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Cross-kingdom mimicry of female insect sex pheromones by sexually deceptive orchids has evolved multiple times.1 Fungus gnats (Diptera) are predicted to be pollinators of hundreds of sexually deceptive orchids,2-4 yet unlike orchids that sexually attract bees and wasps (Hymenoptera),5-11 the chemistry of fungus gnat-pollinated orchids remains unknown. Furthermore, despite the importance of fungus gnats as pollinators, pests, and decomposers of organic material, and evidence for sex pheromones since 1971,12-17 no structure of any fungus gnat sex pheromone has to date been confirmed. In this study, we found a mixture of five hydrocarbons shared between Pterostylis orbiculata orchids and female Mycomya sp. (Mycetophilidae) fungus gnats, which included three alkanes, a C23 diene, and a C23 triene. The triene was an undescribed natural product, which we synthesized and confirmed to be (6Z,9Z)-1,6,9-tricosatriene. Field bioassays with a synthetic blend of the five hydrocarbons elicited attraction and sexual behavior from male gnats. The triene alone elicited attraction and low levels of sexual behavior, but the blend without it was unattractive, suggesting that this compound is a key component of orchid pollinator attraction and the female fungus gnat sex pheromone. In two closely related Pterostylis species, we found related C23 trienes, but not (6Z,9Z)-1,6,9-tricosatriene. These results suggest that unusual long-chain unsaturated hydrocarbons hold the key to sexual deception in Pterostylis orchids, and are an important step toward deciphering female fungus gnat sex pheromones.

The Pompilidae is a cosmopolitan and diverse group of wasps, which commonly feed on nectar. However, pollination systems specialized on pompilids have not been documented in detail outside of southern Africa. Here, we studied Caladenia drummondii (Orchidaceae) where, based on floral traits and preliminary field observations, we predicted pollination by sexual deception of male pompilid wasps. Detailed pollinator observations were undertaken using floral baiting experiments at sites spanning 375 km. Following evidence for nectar on some flowers of C. drummondii, the sugar content on the labellum was analysed by GC-MS. Floral spectral reflectance was measured and compared with Caladenia using other pollination strategies. Males of a single species of pompilid wasp (Calopompilus sp.) were the only visitors capable of pollinating C. drummondii. Attempts to feed from the surface of the labellum were frequent and were associated with removal and deposition of pollinia. GC-MS analysis revealed larger quantities of sugar on the labellum than reported in other Caladenia species. While no sexual or courtship behaviour was observed, the zig-zag and circling flight on approach to the flower is suggestive of odour-based attraction. Floral spectral reflectance was similar to sexually deceptive Caladenia. This study represents the first confirmation of a specialized pompilid pollination system outside of Africa. Although pollination occurs during nectar-foraging, long-distance sexual attraction cannot be ruled out as an explanation for the exclusive male visitation. The similarity in floral spectral reflectance to other Caladenia indicates colour may not impose a constraint on the evolution of pollination by pompilids.

Sexually deceptive orchids achieve pollination by luring male insects to flowers through chemical and sometimes visual mimicry of females. An extreme example of this deception occurs in Cryptostylis, one of only two genera where sexual deception is known to induce pollinator ejaculation. In the present study, bioassay-guided fractionations of Cryptostylis solvent extracts in combination with field bioassays were implemented to isolate and identify floral volatiles attractive to the pollinator Lissopimpla excelsa. ( S)-2-(Tetrahydrofuran-2-yl)acetic acid [( S)-1] and the ester derivatives methyl ( S)-2-(tetrahydrofuran-2-yl)acetate [( S)-2] and ethyl ( S)-2-(tetrahydrofuran-2-yl)acetate [( S)-3], all previously unknown semiochemicals, were confirmed to attract L. excelsa males in field bioassays. Chiral-phase GC and HPLC showed that the natural product 1 comprised a single enantiomer, its S-configuration being confirmed by synthesis of the two enantiomers from known enantiomers of tetrahydrofuran-2-carboxylic acid.

Background and Aims Selection exerted by pollinators on flowers is predicted to occur along two distinct axes. While pollinator attraction to flowers is governed by pollinator preferences, pollen transfer efficiency is mediated by the mechanical fit of pollinators to flower morphology. Although pollinator attraction in sexually deceptive orchids is typically underpinned by floral odour, morphological traits are expected to play a vital role in mechanical fit during floral contact with pollinators. Methods Here we utilize a comprehensive and novel procedure to test for pollinator-mediated selection through mechanical fit with the flower labellum in the orchid Chiloglottis trapeziformis. This approach combines detailed pollinator observations related to plant reproductive fitness with complementary experimental manipulation and phenotypic selection analysis. Key Results Experiments with virgin flowers revealed that pollen removal occurs only during vigorous pseudocopulation. This behaviour involves male wasps that grasp the insectiform callus structure on the labellum while probing the labellum tip in a forward orientation. Both orientation and duration of pseudocopulation were significant predictors of pollen removal, confirming a direct relationship between pollinator behaviour and plant fitness. Controlled floral manipulation that either shortened or elongated the distance between the callus and the labellum tip detected no change in pollinator attraction. The duration of pseudocopulation, however, was significantly reduced on flowers with shortened or elongated callus–tip distances, consistent with stabilizing selection. Phenotypic selection analysis confirmed this prediction in natural populations by uncovering evidence for stabilizing selection on the distance between the callus and the labellum tip. Conclusions Our experimental manipulations and selection analysis in natural populations thus demonstrate stabilizing selection on the distance from the callus to t

Until recently, (methylthio)phenols as natural products had only been reported from bacteria. Now, four representatives of this class of sulfurous aromatic compounds have been discovered as semiochemicals in the orchid Caladenia crebra, which secures pollination by sexual deception. In this case, field bioassays confirmed that a 10:1 blend of 2-(methylthio)benzene-1,4-diol (1) and 4-hydroxy-3-(methylthio)benzaldehyde (2) sexually attracts the male thynnine wasp Campylothynnus flavopictus (Tiphiidae:Thynnineae), the exclusive pollinator of C. crebra. Here we show with field bioassays that another undescribed species of Campylothynnus (sp. A) is strongly sexually attracted to a 1:1 blend of compounds 1 and 2, which elicits very high attempted copulation rates (88%). We also confirm that this Campylothynnus species is a pollinator of Caladenia attingens subsp. attingens. Chemical analysis of the flowers of this orchid revealed two (methylthio)phenols, compound 2 and 2-(methylthio)phenol (3), as candidate semiochemicals involved in pollinator attraction. Thus, (methylthio)phenols are likely to be more widely used than presently known. The confirmation of this Campylothynnus as a pollinator of C. attingens subsp. attingens at our study sites was unexpected, since elsewhere this orchid is pollinated by a different thynnine wasp (Thynnoides sp). In general, sexually deceptive Caladenia only use a single species of pollinator, and as such, this unusual case may offer a tractable study system for understanding the chemical basis of pollinator switching in sexually deceptive orchids.