Evidence for the recruitment of florivorous plant bugs as pollinators.

Angiosperm flowers and their animal visitors have co-evolved for at least 140 Ma, and early flowers were likely used mainly as mating and feeding sites by several groups of insects, including beetles, flies, true bugs, and thrips. Earlier studies suggested that shifts from such neutral or antagonistic relationships toward mutualistic pollination interactions between flowers and insects occurred repeatedly during angiosperm evolution. However, the evolutionary mechanisms and adaptations, which accompanied shifts toward effective pollination, are barely understood, and evidence for such scenarios has been lacking. Here, we show that Syngonium hastiferum (Araceae), a Neotropical representative of an otherwise beetle-pollinated clade, is pollinated by plant bugs (Miridae; Heteroptera), which are florivores of Syngonium schottianum and other Araceae species. We found that S. hastiferum differs in several floral traits from its beetle-pollinated relatives. Scent emission and thermogenesis occur in the morning instead of the evening hours, and its pollen surface is spiny instead of smooth. Furthermore, the floral scent of S. hastiferum includes a previously unknown natural product, (Z)-3-isopropylpent-3-en-1-ol, which we show to have a function in specifically attracting the plant bug pollinators. This is the first known case of a specialized plant bug pollination system and provides clear evidence for the hypothesis that the adoption of antagonistic florivores as pollinators can drive flower diversification. VIDEO ABSTRACT.

Neural and behavioural responses of the pollen-specialist bee Andrena vaga to Salix odours.

An effective means of finding food is crucial for organisms. Whereas specialized animals select a small number of potentially available food sources, generalists use a broader range. Specialist (oligolectic) bees forage on a small range of flowering plants for pollen and use primarily olfactory and visual cues to locate their host flowers. So far, however, little is known about the specific cues oligoleges use to discriminate between hosts and non-hosts and how floral scent compounds of hosts and non-hosts are processed in the bees' olfactory system. In this study, we recorded physiological responses of the antennae (electroantennographic detection coupled to gas chromatography; GC-EAD) and in the brain (optical imaging; GC imaging), and studied host-finding behaviour of oligolectic Andrena vaga bees, a specialist on Salix plants. In total, we detected 37 physiologically active compounds in host and non-host scents. 4-Oxoisophorone, a common constituent in the scent of many Salix species, evoked strong responses in the antennal lobe glomeruli of A. vaga, but not the generalist honeybee Apis mellifera. The specific glomerular responses to 4-oxoisophorone in natural Salix scents reveals a high degree of specialization in A. vaga for this typical Salix odorant component. In behavioural experiments, we found olfactory cues to be the key attractants for A. vaga to Salix hosts, which are also used to discriminate between hosts and non-hosts, and A. vaga demonstrated a behavioural activity for 4-oxoisophorone. A high sensitivity to floral scents enables the specialized bees to effectively find flowers and it appears that A. vaga bees are highly tuned to 4-oxoisophorone at a very low concentration.

The betrayed thief - the extraordinary strategy of Aristolochia rotunda to deceive its pollinators.

Pollination of several angiosperms is based on deceit. In such systems, the flowers advertise a reward that ultimately is not provided. We report on a previously unknown pollination/mimicry system discovered in deceptive Aristolochia rotunda (Aristolochiaceae). Pollinators were collected in the natural habitat and identified. Flower scent and the volatiles of insects (models) potentially mimicked were analyzed by chemical analytical techniques. Electrophysiological and behavioral tests on the pollinators identified the components that mediate the plant-pollinator interaction and revealed the model of the mimicry system. The main pollinators of A. rotunda were female Chloropidae. They are food thieves that feed on secretions of true bugs (Miridae) while these are eaten by arthropod predators. Freshly killed mirids and Aristolochia flowers released the same scent components that chloropids use to find their food sources. Aristolochia exploits these components to deceive their chloropid pollinators. Aristolochia and other trap flowers were believed to lure saprophilous flies and mimic brood sites of pollinators. We demonstrate for A. rotunda, and hypothesize for other deceptive angiosperms, the evolution of a different, kleptomyiophilous pollination strategy. It involves scent mimicry and the exploitation of kleptoparasitic flies as pollinators. Our findings suggest a reconsideration of plants assumed to show sapromyiophilous pollination.

Floral reward, advertisement and attractiveness to honey bees in dioecious Salix caprea.

In dioecious, zoophilous plants potential pollinators have to be attracted to both sexes and switch between individuals of both sexes for pollination to occur. It often has been suggested that males and females require different numbers of visits for maximum reproductive success because male fertility is more likely limited by access to mates, whereas female fertility is rather limited by resource availability. According to sexual selection theory, males therefore should invest more in pollinator attraction (advertisement, reward) than females. However, our knowledge on the sex specific investment in floral rewards and advertisement, and its effects on pollinator behaviour is limited. Here, we use an approach that includes chemical, spectrophotometric, and behavioural studies i) to elucidate differences in floral nectar reward and advertisement (visual, olfactory cues) in dioecious sallow, Salix caprea, ii) to determine the relative importance of visual and olfactory floral cues in attracting honey bee pollinators, and iii) to test for differential attractiveness of female and male inflorescence cues to honey bees. Nectar amount and sugar concentration are comparable, but sugar composition varies between the sexes. Olfactory sallow cues are more attractive to honey bees than visual cues; however, a combination of both cues elicits the strongest behavioural responses in bees. Male flowers are due to the yellow pollen more colourful and emit a higher amount of scent than females. Honey bees prefer the visual but not the olfactory display of males over those of females. In all, the data of our multifaceted study are consistent with the sexual selection theory and provide novel insights on how the model organism honey bee uses visual and olfactory floral cues for locating host plants.

Strong phylogenetic effects on floral scent variation of oil-secreting orchids in South Africa.

PREMISE OF THE STUDY: Evolution involves the interplay between natural selection and phylogenetic constraint. This is particularly evident among the flowering plants where form and diversity of flowers attest to the importance of both pollinator-mediated selection and phylogenetic constraint. Although this has been studied mostly using visible floral characters, invisible volatile chemicals emitted by the flowers should be subject to these same evolutionary forces. Unfortunately, most analyses of floral volatiles have over-emphasized the importance of natural selection and underplayed phylogenetic constraint without quantifying their respective roles in the evolution and composition of floral scents. METHODS: We used multivariate analyses to test the relative importance of pollinators vs. phylogeny in determining the composition of floral scents among oil-secreting orchids in southern Africa. Floral scents of 42 oil-secreting taxa/ecotypes distributed among 12 subclades in the tribe Diseae were sampled using headspace adsorption and gas chromatography-mass spectroscopy. KEY RESULTS: We identified 257 scent compounds distributed over nine different compound classes, with the majority of scents dominated by aliphatic or benzenoid compounds. The only significant predictor of floral scent among these orchids above the species level was phylogeny. Nevertheless, in two of the clades there were differences in scent profiles at the species and ecotype level that corresponded to different pollinators and were thus suggestive of pollinator-mediated selection. CONCLUSIONS: Scent variation was greater than expected and phylogeny was more important than pollinator-mediated selection in predicting the composition of floral scents of oil-secreting orchids, despite the specialized nature of the pollinator reward system.

Non-volatile floral oils of Diascia spp. (Scrophulariaceae).

The floral oils of Diascia purpurea, Diascia vigilis, Diascia cordata, Diascia megathura, Diascia integerrima and Diascia barberae (Scrophulariaceae) were selectively collected from trichome elaiophores. The derivatized floral oils were analyzed by gas chromatography-mass spectrometry (GC-MS), whilst the underivatized samples were analysed by electrospray ionization mass spectrometry (ESI-MS) and Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS). The most common constituents of the floral oils investigated are partially acetylated acylglycerols of (3R)-acetoxy fatty acids (C(14), C(16), and C(18)), as was proven with non-racemic synthetic reference samples. The importance of these oils for Rediviva bees is discussed in a co-evolutionary context.

Inter- and intraspecific variation in floral scent in the genus salix and its implication for pollination.

The floral scent composition of 32 European and two Asian Salix L. species (Salicaceae) was analyzed. Intra- and interspecific variation was compared for a subset of 8 species. All Salix species are dioecious and floral scent was collected from both male and female individuals by using a dynamic headspace MicroSPE method, and analyzed by GC-MS. A total of 48 compounds were detected, most of them being isoprenoids and benzenoids. Commonly occurring compounds included trans-beta-ocimene, cis-beta-ocimene, benzaldehyde, D-limonene, alpha-pinene, cis-3-hexenyl aceatate, linalool, 1,4-dimethoxybenzene, and beta-pinene. Two compounds, 1,4-dimethoxybenzene and trans-beta-ocimene, were responsible for most of the interspecific variation. In a subset of eight extensively sampled species, six had a characteristic floral scent composition; half of the pairwise species comparisons confirmed significant differences. In three of these eight species, intraspecific variability could be explained by sex differences. Variation in Salix floral scent may provide specific signals that guide pollinators and thus contribute to the reproductive isolation of compatible and cooccurring species.

Flower scent of floral oil-producing Lysimachia punctata as attractant for the oil-bee Macropis fulvipes.

Most flowers offer nectar and/or pollen as a reward for pollinators. However, some plants are known to produce mostly fatty oil in the flowers, instead of nectar. This oil is exclusively collected by specialized oil-bees, the pollinators of the oil-plants. Little is known about chemical communication in this pollination system, especially how the bees find their hosts. We collected the floral and vegetative scent emitted by oil-producing Lysimachia punctata by dynamic headspace, and identified the compounds by gas chromatography coupled to mass spectrometry. Thirty-six compounds were detected in the scent samples, several of which were flower-specific. Pentane extracts of flowers and floral oil were tested on Macropis fulvipes in a biotest. Flower and oil extracts attracted the bees, and some of the compounds identified are seldom found in the floral scent of other plants; these may have been responsible for the attraction of the bees.

Stereoisomeric pattern of lilac aldehyde in Silene latifolia, a plant involved in a nursery pollination system.

The monoterpene lilac aldehyde is found in floral scent of several plants species, among them Silene latifolia. This plant is involved in a nursery pollination system, because a noctuid moth, Hadena bicruris, is not only pollinator but also seed predator. Lilac aldehyde is the key floral scent compound of S. latifolia for attracting Hadena. This monoterpene has three stereogenic centers, and eight different isomers are possible. Here, we analysed the ratio of lilac aldehyde isomers from plants originating from 18 different populations of S. latifolia using enantioselective multidimensional GC-MS (enantio-MDGC-MS), and compared resulting variability with variability found in total scent emitted by specimen under study. Though variability in total emitted scent was high, ratio of lilac aldehyde isomers was a more conservative trait. There was no correlation between the ratio of lilac aldehyde isomers and the total emitted floral scent pattern. Both, ratio of stereoisomers and total emitted scent were independent from the geographic origin of the plants. In conclusion, the ratio of lilac aldehyde stereoisomers in S. latifolia is a reliable trait, and may used by the nursery pollinator H. bicruris for host-plant detection.