condition, species are expected to be reproductively isolated through a pre-mating mechanical barrier. Here, the relative contribution of pre- vs. post-mating barriers to gene flow among two Ophrys species that share a common pollinator and can occur in sympatry is studied. METHODS: A natural hybrid zone on Sardinia between O. iricolor and O. incubacea, sharing Andrena morio as pollinator, was investigated by analysing floral traits involved in pollinator attraction as odour extracts both for non-active and active compounds and for labellum morphology. The genetic architecture of the hybrid zone was also estimated with amplified fragment length polymorphism (AFLP) markers, and pollination fitness and seed set of both parental species and their hybrids in the sympatric zone were estimated by controlled crosses. KEY RESULTS: Although hybrids were intermediate between parental species in labellum morphology and non-active odour compounds, both parental species and hybrids produced a similar odour bouquet for active compounds. However, hybrids produced significantly lower fruit and seed set than parental species, and the genetic architecture of the hybrid zone suggests that they were mostly first-generation hybrids. CONCLUSIONS: The two parental species hybridize in sympatry as a consequence of pollinator overlap and weak mechanical isolation, but post-zygotic barriers reduce hybrid frequency and fitness, and prevent extensive introgression. These results highlight a significant contribution of late post-mating barriers, such as chromosomal divergence, for maintaining reproductive isolation, in an orchid group for which pre-mating barriers are often considered predominant.

Floral odour and reproductive isolation in two species of Silene.

Mechanisms preventing interspecific pollination are important in closely related plant species, in particular when post-zygotic barriers are weak or absent. We investigated the role of floral odour in reproductive isolation between the two closely related species Silene latifolia and S. dioica. First, we tested whether floral odour composition and emission differed between the species. We found significant odour differences, but contrary to expectations, both species showed a rhythmic emission of the same compounds between day and night. Second, in a field experiment, odour of the two species was made more similar by applying phenylacetaldehyde to flowers. This manipulation led to higher pollen-analogue transfer between species, revealing that floral odour differences are important for maintaining reproductive isolation. We conclude that differences in single key compounds can reduce pollen transfer across species boundaries by pollinators and demonstrate that odour differences are an important component of premating floral isolation between closely related plant species.

Floral scent in food-deceptive orchids: species specificity and sources of variability.

One third of all orchid species are deceptive and do not reward their pollinators. Such deceptive orchids are often characterised by unusually high variation in floral signals such as colour and scent. In this study, we investigated the scent composition of two Mediterranean food-deceptive orchids Orchis mascula, Orchis pauciflora, and their hybrid, O. x colemanii. Scent was collected IN SITU by headspace sorption and was subsequently analysed with gas chromatography and gas chromatography-mass spectrometry. We compared variation of odour compounds within and between populations as well as species. We identified 35 floral scent compounds, mainly monoterpenes, which were shared by both species. Both quantitative and qualitative variability within and among populations was high. Many individuals within species could be classified to different "odour-types". In spite of high qualitative and quantitative intra- and inter-population variability, the species were clearly differentiated in their scent bouquets, whereas most hybrid individuals emitted an intermediate scent.

Floral evolution and pollinator mate choice in a sexually deceptive orchid.

Sexually deceptive orchids mimic sex pheromones and appearance of female insects to attract males, which pollinate the flowers in an attempted mating. This study examines the effects of pollinator mate choice on orchid floral evolution using the Thynnine wasp Neozeleboria cryptoides (Smith) (Hymenoptera: Tiphiidae), which pollinates the sexually deceptive orchid Chiloglottis trapeziformis Fitzg. (i) When male wasps were given the choice between two female dummies of different sizes and identical amount of synthetic pheromone, they preferentially attempted to copulate with medium-sized dummies over small dummies. (ii) When given the choice between two dummies of identical size but different amounts of pheromone, males preferred the larger amount of pheromone. Larger amounts of pheromone generally attracted more males than smaller amounts. (iii) Orchid flower labella, which mimic a female body, were significantly longer and broader than female wasp bodies, and the flowers also produced on average 10 times more 'pheromone' than females. The evolution and maintenance of these exaggerated mating signals is likely to be mediated by the male pollinator behaviour demonstrated here. (iv) When five dummies were offered simultaneously in a 10 cm circular array, males rarely attempted copulation on more than one dummy during a single visit. This behaviour may foster the evolution or maintenance of clonality in C. trapeziformis, as it will minimize pollen exchange within clones.

Odor compound detection in male euglossine bees.

Male euglossine bees collect fragrances from various sources, which they store and use for as yet unknown purposes. They are attracted, often specifically, to single odor compounds and blends thereof. We used gas chromatography with electroantennographic detection (GC-EAD) and electroantennography (EAG) to investigate the response to 8 odor compounds by males of two euglossine species, Euglossa cybelia Moure and Eulaema polychroma (Mocsàry). In E. cybelia, we recorded EAD reactions in response to 1,8-cineole, methyl benzoate, benzyl actetate, methyl salicylate, eugenol, and methyl cinnamate. E. polychroma responded to the same compounds in EAG experiments, while (1s)(-)alpha-pinene and beta-pinene failed to trigger EAD or EAG responses in the bees. Blends of two compounds triggered larger responses than single compounds in EAG experiments with E. polychroma, however, when alpha-pinene was added, reactions decreased. In the light of existing data on the bees' behavior towards these odor compounds, our work indicates that both peripheral and central nervous processes influence the attraction of euglossine bees to odors.

Sex pheromone mimicry in the early spider orchid (ophrys sphegodes): patterns of hydrocarbons as the key mechanism for pollination by sexual deception.

We investigated the female-produced sex pheromone of the solitary bee Andrena nigroaenea and compared it with floral scent of the sexually deceptive orchid Ophrys sphegodes which is pollinated by Andrena nigroaenea males. We identified physiologically and behaviorally active compounds by gas chromatography with electroantennographic detection, gas chromatography-mass spectrometry, and behavioral tests in the field. Dummies scented with cuticle extracts of virgin females or of O. sphegodes labellum extracts elicited significantly more male reactions than odorless dummies. Therefore, copulation behavior eliciting semiochemicals are located on the surface of the females' cuticle and the surface of the flowers. Within bee and orchid samples, n-alkanes and n-alkenes, aldehydes, esters, all-trans-farnesol and all-trans-farnesyl hexanoate triggered electroantennographic responses in male antennae. Most of the alkanes and alkenes occurred in similar patterns both in the bees and orchids. O. sphegodes leaf extracts contained mostly the same compounds but in different proportions. In behavioral tests with synthetic compounds, blends of alkenes triggered significantly more approaches and pounces of the males whereas alkanes were not more attractive than odorless dummies. Since alkanes and alkenes together were most attractive, we conclude they constitute the bees' sex pheromone as well as the pseudocopulation-behavior releasing orchid-odor bouquet.

Evolution of reproductive strategies in the sexually deceptive orchid Ophrys sphegodes: how does flower-specific variation of odor signals influence reproductive success?

The orchid Ophrys sphegodes Miller is pollinated by sexually excited males of the solitary bee Andrena nigroaenea, which are lured to the flowers by visual cues and volatile semiochemicals. In O. sphegodes, visits by pollinators are rare. Because of this low frequency of pollination, one would expect the evolution of strategies that increase the chance that males will visit more than one flower on the same plant; this would increase the number of pollination events on a plant and therefore the number of seeds produced. Using gas chromatography-mass spectrometry (GC-MS) analyses, we identified more than 100 compounds in the odor bouquets of labellum extracts from O. sphegodes; 24 compounds were found to be biologically active in male olfactory receptors based on gas chromatography with electroantennographic detection (GC-EAD). Gas chromatography (GC) analyses of odors from individual flowers showed less intraspecific variation in the odor bouquets of the biologically active compounds as compared to nonactive compounds. This can be explained by a higher selective pressure on the pollinator-attracting communication signal. Furthermore, we found a characteristic variation in the GC-EAD active esters and aldehydes among flowers of different stem positions within an inflorescence and in the n-alkanes and n-alkenes among plants from different populations. In our behavioral field tests, we showed that male bees learn the odor bouquets of individual flowers during mating attempts and recognize them in later encounters. Bees thereby avoid trying to mate with flowers they have visited previously, but do not avoid other flowers either of a different or the same plant. By varying the relative proportions of saturated esters and aldehydes between flowers of different stem positions, we demonstrated that a plant may take advantage of the learning abilities of the pollinators and influence flower visitation behavior. Sixty-seven percent of the males that visited one flower in an inflorescence returned to visit a second flower of the same inflorescence. However, geitonogamy is prevented and the likelihood of cross-fertilization is enhanced by the time required for the pollinium deposited on the pollinator to complete its bending movement, which is necessary for pollination to occur. Cross-fertilization is furthermore enhanced by the high degree of odor variation between plants. This variation minimizes learned avoidance of the flowers and increases the likelihood that a given pollinator would visit several to many different plants within a population.