While studies of plant‐pollinator communities show that many plant species attract multiple pollinators,1 numerous highly specialized pollination systems have been discovered that are underpinned by chemistry.2a–2c Pollination through sexual deception is a highly specialized pollination strategy, primarily known from orchids, where chemical mimicry of courting female insects leads to the attraction of male pollinators.2 So far there have only been a few studies where the compounds involved have been elucidated and confirmed with field bioassays. These include either blends of alkanes and alkenes, or hydroxy‐ and keto acids in Ophrys orchids, cyclohexane‐1,3‐diones in Chiloglottis orchids, and pyrazines in Drakaea orchids.3

Caladenia is a diverse genus of Australian terrestrial orchids, comprising over 360 species.4 The genus is unique in that it employs multiple pollination strategies including food reward, food deception, and more than 100 cases of sexual deception.5 Until now, the identity of the chemicals for signaling between organisms (semiochemicals) involved in sexual deception has remained elusive for any Caladenia species, although given that many different wasp genera are exploited, multiple chemical systems are expected to operate. Thus, detailed studies of the chemistry of this genus, beyond the promise of revealing new natural products, also offers a unique opportunity to better understand the role of floral volatiles in the evolution of sexual deception as a pollination strategy.

In this work, we investigated the chemistry of sexual deception in Caladenia crebra A.S.George. By combining chemical ecology, analytical chemistry, and organic chemistry methods, we demonstrate that in C. crebra, pollination by the thynnine wasp Campylothynnus flavopictus (Smith, 1859) requires a specific blend of unique aromatic sulfurous compounds in precise ratios. In the exceptional mating behavior of thynnine wasps, the larger, winged males carry the smaller, wingless females in copula to a food source for mating and feeding.3a While conducting field bioassays with artificially presented orchid flowers, on three occasions, thynnine wasp pairs were observed arriving at the flower in copula, a behavior not previously reported from sexually deceptive orchids. Furthermore, in one of these cases, which was captured on video (Video S3 in the Supporting Information), the female was released during vigorous attempted copulation by the male with the flower. These unprecedented observations, despite the limited number of observations, confirm the extreme sexual attractiveness of the flower, through its semiochemicals, to the male pollinators.

Despite success in other thynnine systems,3a,3b gas chromatography/electroantennography detection (GC–EAD), where insect antennae are employed as the chemical detector, failed to detect any electrophysiologically active compounds in the C. flavopictus/C. crebra system. Variation of GC conditions, columns, and floral extraction methods did not aid detection, thus indicating that the limitation is either suboptimal chromatographic properties for these compounds (broad peaks) or problems related to the specific antennal receptors. Furthermore, GC–MS screening for cyclohexane‐1,3‐dione‐ and pyrazine‐based compounds known in other Australian orchid/thynnine wasp pollination systems,2 as well as structurally related compounds, revealed none of these compounds. A final set of semiochemical candidates was defined by their presence in both the active lateral sepal tips (clubs, Figure 1) and female wasp extracts, but not in the inactive floral remains. High‐resolution GC–MS (GC‐HRMS) revealed compounds with molecular formulae C 7 H 8 OS (m/z: 140.0296, calcd 140.0296), C 7 H 8 O 2 S (m/z: 156.0251, calcd 156.0245), C 8 H 8 O 2 S (m/z: 168.0240, calcd 168.0245) and C 8 H 10 O 2 S (m/z: 170.0399, calcd 170.0402), all of which were more abundant in highly polar extracts (methanol) than in moderately polar extracts (dichloromethane).

Figure 1 Open in figure viewer PowerPoint Caladenia crebra, Campylothynnus flavopictus, and the identified semiochemicals. a) C. crebra with floral parts labelled, the pollinator C. flavopictus pseudocopulating with a pin spiked with synthetic semiochemicals and a flower. The structures of the four (methylthio)phenol semiochemicals are shown. b) Structurally or biologically related sulfur‐containing natural products in other species.10, 11, 12, 13, 15, 16, 19 Semiochemicals=pheromones or other chemicals involved in signaling between organisms. Photographs: Rod Peakall.

The mass spectra and molecular formulae indicated that all four candidates were phenols with sulfur‐containing substituents. The identification process was expedited by the occurrence of the mass spectrum of our simplest candidate in the NIST‐11 mass spectral library. This compound (1; Figure 2), previously found in bacteria,6 was confirmed by co‐injection of the biological extracts with a commercially available reference compound. Based on similarities in molecular formulae and mass spectra, we proposed several structurally related compounds for the remaining three candidates, including hydroxy, hydroxymethyl and formyl compounds, which were all prepared synthetically and from which the matching compounds were successfully identified. Compounds 2–4 (Figure 2), all ortho‐(methylthio)phenols, were confirmed to be the natural products by co‐injection with the extracts.7

Figure 2 Open in figure viewer PowerPoint Mass spectra of the four (methylthio)phenols 1–4 in C. crebra and C. flavopictus.

In the analysis of solvent extracts of wasp body parts, compounds 2 and 3 were successfully extracted from heads with ethanol or dichloromethane as solvents. The ratios of compounds 2 and 3 differed between the ethanol flower extracts (ca. 10‐fold excess of 3) and the two ethanol female wasp extracts examined (ca. 10‐fold excess of 2).

Compound 3 was prepared from 5 through an S‐methylation, Vilsmeier–Haack formylation and O‐demethylation (Scheme 1).8 The hydroxymethyl compound 4 was prepared from 3 through a sodium borohydride reduction while the hydroquinone 2 could be prepared by oxidizing the commercially available phenol 1 using Fremy's salt in a buffered system (Scheme 1).9

Scheme 1 Open in figure viewer PowerPoint Synthesis of (methylthio)phenols 1–4 identified from C. crebra and C. flavopictus.

Across two years of field bioassays with these compounds, a total of 923 C. flavopictus male wasp responses were recorded. Through a combination of additive and subtractive bioassays, we showed that the two thiophenols 2 and 3 are required to achieve strong sexual attraction in this system, including frequent attempted copulations at simplistic female dummies. Neither compounds 1 or 2 on their own were attractive, while compound 3 did elicit weak attraction. Compounds 1 and 4 appear to be unnecessary for eliciting strong sexual behavior, but are not inhibitory in blends with 2 and 3 (Figure 3 and Figures S1 and S2 in the Supporting Information).

Figure 3 Open in figure viewer PowerPoint Outcomes of bioassays showing the responses of male Campylothynnus flavopictus thynnine wasps to four different blends of synthetic thiophenols (1–4). a) Compounds 2–4 at 100:10:1 as a control blend. b) Compounds 2–4 at 100:100:1. c) Compounds 1–4 at 10:100:10:1. d) Compounds 2 and 3 at 100:10 (control blend without 4). Wasp responses are shown as mean proportions of the total (±SEM), further partitioned into approaches (A, black bars), lands (L, dark grey bars), and attempted copulations (C, light grey bars). G‐test results shown are relative to the control blend shown in (a). Note that there was no response in replicate experiments with a blend of 2 and 4 at 100:1 (control blend without 3) and a blend of 3 and 4 at 10:1 (control blend without 2). See Figures S1 and S2 for details and the outcomes of additional bioassays.

Compounds 2, 3, and 4 have not been previously found in nature. Sulfur‐containing phenols have to our knowledge only recently been discovered in marine bacteria.6 Other aromatic sulfurous compounds identified from plants or animals, apart from orchids, are limited to thiophenes,10 thiazoles,11 and benzyl methyl sulfide (Figure 1).12 Sulfurous floral volatiles are typically rare13 and their function often unknown.14 Within orchids, the only examples are carbon disulfide,15 dimethyl disulfide,13 S‐methyl thioacetate,16 and 2‐aminothiophenol,13 each only reported once (Figure 1). Furthermore, we appear to have discovered the first volatile sulfur‐containing pheromone in Hymenoptera17 and the second in any animal18 after methyl 2‐(methylthio)benzoate was identified as a sex pheromone for two beetle species (Figure 1).19

The challenges in detecting, identifying, synthesizing, and confirming the function of new and unusual semiochemicals suggest that there is likely to be a strong bias towards reporting pollination studies involving common compounds. Accordingly, it is also very likely that pollination systems employing novel compounds are underrepresented in the literature, since advanced chemistry studies capable of identifying new, unusual, and EAD‐challenging compounds are rarely conducted. Prioritizing such studies would certainly lead not only to new semiochemical discoveries, but also to new insight into the multiple routes to specialization and the biosynthetic pathways involved.

Acknowledgements B.B., G.R.F., R.D.P., R.A.B. and R.P. acknowledge the Australian Research Council for funding (DE160101313, FT110100304, DE15010720 and LP130100162). The authors acknowledge the facilities and the scientific and technical assistance of the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy, Characterization & Analysis, The University of Western Australia, a facility funded by the University, State, and Commonwealth Governments. Pollinators were identified by Graham Brown from the Northern Territory Museum and Art Gallery. Alyssa Weinstein is thanked for laboratory assistance.