Received 19 August 2002 FirstCite Accepted 12 November 2002 e-publishing Published online Pollinator attraction in a sexually deceptive orchid by means of unconventional chemicals Manfred Ayasse1*, Florian P. Schiestl1†, Hannes F. Paulus1, Fernando Ibarra2 and Wittko Francke2 1Institute of Zoology, Department of Evolutionary Biology, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria 2Department of Organic Chemistry, Universita¨t Hamburg, Martin Luther King Platz 6, D-20146 Hamburg, Germany Ophrys flowers mimic virgin females of their pollinators, and thereby attract males for pollination. Stimu- lated 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 (ω-1)-hydroxy and (ω-1)-oxo acids, especially 9-hydroxydecanoic acid. These com- pounds, 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-Hydroxyde- canoic 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. Keywords: Campsoscolia ciliata; Ophrys speculum; pollination by sexual deception; chemical mimicry; oxygenated acids 1. INTRODUCTION 1916; Pouyanne 1917). Pseudocopulation was sub- sequently studied intensively by Kullenberg (1961) who Ophrys orchids have evolved ‘one of the most remarkable suggested that Ophrys orchids mimic the females’ sex pher- pollination mechanisms found in any plants’ (Proctor et al. omone of the pollinators to dupe males into visiting their 1996, p. 205). They grow especially around the Mediter- flowers, and thereby pollinating them. ranean, and their pollinators are mostly bees (Andrenidae, Ophrys flowers, and also females of their pollinator spec- Colletidae, Megachilidae and Apidae). Sphecid and scoliid ies, produce complex odour bouquets of more than 100 wasps and beetles (Elateridae and Scarabaeidae) are volatiles (Borg-Karlson 1987, 1990). Behavioural experi- involved in a few cases (Kullenberg 1961; Borg-Karlson ments using synthetic copies of the compounds produced 1990). Ophrys flowers mimic virgin females of their pollina- by Ophrys flowers have shown that only certain volatiles tors, and male insects are lured to the orchid by volatile are active in stimulating mating behaviour in the males semiochemicals and visual cues. At close range, chemical (Kullenberg & Bergstro¨m 1976; Tengo¨ 1979; Borg-Karl- signals from the flowers elicit sexual behaviour in males, son 1990). Furthermore, these experiments demonstrated which try to copulate with the flower labellum and respond that those compounds identified only in insect glandular as if in the presence of female sex pheromones. Thereby secretions had a greater capacity to attract males than did the male touches the gynostemium, and the pollinia may volatiles identified in flowers. Borg-Karlson (1990) there- become attached to his head or, in some species, to the tip fore concluded that Ophrys flowers produce a set of of his abdomen. His copulatory attempts with another ‘second-class attractivity compounds’, which attract only flower ensure that the pollinia are transferred to the flower’s that part of the pollinator population with a low threshold stigmatic surface and pollination is ensured. This phenom- for sexual stimuli in the absence of females. However, the enon is termed ‘Pouyannian mimicry’ (Pasteur 1982), synthetic compounds tested in these experiments rarely because Pouyanne was the first to describe the behaviour of triggered highly intensive male mating behaviour males of the scoliid wasp Campsoscolia ciliata on O. speculum (pseudocopulation) that would ensure pollination. A bio- flowers as a ‘pseudocopulation’ (Correvon & Pouyanne test comparing the attractiveness of floral fragrances pro- duced by Ophrys flowers with the pheromonal secretions of the respective female insects should be preferably per- * Author and address for correspondence: Department of Experimental formed using those compounds that actually elicit cop- Ecology, Albert-Einstein Allee 11, D-89069 Ulm, Germany ulatory behaviour. ([email protected]). † Present address: Geobotanical Institute ETH, Zollikerstrasse 107, CH- Using coupled gas chromatography–electroantenno- 8008 Zu¨rich, Switzerland. graphic detection (GC–EAD), gas chromatography–mass Proc. R. Soc. Lond. B 02pb0717.1 2003 The Royal Society DOI 10.1098/rspb.2002.2271 02pb0717.2 M. Ayasse and others Pollinator attraction in a sexually deceptive orchid spectrometry (GC–MS) and behavioural field tests, we males were not the same, the male reactions were standardized identified behaviour-mediating compounds in the orchid by measuring the flight activity. A 1 m long wire was mounted O. speculum and its pollinator, C. ciliata. Furthermore, we in the experimental area. The number of males crossing the wire tested the hypothesis that Ophrys flowers produce only over a period of 1 min was counted at least every 30 min. For ‘second-class attractivity compounds’ (Borg-Karlson each 3 min test, individual reactions of the males were divided 1990) and are less attractive to males than are their own by the mean male flight activity and multiplied by 100. females. In a second set of experiments we tested the hypothesis that Ophrys flowers are less attractive to males than are their own females. In two behavioural tests we compared the attractiveness 2. MATERIAL AND METHODS of female wasps and Ophrys flowers. In the first test frozen virgin (a) Sample collection female wasps were brought to the field in a container with ice. Campsoscolia ciliata females and O. speculum flower labella A randomly collected individual Ophrys flower and a dead were collected at various locations near C’an Picaford (Mallorca, female were fixed on insect pins and positioned 1.5 cm above the Balearic Islands). Individual Ophrys labella were cut from the ground (the height at which C. ciliata males are patrolling flowers and extracted in 0.5 ml diethyl ether (Merk Uvasol) at and searching for females; Paulus & Gack 1980) and 5 cm apart. 20 °C for 2 days. Virgin C. ciliata females attractive to patrolling In a dual-choice experiment, copulation attempts with the males were killed by freezing. We checked for emptiness of the female and the flower were recorded during a 3 min test period. spermatheca by dissections and found all of the attractive Each couple of flower and female was tested only once in the females to be unmated. The following solvent extracts were same location. obtained from individual females: cuticle extracts—single frozen To exclude the possibility that the use of dead females influ- females were extracted for 5 min in 400 µl diethyl ether (Merk enced the result of the first test, we conducted a second experi- Uvasol); and head extracts—after decapitation, heads were ment using live females. Fresh flowers were collected at random. extracted in 200 µl diethyl ether for 24 h. Samples were stored In addition, virgin C. ciliata females were collected, which were in the freezer until chemical analyses or behavioural experiments highly attractive to patrolling males. Dead Soxhlet-extracted were performed. female wasps were used as dummies and placed in a male patrol- ling area. In a 3 min test period the scent of either an attractive (b) Electrophysiology live wasp or an Ophrys flower confined in a glass vial was con- To detect electrophysiologically active compounds in C. ciliata tinuously blown over the dummy. The surface of the vial was cuticle extracts or O. speculum labellum extracts, GC–EAD previously chemically deactivated (Sylon CT, Supelco Inc.). In analyses were performed as described in Ayasse et al. (2000) and this experiment the visual cues were always the same and male Schiestl et al. (2000). behaviour was influenced only by the scent emitted by the flower or by the female wasp. (c) Behavioural assays Our bioassays were conducted at the same locations near C’an (d) Chemical analyses Picaford as those where we also collected Ophrys and wasp Samples of volatiles were analysed using a gas chromatograph samples. All behavioural experiments were performed between HP 5890 (Hewlett–Packard) equipped with a DB5 capillary col- 10.00 and 14.00, when the flight activity of mate-searching male umn (30 m × 0.32 mm inside diameter). The GC was operated wasps was highest. splitless at 120 °C for 30 s, before a programmed increase to In a first experiment we tested the behavioural responses of 280 °Cat4°CminϪ1. For quantitative analysis, n-octadecane C. ciliata males to individual female wasps, to O. speculum flow- was added as an internal standard. Response factors were ers, to odour samples of individual female wasps and Ophrys determined by single level calibration.