An unusual tricosatriene is crucial for male fungus gnat attraction and exploitation by sexually deceptive Pterostylis orchids.

Cross-kingdom mimicry of female insect sex pheromones by sexually deceptive orchids has evolved multiple times. Fungus gnats (Diptera) are predicted to be pollinators of hundreds of sexually deceptive orchids, yet unlike orchids that sexually attract bees and wasps (Hymenoptera), 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, 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 C diene, and a C 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 C 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.