Looks matter: changes in flower form affect pollination effectiveness in a sexually deceptive orchid.

Many species of the sexually deceptive genus Ophrys are characterized by insect-like flowers. Their form has been traditionally considered to play an important role in pollinator attraction and manipulation. Yet, the evolution of the floral form remains insufficiently understood. We hypothesize that pollinator-mediated selection is essential for driving floral form evolution in Ophrys, but that form components are being subjected to varying selection pressures depending on their role in mediating interactions with pollinators. By using the Eucera-pollinated Ophrys leochroma as a model, our aim has been to assess whether and in what manner pollination effectiveness is altered by experimental manipulation of the flower form. Our results show that floral form plays an essential and, so far, underestimated role in ensuring effective pollination by mechanically guiding pollinators towards the reproductive structures of the flower. Pollinators are significantly less effective in interacting with flowers having forms altered to resemble those of species pollinated by different hymenopteran genera. Further, those components used by pollinators as gripping points were found to be more effective in ensuring pollinia transfer than those with which pollinators do not directly interact. Thus, mechanically active and inactive components appear to be under different selection pressures. As a consequence, mechanically active components of the flower form could reflect adaptations to the interaction with particular pollinator groups, whereas mechanically inactive components can vary more freely. Disentangling selection patterns between the functionally different components of flower form may provide valuable insights into the mechanisms driving the morphological diversification of sexually deceptive pollination systems.

Visual discrimination between two sexually deceptive Ophrys species by a bee pollinator.

Almost all species of the orchid genus Ophrys are pollinated by sexual deception. The orchids mimic the sex pheromone of receptive female insects, mainly hymenopterans, in order to attract males seeking to copulate. Most Ophrys species have achromatic flowers, but some exhibit a coloured perianth and a bright, conspicuous labellum pattern. We recently showed that the pink perianth of Ophrys heldreichii flowers increases detectability by its pollinator, males of the long-horned bee Eucera berlandi. Here we tested the hypothesis that the bright, complex labellum pattern mimics the female of the pollinator to increase attractiveness toward males. In a dual-choice test we offered E. berlandi males an O. heldreichii flower and a flower from O. dictynnae, which also exhibits a pinkish perianth but no conspicuous labellum pattern. Both flowers were housed in UV-transmitting acrylic glass boxes to exclude olfactory signals. Males significantly preferred O. heldreichii to O. dictynnae flowers. In a second experiment, we replaced the perianth of both flowers with identical artificial perianths made from pink card, so that only the labellum differed between the two flower stimuli. Males then chose between both stimuli at random, suggesting that the presence of a labellum pattern does not affect their choice. Spectral measurements revealed higher colour contrast with the background of the perianth of O. heldreichii compared to O. dictynnae, but no difference in green receptor-specific contrast or brightness. Our results show that male choice is guided by the chromatic contrast of the perianth during the initial flower approach but is not affected by the presence of a labellum pattern. Instead, we hypothesise that the labellum pattern is involved in aversive learning during post-copulatory behaviour and used by the orchid as a strategy to increase outcrossing.

Ontogenetic shifts in intraguild predation on thrips by phytoseiid mites: the relevance of body size and diet specialization.

In greenhouse agroecosystems, a guild of spider mite predators may consist of the oligophagous predatory mite Phytoseiulus persimilis Athias-Henriot, the polyphagous predatory mite Neoseiulus californicus McGregor (both Acari: Phytoseiidae) and the primarily herbivorous but facultatively predatory western flower thrips Frankliniella occidentalis Pergande (Thysanoptera: Thripidae). Diet-specialization and the predator body size relative to prey are crucial factors in predation on F. occidentalis by P. persimilis and N. californicus. Here, it was tested whether the relevance of these factors changes during predator ontogeny. First, the predator (protonymphs and adult females of P. persimilis and N. californicus): prey (F. occidentalis first instars) body size ratios were measured. Second, the aggressiveness of P. persimilis and N. californicus towards F. occidentalis was assessed. Third, survival, development and oviposition of P. persimilis and N. californicus with F. occidentalis prey was determined. The body size ranking was P. persimilis females > N. californicus females > P. persimilis protonymphs > N. californicus protonymphs. Neoseiulus californicus females were the most aggressive predators, followed by highly aggressive N. californicus protonymphs and moderately aggressive P. persimilis protonymphs. Phytoseiulus persimilis females did not attack thrips. Frankliniella occidentalis larvae are an alternative prey for juvenile N. californicus and P. persimilis, enabling them to reach adulthood. Females of N. californicus but not P. persimilis sustained egg production with thrips prey. Within the guild studied here, N. californicus females are the most harmful predators for F. occidentalis larvae, followed by N. californicus and P. persimilis juveniles. Phytoseiulus persimilis females are harmless to F. occidentalis.

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.