Pollinator attraction in a sexually deceptive orchid by means of unconventional chemicals

Ophrys flowers mimic virgin females of their pollinators, and thereby attract males for pollination. Stimulated by scent, the males attempt to copulate with flower labella and thereby ensure pollination. Here, we show for the first time, to our knowledge, that pollinator attraction in sexually deceptive orchids may be based on a few specific chemical compounds. Ophrys speculum flowers produce many volatiles, including trace amounts of (omega-1)-hydroxy and (omega-1)-oxo acids, especially 9-hydroxydecanoic acid. These compounds, which are novel in plants, prove to be the major components of the female sex pheromone in the scoliid wasp Campsoscolia ciliata, and stimulate male copulatory behaviour in this pollinator species. The specificity of the signal depends primarily on the structure and enantiomeric composition of the oxygenated acids, which is the same in wasps and in the orchids. The overall composition of the blend differs significantly between the orchid and its pollinator and is of secondary importance. 9-Hydroxydecanoic acid is a rarely occurring compound that until now has been identified only in honeybees. Contrary to the standard hypothesis that Ophrys flowers produce only 'second-class attractivity compounds' and are neglected once the pollinator females are present, we show that flowers are more attractive to the males than are their own females.     

Pollination biology of the deceptive orchid Changnienia amoena

The pollination biology of Changnienia amoena , an endangered terrestrial orchid, was investigated at two sites in the Shennongjia Mountains, Hubei, central China. The results show that the orchid is exclusively pollinated by bumblebees. In Longmenhe, Bombus ( Diversobombus ) trifasciatus is the primary effective pollinator, whereas B.  ( Tricornibombus ) imitator is the only pollinator in Guanmenshan. These two bumblebees can be treated as a functional group because they carry pollinaria on the same position on their bodies and have similar pollinating behaviours. The morphological traits of the flower adapt precisely to the pollinators. Visitation by bumblebees is mainly in the first half of the flowering period of C. amoena . Pollinarium removal and pollinia deposition take place when bumblebees withdraw from the flower. Crossing experiments show that C. amoena is a self-compatible and outcrossing species. Fruit set in this species is low and may result from limited pollinators because 87.5% of individuals set fruits under hand-pollination, but only 6–12% of individuals set fruits under natural conditions. These results imply that it is important to conserve the pollinator community together with the plants when conservation management for this endangered species is undertaken. Artificial pollination would also be a useful measure to facilitate restoration of the small populations.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 150 , 165–175.     

The genetic basis of pollinator adaptation in a sexually deceptive orchid

In plants, pollinator adaptation is considered to be a major driving force for floral diversification and speciation. However, the genetic basis of pollinator adaptation is poorly understood. The orchid genus Ophrys mimics its pollinators' mating signals and is pollinated by male insects during mating attempts. In many species of this genus, chemical mimicry of the pollinators' pheromones, especially of alkenes with different double-bond positions, plays a key role for specific pollinator attraction. Thus, different alkenes produced in different species are probably a consequence of pollinator adaptation. In this study, we identify genes that are likely involved in alkene biosynthesis, encoding stearoyl-acyl carrier protein (ACP) desaturases (SAD), in three closely related Ophrys species, O. garganica, O. sphegodes, and O. exaltata. Combining floral odor and gene expression analyses, two SAD homologs (SAD1/2) showed significant association with the production of (Z)-9- and (Z)-12-alkenes that were abundant in O. garganica and O. sphegodes, supporting previous biochemical data. In contrast, two other newly identified homologs (SAD5/6) were significantly associated with (Z)-7-alkenes that were highly abundant only in O. exaltata. Both molecular evolutionary analyses and pollinator preference tests suggest that the alkenes associated with SAD1/2 and SAD5/6 are under pollinator-mediated divergent selection among species. The expression patterns of these genes in F(1) hybrids indicate that species-specific expression differences in SAD1/2 are likely due to cis-regulation, while changes in SAD5/6 are likely due to trans-regulation. Taken together, we report a genetic mechanism for pollinator-mediated divergent selection that drives adaptive changes in floral alkene biosynthesis involved in reproductive isolation among Ophrys species.     

Metapopulation pollination in the deceptive orchid Anacamptis pyramidalis

Pollen dispersal between local plant populations within a range of 6 km in a geographically defined metapopulation of the lepidopteran-pollinated deceptive orchid Anacamptis pyramidalis was studied on the island Öland in the Baltic Sea. Local A. pyramidalis populations were examined for pollinators, flowering individuals, and fruit set. Population sizes of pollen vector species were estimated using a mark–release–recapture technique. As pollen vectors, the burnet moth Zygaena minos and the butterfly Aporia crataegi dominated. 205 out of 745 marked lepidopterans were pollinarium carriers. The proportion carriers of the total was considerably higher in Z. minos (50.3%), than in A. crataegi (21.5%) and nymphalidae (8.2%). Furthermore, Z. minos moved much shorter distances than A. crataegi did, while no difference in potential pollen dispersal distances were found between males and females. The number of individual vectors recaptured in another local population of A. pyramidalis was low: A. crataegi (8) and Z. minos (1). The ratio of pollinaria transferred to another local A. pyramidalis population compared to pollinaria remaining within the same local population was 1:41. This study highlights that pollen dispersal distances vary between pollen vector species.     

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.     

Floral visual signal increases reproductive success in a sexually deceptive orchid

Sexually deceptive orchids mimic signals emitted by female insects in order to attract mate-searching males. Specific attraction of the targeted pollinator is achieved by sex pheromone mimicry, which constitutes the major attraction channel. In close vicinity of the flower, visual signals may enhance attraction, as was shown recently in the sexually deceptive orchid Ophrys heldreichii. Here, we conducted an in situ manipulation experiment in two populations of O. heldreichii on Crete to investigate whether the presence/absence of the conspicuous pink perianth affects reproductive success in two natural orchid populations. We estimated reproductive success of three treatment groups (with intact, removed and artificial perianth) throughout the flowering period as pollinaria removal (male reproductive success) and massulae deposition (female reproductive success). Reproductive success was significantly increased by the presence of a strong visual signal??the conspicuous perianth??in one study population, however, not in the second, most likely due to the low pollinator abundance in the latter population. This study provides further evidence that the coloured perianth in O. heldreichii is adaptive and thus adds to the olfactory signal to maximise pollinator attraction and reproductive success.     

Differentiation in a tropical deceptive orchid: colour polymorphism and beyond

Colour polymorphism has often been described among individuals of deceptive orchid species, and several studies have investigated reproductive success variations among colour morphs. However, whether colour morphs differed in other traits has received little attention in previous studies. Here, we report the case of a tropical deceptive orchid in Reunion Island that exhibits three different colour varieties. We investigated patterns of trait variation among colour varieties and found significant differences in floral and inflorescence morphology. Interestingly, we found that most populations included only individuals of a single variety, and that strong differences exist among varieties in spatial distribution and flowering phenology. This situation differs from previously reported cases of floral polymorphism in deceptive orchids where colour morphs co-occur and flower in the same populations. The spatio-temporal variation in flowering suggests that colour varieties have independent reproductive ecologies and are adapted to local conditions. We propose that potential variations in pollinator species abundance or diversity and the co-occurrence of nectar-producing species in the community may have driven the adaptation of each variety to its current pollination niche.     

Ecological interactions of the sexually deceptive orchid Orchis galilaea

Plant species dependent on highly specific interactions with pollinators are vulnerable to environmental change. Conservation strategies therefore require a detailed understanding of pollination ecology. This two-year study examined the interactions between the sexually deceptive orchid, Orchis galilaea, and its pollinator Lasioglossum marginatum. Relationships were investigated across three different habitats known to support O. galilaea (garrigue, oak woodland, and mixed oak/pine woodland) in Lebanon. Visitation rates to flowers were extremely low and restricted to male bees. The reproductive success of O. galilaea under ambient conditions was 29.3% (±2.4), compared to 89.0% (±2.1) in plants receiving cross-pollination by hand. No difference in reproductive success was found between habitat types, but values of reproductive success were positively correlated to the abundance of male bees. Pollination limitation can have negative impacts on the population growth of orchids, and this study provides clear evidence for more holistic approaches to habitat conservation to support specific interactions.     

Chemical communication in the sexually deceptive orchid genus Cryptostylis

Pollination by sexual deception is among the most intriguing of orchid pollination syndromes. Odours are well established as the primary stimuli for sexually attracting the male insect pollinators in these orchids. We applied gas chromatography with electroantennographic detection (GC-EAD) to investigate chemical communication between the sympatric, but morphologically distinct, orchids Cryptostylis erecta and C. subulata and their pollinators. Cryptostylis is unusual among sexually deceptive orchid genera in that all five Australian species share the same pollinator, the ichneumonid wasp Lissopimpla excelsa , but hybrids are unknown. We show that volatile odour compounds are not produced in detectable amounts in either species. Floral extracts containing many low-volatility compounds showed considerable differences in composition between C. erecta and C. subulata . By contrast, GC-EAD revealed the male wasp pollinators are electrophysiologically responsive to the same GC peak on two different kinds of GC column in both orchids. This leads us to predict that a single compound is the sexual attractant in all Australian Cryptostylis . The apparent conservation of chemical signals among distinct species contrasts with that of other sexually deceptive orchids that are often morphologically similar but reproductively isolated by their different chemical signals.  © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society , 2004, 144 , 199–205.     

Scent variation and hybridization cause the displacement of a sexually deceptive orchid species

In the sexually deceptive orchid genus Ophrys, reproductive isolation is based on the specific attraction of males of a single pollinator species, mostly bees, by mimicking the female sex pheromone of this species. Changes in the floral odor can lead to hybridization, introgression, and possibly speciation. We investigated hybrid swarms of O. lupercalis and O. iricolor on Sardinia using behavioral, electrophysiological (GC-EAD), chemical, morphological, and genetic methods (AFLPs). In behavioral experiments, approximately 20% of the flowers from both species and hybrids were attractive to the "wrong" or both pollinator species. Analysis of the EAD-active hydrocarbons in the floral odor showed an overlap in the two species, whereby hybrid individuals could not be separated from O. iricolor. The genetic analysis confirmed the hybridization of the species. Plants of O. iricolor and hybrids are genetically indistinguishable and form an O. iricolor × lupercalis hybrid population. Remaining plants of O. lupercalis will possibly be displaced by the O. iricolor × lupercalis hybrid population in the future. Our study showed that in deceptive orchids, variation in the pollinator attracting cues, in this case, scent, can be the first step for speciation and at the same time cause the displacement of a species.     

Geographic variation of floral scent in a highly specialized pollination mutualism

Floral scents are important signals for communication between plants and pollinators. Several studies have focused on interspecific variation of these signals, but little is known about intraspecific variation in flower scent, particularly for species with wide geographic distributions. In the highly specific mutualism between Ficus species and their pollinating wasps, chemical mediation is crucial for partner encounter. Several studies show that scents, i.e. blends of volatiles, are species-specific, but no studies address interpopulation variation of scents in fig pollination mutualisms, which often have broad geographic distributions. In this study, using absorption/desorption headspace techniques, we analyzed variation in floral scent composition among three populations of each of two widely distributed Asian Ficus species. We identified more than 100 different volatile organic compounds, predominantly terpenes. In both species, significant differences were found between scent bouquets of East Asian and Indian populations. These differences are discussed in relation to geographical barriers that could disrupt gene exchange between these two areas, thereby isolating Indian populations from those of Eastern Asia.     

Pollinator limitaton in a deceptive orchid, Pogonia japonica, on a floating peat mat

Deceptive orchid species that offer no floral rewards to pollinators often experience pollinator limitation because they. The breeding system and fruit set were investigated in order to examine pollinator limitation for a population of a deceptive orchid, Pogonia japonica , on a floating peat mat in Mizorogaike Pond, western Japan. Fruit sets for outcross-pollinated, self-pollinated and pollinator-excluded flowers were 75%, 80%, and 0%, respectively. Thus, this species was self-compatible but neither autogamous nor apogamous. The fruit set for open-pollinated flowers was 22.9% and 17.4% in 1994 and 1995, suggesting that pollinator limitation did occur in the field. Larger flowers were more likely to develop into fruit than smaller ones in open pollination (36% vs 8%). Thus, flower size seems to affect fruiting of this species. No effective visits by major pollinators such as bees, which could carry pollinia, were observed during 16.5 h of observation during daytime. Instead, thrips were often found moving on gynostemium or pollinia and circumstantial evidence that suggests thrips may partly transfer pollen in pieces out of a granular pollinium of P. japonica was obtained. Pogonia japonica should come to use thrips as a pollinator to supplement low fruit set under limited flower visitation by major pollinators.     

Variable selection patterns on the labellum shape of Geoblasta pennicillata,a sexually deceptive orchid

By mimicking shape and female mating pheromones, flowers of sexually deceptive orchids attract sexually excited males which pollinate them while trying to copulate. Although many studies have demonstrated the crucial importance of odour signals in these systems, most flowers pollinated by pseudocopulation resemble, at least superficially, an insect body and these visual cues may be important to cheat pollinators. In this 2‐year study, we show that the shape of the labellum of Geoblasta pennicillata is a target of pollinator‐mediated natural selection. Contrary to our expectations, plants with a labellum shape more similar to female wasps were not favoured. The strength and pattern of phenotypic selection varied between study years and sexual functions. Although selection through female success was probably associated to the fine‐tuning of the mechanical fit between flower form and male wasp, shape was the target of natural selection through male success in both study years indicating that male wasps use this trait when choosing flowers. The imperfect mimicry and patterns of selection observed indicated that an exact imitation is not needed to attract and deceive the pollinators and they suggested a receiver perceptual bias towards uncommon phenotypes.     

Thynnine wasps discriminate among heights when seeking mates: tests with a sexually deceptive orchid

The flower of a sexually deceptive orchid, Chiloglottis reflexa, mimics both the sex pheromone and the appearance of a female thynnine wasp (Neozeloboria nr. proxima). The flower is pollinated when visited by male wasps, who attempt mating with the flower. We have used these mimetic flowers to investigate mating behavior of the male wasps. In field choice experiments, males strongly prefer to visit flowers that are very low in the habitat, 15 cm, vs. flowers that are placed at 55 or 105 cm. These studies suggest that male precopulatory response is strongly dependent on the microlocation of the female (or female mimic). Other insect-mimicking orchids, which together attract several groups of Hymenoptera, may be useful in analogous experiments on mating behavior. Additionally, these experiments help elucidate features of the mimetic flowers, particularly stature, that act to efficiently attract potential pollinators.     

The production of a key floral volatile is dependent on UV light in a sexually deceptive orchid

Background and Aims

Plants use a diverse range of visual and olfactory cues to advertize to pollinators. Australian Chiloglottis orchids employ one to three related chemical variants, all 2,5-dialkylcyclohexane-1,3-diones or ‘chiloglottones’ to sexually attract their specific male pollinators. Here an investigation was made of the physiological aspects of chiloglottone synthesis and storage that have not previously been examined.

Methods

The location of chiloglottone production was determined and developmental and diurnal changes by GC-MS analysis of floral tissue extracts was monitored in two distantly related Chiloglottis species. Light treatment experiments were also performed using depleted flowers to evaluate if sunlight is required for chiloglottone production; which specific wavelengths of light are required was also determined.

Key Results

Chiloglottone production only occurs in specific floral tissues (the labellum calli and sepals) of open flowers. Upon flower opening chiloglottone production is rapid and levels remain more or less stable both day and night, and over the 2- to 3-week lifetime of the flower. Furthermore, it was determined that chiloglottone production requires continuous sunlight, and determined the optimal wavelengths of sunlight in the UV-B range (with peak of 300 nm).

Conclusions

UV-B light is required for the synthesis of chiloglottones – the semiochemicals used by Chiloglottis orchids to sexually lure their male pollinators. This discovery appears to be the first case to our knowledge where plant floral odour production depends on UV-B radiation at normal levels of sunlight. In the future, identification of the genes and enzymes involved, will allow us to understand better the role of UV-B light in the biosynthesis of chiloglottones.     

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.     

Microsatellite markers for evolutionary studies in the sexually deceptive orchid genus Chiloglottis

The orchids in the genus Chiloglottis are pollinated exclusively by sexual deception. We describe 20 variable microsatellite loci that will facilitate the study of the evolution and ecology of this specialized pollination system. Two loci were isolated from Chiloglottis trilabra, with the rest derived from Chiloglottis valida. All loci were polymorphic in C. valida (H_E 0.07–0.87). Most loci amplified, and were variable within close relatives of C. valida, and a number amplified more broadly, including outside the genus.     

Colour similarity to rewarding model plants affects pollination in a food deceptive orchid, Orchis boryi

We studied the pollination of Orchis boryi at five different locations on the Greek mainland. Orchis boryi is food deceptive and obligatorily insect pollinated. Primary pollinators were Apis mellifera and Bombus spp., which foraged on rewarding plant species nearby and visited O. boryi in between. To analyse floral colour similarity among rewarding plants and O. boryi as perceived by bees, a model of bee colour vision was employed. For each food plant an index was calculated that described the probability of a bee foraging on it to subsequently choose an orchid flower. This choice probability correlated to colour distance according to the model of bee colour vision, indicating that bees chose the deceptive orchid more frequently if they foraged on more similarly coloured species. At different sites different plant species served as models. Bees foraging on food plants from which a high choice rate to the orchid was observed visited the orchid less often after approaching it than other bees, which is likely to reflect avoidance learning. In general, the pollination syndrome appears to be a generalized form of Batesian mimicry, in which similarity to rewarding plants determines reproductive success. As expected by negative density-dependent selection, individual fruit set and pollinia export rate correlated negatively with orchid density, but were unaffected by food plant density, orchid frequency, individual variation of labellum colour, labellum size, or mouth width of the flowers.     

Strong pollinator‐mediated selection for increased flower brightness and contrast in a deceptive orchid

Contrasting flower color patterns that putatively attract or direct pollinators toward a reward are common among angiosperms. In the deceptive orchid Anacamptis morio, the lower petal, which makes up most of the floral display, has a light central patch with dark markings. Within populations, there is pronounced variation in petal brightness, patch size, amount of dark markings, and contrast between patch and petal margin. We tested whether pollinators mediate selection on these color traits and on morphology (plant height, number of flowers, corolla size, spur length), and whether selection is consistent with facilitated or negative frequency‐dependent pollination. Pollinators mediated strong selection for increased petal brightness (Δβ_poll = 0.42) and contrast (Δβ_poll = 0.51). Pollinators also tended to mediate stabilizing selection on brightness (Δγ_poll = –0.27, n.s.) favoring the most common phenotype in the population. Selection for reduced petal brightness among hand‐pollinated plants indicated a fitness cost associated with brightness. The results demonstrate that flower color traits influence pollination success and seed production in A. morio, indicating that they affect attractiveness to pollinators, efficiency of pollen transfer, or both. The documented selection is consistent with facilitated pollination and selection for color convergence toward cooccurring rewarding species.