Are tetraploids more successful? Floral signals, reproductive success and floral isolation in mixed-ploidy populations of a terrestrial orchid

Background and Aims Polyploidization, the doubling of chromosome sets, is common in angiosperms and has a range of evolutionary consequences. Newly formed polyploid lineages are reproductively isolated from their diploid progenitors due to triploid sterility, but also prone to extinction because compatible mating partners are rare. Models have suggested that assortative mating and increased reproductive fitness play a key role in the successful establishment and persistence of polyploids. However, little is known about these factors in natural mixed-ploidy populations. This study investigated floral traits that can affect pollinator attraction and efficiency, as well as reproductive success in diploid and tetraploid Gymnadenia conopsea (Orchidaceae) plants in two natural, mixed-ploidy populations. Methods Ploidy levels were determined using flow cytometry, and flowering phenology and herbivory were also assessed. Reproductive success was determined by counting fruits and viable seeds of marked plants. Pollinator-mediated floral isolation was measured using experimental arrays, with pollen flow tracked by means of staining pollinia with histological dye. Key Results Tetraploids had larger floral displays and different floral scent bouquets than diploids, but cytotypes differed only slightly in floral colour. Significant floral isolation was found between the two cytotypes. Flowering phenology of the two cytotypes greatly overlapped, and herbivory did not differ between cytotypes or was lower in tetraploids. In addition, tetraploids had higher reproductive success compared with diploids. Conclusions The results suggest that floral isolation and increased reproductive success of polyploids may help to explain their successful persistence in mixed-ploidy populations. These factors might even initiate transformation of populations from pure diploid to pure tetraploi

Evolution of sexual mimicry in the orchid subtribe orchidinae: the role of preadaptations in the attraction of male bees as pollinators

BACKGROUND: Within the astonishing diversity of orchid pollination systems, sexual deception is one of the most stunning. An example is the genus Ophrys, where plants attract male bees as pollinators by mimicking female mating signals. Unsaturated hydrocarbons (alkenes) are often the key signal for this chemical mimicry. Here we investigate the evolution of these key compounds within Orchidinae by mapping their production in flowers of selected species onto their estimated phylogeny. RESULTS: We found that alkenes, at least in trace amounts, were present in 18 of 20 investigated species together representing 10 genera. Thus, the reconstruction of ancestral state for alkene-production showed that this is a primitive character state in Ophrys, and can be interpreted as a preadaptation for the evolution of sexual deception. Four of the investigated species, namely Ophrys sphegodes, Serapias lingua, S. cordigera, and Anacamptis papilionacea, that are pollinated primarily by male bees, produced significantly larger amounts and a greater number of different alkenes than the species pollinated either primarily by female bees or other insects. CONCLUSION: We suggest that high amounts of alkenes evolved for the attraction of primarily male bees as pollinators by sensory exploitation, and discuss possible driving forces for the evolution of pollination by male bees

On the roles of colour and scent in a specialized floral mimicry system

Background and Aims Sexually deceptive orchids achieve cross-pollination by mimicking the mating signals of female insects, generally hymenopterans. This pollination mechanism is often highly specific as it is based primarily on the mimicry of mating signals, especially the female sex pheromones of the targeted pollinator. Like many deceptive orchids, the Mediterranean species Ophrys arachnitiformis shows high levels of floral trait variation, especially in the colour of the perianth, which is either green or white/pinkinsh within populations. The adaptive significance of perianth colour polymorphism and its influence on pollinator visitation rates in sexually deceptive orchids remain obscure. Methods The relative importance of floral scent versus perianth colour in pollinator attraction in this orchid pollinator mimicry system was evaluated by performing floral scent analyses by gas chromatography-mass spectrometry (GC-MS) and behavioural bioassays with the pollinators under natural conditions were performed. Key Results The relative and absolute amounts of behaviourally active compounds are identical in the two colour morphs of O. arachnitiformis. Neither presence/absence nor the colour of the perianth (green versus white) influence attractiveness of the flowers to Colletes cunicularius males, the main pollinator of O. arachnitiformis. Conclusion Chemical signals alone can mediate the interactions in highly specialized mimicry systems. Floral colour polymorphism in O. arachnitiformis is not subjected to selection imposed by C. cunicularius males, and an interplay between different non-adaptive processes may be responsible for the maintenance of floral colour polymorphism both within and among population

Scent releasing silicone septa: A versatile method for bioassays with volatiles

Volatile organic compounds are of great importance for communication within biological systems. For the experimental investigation of the functions of volatiles, methods for experimental manipulation are needed. Based on scent-release methods from pheromone research, we describe a simple and cheap method for scent manipulation using silicone rubber (i.e. a silicone septum). Volatile compounds are applied to the septum by soaking the septa for 1 h in a solvent/volatile solution. After removal of the septum from the solution and a drying period of 1 h to allow for evaporation of the solvent, the silicone emits the volatiles at a continuously decreasing rate for a minimum of 24 h. In this study, we measure the variability of the emission and quantify the emission of 22 common floral scent compounds at four different time points and in four different soaking concentrations. Our results show that for the same compound and soaking concentration, variability of volatile emission was low, showing the method leads to repeatable emission rates and can be fine-tuned to the desired emission rate. We provide a calculation tool based on linear regression to allow an experimenter to calculate soaking concentration for each of the 22 compounds to achieve a desirable emission from the septa, as well as to estimate the emission rate of a volatile from the septa after a given time

Ecological role of volatiles produced by Epichloë: differences in antifungal toxicity

Species of Epichloë (Ascomycota, Clavicipitaceae) are endophytic symbionts of pooid grasses. Sexual reproduction of the fungus depends on gamete-transferring Botanophila flies, which in earlier studies were shown to be specifically attracted by the fungal volatiles chokol K and methyl (Z)-3-methyldodec-2-enoate. As several Epichloë volatiles are known to have antimicrobial properties, it was hypothesised that the original function of insect-attracting volatiles is microbial deterrence. However, the origin of volatile compounds and their toxicity within an ecological context has not yet been clarified. We examined the inhibitory effect of chokol K and methyl (Z)-3-methyldodec-2-enoate on mycoparasites, plant pathogenic fungi and on Epichloë itself at ecologically relevant concentrations, and assessed volatile production in pure cultures of Epichloë on complex and defined media supplemented with inorganic sources of carbon and nitrogen. Chokol K reduced the spore germination of all tested fungi, whereas methyl (Z)-3-methyldodec-2-enoate had no inhibitory effect. Moreover, only chokol K was produced in culture, confirming its fungal origin. Our findings are consistent with the proposed scenario that fungal volatile substances have followed an evolutionary pathway from defence to attractio

Floral adaptation to local pollinator guilds in a terrestrial orchid

Background and Aims Studies of local floral adaptation in response to geographically divergent pollinators are essential for understanding floral evolution. This study investigated local pollinator adaptation and variation in floral traits in the rewarding orchid Gymnadenia odoratissima, which spans a large altitudinal gradient and thus may depend on different pollinator guilds along this gradient. Methods Pollinator communities were assessed and reciprocal transfer experiments were performed between lowland and mountain populations. Differences in floral traits were characterized by measuring floral morphology traits, scent composition, colour and nectar sugar content in lowland and mountain populations. Key Results The composition of pollinator communities differed considerably between lowland and mountain populations; flies were only found as pollinators in mountain populations. The reciprocal transfer experiments showed that when lowland plants were transferred to mountain habitats, their reproductive success did not change significantly. However, when mountain plants were moved to the lowlands, their reproductive success decreased significantly. Transfers between populations of the same altitude did not lead to significant changes in reproductive success, disproving the potential for population-specific adaptations. Flower size of lowland plants was greater than for mountain flowers. Lowland plants also had significantly higher relative amounts of aromatic floral volatiles, while the mountain plants had higher relative amounts of other floral volatiles. The floral colour of mountain flowers was significantly lighter compared with the lowland flowers. Conclusions Local pollinator adaptation through pollinator attraction was shown in the mountain populations, possibly due to adaptation to pollinating flies. The mountain plants were also observed to receive pollination from a greater diversity of pollinators than the lowland plants. The different floral phenotypes of the altitudinal regions are likely to be the consequence of adaptations to local pollinator guild

The effects of becoming taller: direct and pleiotropic effects of artificial selection on plant height in Brassica rapa

Plant height is an important trait for plant reproductive success. Plant height is often under pollinator-mediated selection, and has been shown to be correlated with various other traits. However, few studies have examined the evolutionary trajectory of plant height under selection and the pleiotropic effects of plant height evolution. We conducted a bi-directional artificial selection experiment on plant height with fast cycling Brassica rapa plants to estimate its heritability and genetic correlations, and to reveal evolutionary responses to artificial selection on height and various correlated traits. With the divergent lines obtained through artificial selection, we subsequently conducted pollinator-choice assays and investigated resource limitation of fruit production. We found that plant height variation is strongly genetically controlled (with a realized heritability of 41-59%). Thus, plant height can evolve rapidly under phenotypic selection. In addition, we found remarkable pleiotropic effects in phenology, morphology, floral scent, color, nectar and leaf glucosinolates. Most traits were increased in tall-line plants, but flower size, UV reflection and glucosinolates were decreased, indicating potential trade-offs. Pollinators preferred plants of the tall selection lines over the short selection lines in both greenhouse experiments with bumblebees and field experiment with natural pollinators. We did not detect any differences in resource limitation between plants of the different selection lines. Overall, our study predicts that increased height should evolve under positive pollinator-mediated directional selection with potential trade-offs in floral signals and herbivore defense

Scent releasing silicone septa: A versatile method for bioassays with volatiles

Volatile organic compounds are of great importance for communication within biological systems. For the experimental investigation of the functions of volatiles, methods for experimental manipulation are needed. Based on scent-release methods from pheromone research, we describe a simple and cheap method for scent manipulation using silicone rubber (i.e. a silicone septum). Volatile compounds are applied to the septum by soaking the septa for 1 h in a solvent/volatile solution. After removal of the septum from the solution and a drying period of 1 h to allow for evaporation of the solvent, the silicone emits the volatiles at a continuously decreasing rate for a minimum of 24 h. In this study, we measure the variability of the emission and quantify the emission of 22 common floral scent compounds at four different time points and in four different soaking concentrations. Our results show that for the same compound and soaking concentration, variability of volatile emission was low, showing the method leads to repeatable emission rates and can be fine-tuned to the desired emission rate. We provide a calculation tool based on linear regression to allow an experimenter to calculate soaking concentration for each of the 22 compounds to achieve a desirable emission from the septa, as well as to estimate the emission rate of a volatile from the septa after a given time

Variability in Floral Scent in Rewarding and Deceptive Orchids: The Signature of Pollinator-imposed Selection?

Background and Aims A comparative investigation was made of floral scent variation in the closely related, food-rewarding Anacamptis coriophora and the food-deceptive Anacamptis morio in order to identify patterns of variability of odour compounds in the two species and their role in pollinator attraction/avoidance learning. Methods Scent was collected from plants in natural populations and samples were analysed via quantitative gas chromatography and mass spectrometry. Combined gas chromatography and electroantennographic detection was used to identify compounds that are detected by the pollinators. Experimental reduction of scent variability was performed in the field with plots of A. morio plants supplemented with a uniform amount of anisaldehyde. Key Results Both orchid species emitted complex odour bouquets. In A. coriophora the two main benzenoid compounds, hydroquinone dimethyl ether (1,4-dimethoxybenzene) and anisaldehyde (methoxybenzaldehyde), triggered electrophysiological responses in olfactory neurons of honey-bee and bumble-bee workers. The scent of A. morio, however, was too weak to elicit any electrophysiological responses. The overall variation in scent was significantly lower in the rewarding A. coriophora than in the deceptive A. morio, suggesting pollinator avoidance-learning selecting for high variation in the deceptive species. A. morio flowers supplemented with non-variable scent in plot experiments, however, did not show significantly reduced pollination success. Conclusions Whereas in the rewarding A. coriophora stabilizing selection imposed by floral constancy of the pollinators may reduce scent variability, in the deceptive A. morio the emitted scent seems to be too weak to be detected by pollinators and thus its high variability may result from relaxed selection on this floral trai