J Chem Ecol (2016) 42:17–23 DOI 10.1007/s10886-015-0660-0

Parapheromones for Thynnine Wasps

Björn Bohman1,2,3 & Amir Karton3 & Ruby C. M. Dixon1 & Russell A. Barrow1 & Rod Peakall2,3

Received: 3 August 2015 /Revised: 17 November 2015 /Accepted: 2 December 2015 /Published online: 14 December 2015 # Springer Science+Business Media New York 2015

Abstract Sexually deceptive orchids produce floral volatiles 5-dimethylpyrazine. All blends tested stimulated approaches by that attract male insect pollinators. This interaction between male wasps, with some also eliciting landing and attempted flower and pollinator normally is highly specific. In the few copulation. High-level calculations (G4(MP2)) showed the cases where the chemical composition of the volatiles is energy differences between the structural isomers were known, the compounds have been found to be identical to small, although the degree of sexual attraction varied, those that comprise the sex pheromone of the female wasp. indicating the importance of structural factors for activity. In this study, we investigated whether there is potential for One of the parapheromones, 2-hydroxymethyl-3,5-dimethyl- flexibility in the molecular structure of the chemical cues used 6-ethylpyrazine, elicited similar proportions of approaches, to mediate these specific interactions. Specifically, we asked landings, and attempted copulations as the sex pheromone at whether strong sexual attraction can be maintained with struc- the ratio and dose tested. The findings suggest that there is tural modifications of sex pheromone components. Our study potential for chemical flexibility in the evolution of sexual focused on the orchid, glyptodon, which is pollinated deception. by males of the thynnine wasp, Zaspilothynnus trilobatus. Three alkylpyrazines and a unique hydroxymethylpyrazine Keywords Drakaea . Sexual deception . Pyrazine . are components of the female produced sex pheromone of Z. Parapheromone . Specificity trilobatus, and also the semiochemicals produced by the or- chid that lures the males as pollinators. A blend of 2-butyl-3,5- dimethylpyrazine and 2-hydroxymethyl-3,5-diethyl-6- Introduction methylpyrazine (3:1) is as attractive as the full blend of four compounds. Therefore, in this study we substituted 2-hy- Sex pheromones mediate chemical communication between droxymethyl-3,5-diethyl-6-methylpyrazine with one of five the sexes of a species in order to assist reproduction. At a local structurally related parapheromones in a blend with 2-butyl-3, scale, sex pheromone signals typically are highly specific. At the chemical level, this specificity is achieved either by using unique chemicals or by specific blends of more common chemicals (Roelofs and Comeau 1969). In Lepidoptera, the * Björn Bohman [email protected] group that has been the subject of the great majority of studies of insect chemical communication, variable blends of struc- turally similar compounds are common as female sex phero- mones (Francke and Schulz 2010). However, some exceptions 1 Research School of Chemistry, The Australian National University, are known, such as the case of the single compound Canberra, ACT 0200, Australia disparlure, which is the sex pheromone of the gypsy moth, 2 Research School of Biology, The Australian National University, Lymantria dispar (Bierl et al. 1970). Given the usually high Canberra, ACT 0200, Australia specificity of sex pheromones or sex pheromone blends, it is 3 School of Chemistry and Biochemistry, The University of Western of interest to know whether some components can be Australia, Crawley, WA 6009, Australia substituted with different but structurally similar compounds. 18 J Chem Ecol (2016) 42:17–23

The possibility that sex pheromone components could be the Asteraceae and Iridaceae (Ayasse et al. 2011; Borg- substituted appears not to have been widely explored, at least Karlson 1985;Frankeetal.2009;Gaskett2011; Phillips et in part because in the few cases reported the authentic phero- al. 2014; Schiestl et al. 2003). Multiple unrelated orchid gen- mones show by far the strongest activity (Cardé 1990; Renou era have independently evolved this strategy and Guerrero 2000; Schneider et al. 1974). on at least four continents (Ayasse et al. 2011; Gaskett Despite the specific nature of sex pheromones, it some- 2011; Phillips et al. 2014). In sexually-deceptive orchids, pol- times is possible to attract several species with a common lination occurs during either a pre-copulatory routine, or mixture of compounds. For example, 52 species of moths attempted copulation with the flower (Paulus and Gack were attracted to a two- or three-component blend of alkenals, 1990; Peakall 1990). Despite comprehensive early studies alkenols, and alkenol acetates in a screening experiment on the pollination of sexually-deceptive orchids in Europe by Ando et al. (1981). Cis-7,8-epoxy-3-methyloctadecane, a and Australia (Coleman 1929, Kullenberg 1950), the chemis- parapheromone with close structural resemblance to try of these interactions is only now beginning to be elucidat- disparlure (cis-7,8-epoxy-2-methyloctadecane), effectively ed, with progress spanning several different orchid/pollination attracted male gypsy moths, as well as male Lymantria systems on both continents (Borg-Karlson 1987, 1990; monarcha in field experiments (Schneider et al. 1974). Schiestl et al. 2003). There also are examples where authentic sex pheromones Although the number of fully investigated cases is limited, have been successfully modified to ease preparation, reduce so far the orchid semiochemical components identified match cost, or improve applicability. For example, as early as 1968 it closely the components of the sex pheromone of the pollina- was demonstrated that when various synthetic compounds tors. For example, in European Ophrys orchids, the bee- wereaddedto(E)-11-tetradecenyl acetate, the female sex pollinated O. sphegodes utilises a similar blend of alkanes pheromone of the red-banded leaf roller, Argyrotaenia and alkenes as the pollinator Andrena nigroaenea (Schiestl velutinana, different effects were observed (Roelofs and et al. 1999, 2000), and the scoliid wasp pollinated O. Comeau 1968). The Z-isomer of the sex pheromone strongly speculum shares the attractive oxycarboxylic acids and inhibited the attraction of males, while the analogue dodecyl hydroxycarboxylic acids with its pollinator Campsoscolia acetate exhibited synergistic effects, increasing male attraction ciliata (Ayasse et al. 2003). In Australia, Chiloglottis to traps (Roelofs and Comeau 1968). trapeziformis shares the same cyclohexane-1,3-dione with its In this study, we focused on an example drawn from thynnine wasp pollinator, Neozeleboria cryptoides (Peakall et Hymenoptera (ants, bees, and wasps). Despite possessing a al. 2010;Schiestletal.2003). uses the similar number of species to Lepidoptera, the sex pheromones same pyrazine compounds to lure male Zaspilothynnus of Hymenoptera are considerably less well known. Ayasse et trilobatus wasps as the females of the corresponding species. al. (2001) noted that Hymenopteran sex attractants, which are To date, all these orchid species are believed to utilize the typically emitted by females, normally consist of just one or a same compound(s) found in the female sex pheromones of few compounds, although they also queried whether that was their pollinators. because more complex systems had not been extensively stud- In the present study, our aim was to investigate whether it is ied at the time (Ayasse et al. 2001). However, with the inclu- possible for a critical component of the sex pheromone of Z. sion of more recent studies (e.g., Bohman et al. 2014b; trilobatus to be substituted, and to determine if there is a Niehuis et al. 2013), this trend still appears to hold, even if relationship between the structural properties of the investigat- exceptions are known where male-emitted pheromones and ed parapheromones and biological activity. We conclude by complex blends are used (e.g., Stökl et al. 2014). Species of discussing the implications of the findings for understanding ants and bees, which exhibit complex social interactions, con- the evolution of sexual deception. stitute one group of Hymenoptera where sex pheromone com- plexity could be expected. In these species, it is common for both males and females to emit semiochemicals, and the iden- Methods and Materials tification of specific sex pheromones has proven difficult (Beani et al. 2014). In a study of sex pheromone specificity Test Compounds We previously identified the sex phero- in a semi-social Hymenopteran species of Vespa in Japan, mone blend for Z. trilobatus as consisting of three cross attraction among all six species was observed when trialkylpyrazines (1–3) and 2-hydroxymethyl-3,6-diethyl-5- sexually mature males were exposed to queens or queen ex- methylpyrazine (4) (Bohman et al. 2014b)(Fig.1). tracts from other species (Ono and Sasaki 1987). However, a blend of 3 and 4 (ratio 3:1) was as attractive as In sexual deception, potential insect pollinators such as the full blend of all four compounds, i.e., compounds 1 and 2 bees and wasps are sexually lured to flowers by sex phero- were not absolutely necessary. In this study, we substituted 4 mone mimicry. Pollination by sexual deception is most prev- with one of five structural analogues, while holding 3 constant alent in the , although it has been documented in and at the same proportion (ratio 3:1) as in the natural blend J Chem Ecol (2016) 42:17–23 19

Fig. 1 Substituted pyrazine derivatives (compounds 1–9)and optimized structures of compounds 4–9,obtainedatthe B3LYP/6-31G(2df,p) level of theory. Atomic color scheme: H, white;C,grey;N,blue;O,red

(Fig. 1). Compared to the collectively structurally similar described previously as well as the protocols for identification trialkylpyrazines 1–3,compound4 was structurally distinc- and confirmation of all semiochemicals used (Bohman et al. tive, not previously identified as a natural product, and critical 2012a, b, 2014a, b). All compounds were synthesized using for stimulating landings and attempted copulation with spiked Minisci type chemistry to >97 % purity, confirmed by GC and dummies in field experiments. Compound 4 also is predicted NMR (Bohman et al. 2014a). to be the key to the species-specific communication between Z. trilobatus and D. glyptodon. As the study system is Field Tests At both sexually deceptive orchid flowers (Peakall constrained in opportunities to conduct bioassays (Bohman 1990; Peakall et al. 2010) and in bioassays involving con- et al. 2014b), by substituting compound 4 rather than the more firmed sex pheromone components (Bohman et al. 2014b; generic alkylpyrazines 1–3, we could increase the number of Peakall et al. 2010; Schiestl et al. 2003), individual male wasp replicates and ensure a consistent data set. In order to evaluate responses vary in the degree of sexual stimulation, with most the degree of pollinator sexual attraction in field bioassays, individuals approaching or landing, and a smaller proportion compounds 4–9 were tested individually in blends with 2- then attempting copulation. To assess whether the degree of butyl-3,5-dimethylpyrazine (3)(Fig.1). Details covering sexual response varied among blends, replicate experiments and insect collection, extraction and isolation have been were conducted in October 2014 employing baited dummies 20 J Chem Ecol (2016) 42:17–23 consisting of black plastic dressmakers pins (4 mm diam pin- 31G(2df,p) level of theory. Zero-point vibrational energy, head) attached to bamboo skewers (25 cm in height). For all enthalpic, and entropic corrections have been obtained from bioassays, blends of 2-butyl-3,5-dimethylpyrazine (3)(75μg) such calculations. The equilibrium structures were verified to and the test hydroxymethylpyrazine (25 μg) were applied to have all real harmonic frequencies. Gas-phase Gibbs free en- the pinhead by syringe as 10 μg/μl solutions in dichlorometh- ergies at 298 K were obtained using the G4(MP2) thermo- ane, and the solvent was allowed to evaporate. In preliminary chemical protocol (Curtiss et al. 2007). experiments, charged beads remained active for at least 24 h, but in the experiments here they were used only for a maxi- mum of 4 h (Bohman et al. 2014b). Results Experiment 1 involved three combinations of diethyl- methyl-hydroxymethylpyrazines (4–6)inblendswith Field Tests Experiment 1 was replicated twice over two days alkylpyrazine 3 (i.e., 3 + 4, 3 + 5,and3 + 6) in a ratio of 3:1 with a total of 247 wasp responses scored over 47 trials. with 3 as the major component. Experiment 2 involved four Experiment 2 was replicated three times over two days with combinations of dimethyl-ethyl-hydroxymethylpyrazines a total of 158 wasp responses scored over 25 trials. The pro- (7–9) and the pheromone component 4 (control) in blends portion of approaches, landings, and attempted copulations with 3 (i.e., 3 + 7, 3 + 8, 3 + 9, and 3 + 4) in a ratio of 3:1 elicited by blends with compound 4 were similar across with 3 as the major component. Experiments 1 and 2 (G =1.04,df =2,P =0.596,N =105, At the study site, where male wasps were known to be Fig. 2a, d). patrolling for mates, each experiment consisted of a series of In Experiment 1, blends with compounds 5 and 6 elicited sequential trials in which a single blend was presented for significantly weaker sexual responses, with fewer landings 3 min, during which time the number of male wasp ap- and attempted copulations than observed for the control blend proaches, landings, and attempted copulations were recorded. of 3 + 4. The blend with compound 6 in particular elicited few Each trial was conducted at least 15 m from a previously used landings and attempted copulations (Fig. 2c). location, with treatments randomly varied between trials. In Experiment 2, the blend with compound 7 elicited a Trials that failed to attract one or more male wasps within similar behavioral response compared to the control blend of 2 min were aborted, and restarted at a new location. An ex- 3 + 4, with no significant differences detected in the propor- periment was completed when a minimum of three successful tion of approaches, landings, or attempted copulations trials for each of the treatments was achieved. Replicate ex- (G =3.04,df =2,P =0.218,N = 91, Fig. 2d, e). In contrast, periments were conducted on different days with new the blends with compounds 8 and 9 barely elicited any dummies and chemical preparations. attempted copulation (8:0%,9: 3 %, Fig. 2f, g). Given the characteristic rapid decline in pollinator response over the 3 min trials, and results from mark-recapture experi- Ab initio Calculations In order to exclude the possibility that ments showing that wasps do not revisit baits on the same day, energetic factors influence the activity of the parapheromones, the risk of pseudo-replication, both within and between trials, high-level calculations using the G4(MP2) thermochemical was deemed unlikely (Bohman et al. 2014b; Peakall 1990). protocol were performed in order to characterize structurally The total number of approaches, landings, and attempted cop- and energetically the pyrazine derivatives 4–9 (Fig. 1). A ulations were pooled across trials for each treatment. G-tests common structural feature of compounds 4–9 is that they all were performed using GenAlEx 6.5 (Peakall and Smouse have a hydroxymethyl group at position 2 of the ring. The 2006, 2012) to test the null hypothesis that there was no dif- O–H bond of the hydroxymethyl group forms a hydrogen ference in the proportion of approaches, landings, and bond with the nitrogen center of the pyrazine (Fig. 1). The attempted copulations between the control (3 + 4) and each hydrogen bond distances are similar in all compounds, rang- of the other treatments. To enable visual comparisons among ing from 1.971 Å (compounds 5 and 9) to 1.976 Å (compound experiments and treatments, the wasp responses of ap- 7). The differences between compounds 4–9 are the numbers proaches, landings, and attempted copulations are shown in and arrangements of the methyl and ethyl substituents at po- Fig. 2 as mean proportions of the total, with sample sizes sitions 3, 5, and 6 of the pyrazine ring. Compounds 4–6 are given in the legend. structural isomers with one methyl and two ethyl substituents, while compounds 7–9 are structural isomers with two methyl Ab initio Calculations In order to characterize structurally and one ethyl groups (Fig. 1). Both sets of isomers span a and energetically the pyrazine derivatives and relate these narrow energetic range of about 1 kJ mol−1.Inparticular,at properties to biological activity, high-level ab initio and den- the G4(MP2) level we obtain the following heats of formation sity functional theory calculations were performed using the at 298 K (ΔHf,298): –155.24 (4), −155.08 (5), −154.82 (6), Gaussian 09 program suite (Frisch et al. 2009). Geometries −135.35 (7), −135.00 (8), and −135.17 (9)kJmol−1. Thus, and vibrational frequencies were obtained at the B3LYP/6- for compounds 4–6 we obtain relative isomerization energies J Chem Ecol (2016) 42:17–23 21

Fig 2 Outcomes of bioassays to ad blends of synthetic pyrazines. Experiment 1: a Control - com- pounds 3 + 4, N =65,b com- pounds 3 + 5, N =81,c com- pounds 3 + 6, N =101.Experi- ment 2: d Control – compounds 3 + 4, N =40,e compounds 3 + 7, N =51,f compounds 3 + 8, N =36,andg compounds 3 + 9, N = 31. Pollinator responses are be shown as mean proportions of the total (± standard error, SE), fur- ther partitioned into approaches (A, black bars), landings (L, dark grey bars), and attempted copu- lations (C, light grey bars). All bioassays were performed using individual blends of hydroxymethylpyrazines 4–9 cf with alkylpyrazine 3 in a ratio of 3:1 with 3 as the major compo- nent. G-test results shown are rel- ative to the control for each ex- periment, which is shown in the top graph of the respective panel

g

−1 (ΔHisomer) of: 0.00 (4), 0.16 (5), and 0.42 (6)kJmol ,while deceptive Chiloglottis orchids, which are typically pollinated for compounds 7–9, we obtain relative ΔHisomer values of: by thynnine wasps of the Neozeleboria, extensive field 0.00 (7), 0.35 (8), and 0.18 (9)kJmol−1. The small energy bioassays have confirmed that the extreme pollinator specific- differences between the isomers suggest that other aspects ity of this orchid genus (with an average of 1.1 pollinator such as steric factors are responsible for the differences in species per orchid species) is strongly chemically based. In activities demonstrated for the isomers. this case, specificity among sympatric pollinators (and or- chids) is achieved via either different single chiloglottone compounds (2,5-dialkylcyclohexane-1,3-diones), or variation Discussion in a blend of two chiloglottones. Interestingly, even close structural isomers fail to elicit attraction (Peakall et al. 2010; Most of the studies on the capacity to substitute chemicals, Whitehead and Peakall 2014). Contrary to this, in a recent both of semiochemicals generally and pheromones more spe- study of another orchid/wasp system, we reported that the cifically, have been related to structure − activity studies for semiochemical 3-(3-methylbutyl)-2,5-dimethylpyrazine was pest insects, mainly within Lepidoptera. In these applications, equally attractive to the Catocheilus pollinator of the orchid the primary aim often has been to attract insects, and rarely has as the structurally closely related floral attrac- there been distinction between attractants and sex phero- tant 2-(3-methylbutyl)-3,5,6-trimethylpyrazine (Bohman and mones. This has made the results difficult to interpret in terms Peakall 2014). Beyond our own reports of thynnine wasp of sex pheromone specificity. pollinators, we are not aware of any studies that have directly In sexual-deceptive pollination systems, studies have indi- investigated the chemical basis of sex pheromone specificity cated that extreme plant- pollinator specificity is the norm of Hymenoptera. (Gaskett 2011; Paulus and Gack 1990; Peakall et al. 2010; In the present study, we confirmed that the substitution Phillips et al. 2009). Furthermore, in the case of sexually- of sex pheromone components with structurally similar 22 J Chem Ecol (2016) 42:17–23 parapheromones is possible, with the synthetic analogue 7 in exact match as previously expected (Schlüter and Schiestl combination with compound 3 eliciting the same normal 2008). For a system in the early stages of evolution, such sexual behavioral repertoire of approach, landing, and evolutionary flexibility may be important. Similarly, to be able attempted copulation as the natural pheromone component to substitute semiochemicals could be important in the early 4. Substitution of chemicals did not alter the extreme specific- stages of pollinator switching, which is known to be often ity of the interaction at our study site. Across the two experi- correlated with speciation in sexually deceptive orchids ments, we attracted more than 400 individual male wasps, all (Peakall et al. 2010; Whitehead and Peakall 2014). Further of which possessed the characteristic morphology of Z. studies of the chemical basis of specificity and the scope for trilobatus. While we cannot conclusively rule out the possi- substitutability in other sexually deceptive systems, and for bility of morphologically cryptic species, which are known to the relevant pollinator sex pheromone systems more generally, exist within some groups of thynnine wasps (Griffiths et al. will be required to fully understand the evolution of sexual 2011;Menzetal.2015), we did not encounter any morpho- deception. logically distinct wasps in the bioassays. Thus, the extreme specificity of the interaction appears to hold, even with the Acknowledgments We thank Ryan Phillips and Alyssa Weinstein for substitution of related compounds. In the European sexually- insightful comments on the manuscript. Funding was provided by the Australian Research Council (LP0989338 and LP130100162 to RP and deceptive Ophrys orchids, pollinator specificity also can be RAB). maintained in spite of some variation in the chemical blend. For example, in Ophrys exaltata, which is pollinated by the bee Colletes cunicularius, quantitative differences in the com- References plex alkene/alkane blend have been reported between the mimic and the natural sex pheromone (Vereecken and Ando T, Kuroko H, Nakagaki S, Saito O, Oku T, Takahashi N (1981) Schiestl 2008). Multi-component sex attractants in systematic field tests of male We suggest the differences we observed in the degree Lepidoptera. Agric Biol Chem 45:487–495 of sexual response elicited in Z. trilobatus by the Ayasse M, Dötterl S (2014) The role of preadaptations or evolutionary novelties for the evolution of sexually deceptive orchids. New hydroxymethylpyrazine analogues are due to differences in Phytol 203:710–712 the steric properties of the compounds. We note that a com- Ayasse M, Paxton R, Tengö J (2001) Mating behavior and chemical mon structural feature linking compounds 4 and 7 (which is communication in the order Hymenoptera. Annu Rev Entomol 46: not present in the other isomers) is the presence of the 5- 31–78 methyl-6-ethyl substitution of the pyrazine ring. Ayasse M, Schiestl FP, Paulus HF, Ibarra F, Francke W (2003) Pollinator attraction in a sexually deceptive orchid by means of unconventional How sexual deception evolves, and why it has evolved chemicals. Proc R Soc B 270:517–522 repeatedly, remains a mystery (Ayasse and Dötterl 2014; Ayasse M, Stökl J, Francke W (2011) Chemical ecology and pollinator- Bohman et al. 2014b). A key impediment to the evolution of driven speciation in sexually deceptive orchids. Phytochemistry 72: – sexual deception would appear to be the highly specific nature 1667 1677 Beani L, Dessì-Fulgheri F, Cappa F, Toth A (2014) The trap of sex in of sex pheromones (Symonds and Elgar 2008). This problem social insects: from the female to the male perspective. Neurosci appears to be all the more puzzling in cases such as Biobehav Rev 46:519–533 Chiloglottis and Drakaea orchids, where the critical chemicals Bierl BA, Beroza M, Collier C (1970) Potent sex attractant of the gypsy involved in the pollinator interaction are not known moth: its isolation, identification, and synthesis. Science 170:87–89 Bohman B, Peakall R (2014) Pyrazines attract Catocheilus thynnine elsewhere in , and appear to have no obvious prior func- wasps. Insects 5:474–487 tion (Ayasse and Dötterl 2014; Bohman et al. 2014b). In Bohman B, Jeffares L, Flematti G, Byrne LT, Skelton BW, Phillips RD, Ophrys species that secure male bee pollination via specific Dixon KW, Peakall R, Barrow RA (2012a) Discovery of blends of alkenes/alkanes, these same compounds are found tetrasubstituted pyrazines as semiochemicals in a sexually deceptive – widely across the flowers of other related orchid genera, and a orchid. J Nat Prod 75:1589 1594 Bohman B, Jeffares L, Flematti G, Phillips RD, Dixon KW, Peakall R, prior function of desiccation tolerance has been hypothesized Barrow RA (2012b) The discovery of 2-hydroxymethyl-3-(3- (Schiestl and Cozzolino 2008; Schlüter and Schiestl 2008). methylbutyl)-5-methylpyrazine: a semiochemical in orchid pollina- Thus, evolution of sexual deception via preadaptation appears tion. Org Lett 14:2576–2578 to be plausible in this genus. Bohman B, Berntsson B, Dixon RCM, Stewart CD, Barrow RA (2014a) Alkylations and hydroxymethylations of pyrazines via green Our finding in the present study of substitutability among Minisci-type reactions. Org Lett 16:2787–2789 analogues of the key sex pheromone component of Z. Bohman B, Phillips RD, Menz MHM, Berntsson BW, Flematti GR, trilobatus suggests there may be potential for flexibility in Barrow RA, Dixon KW, Peakall R (2014b) Discovery of the evolution of sexual deception. That is, despite the apparent pyrazines as pollinator sex pheromones and orchid semio- chemicals: implications for the evolution of sexual deception. extreme specificity and precise matching of chemicals that New Phytol 203:939–953 characterize all of the studied systems to date, it is possible Borg-Karlson A-K (1985) Chemical basis for the relationship between that sexual deception might not be as tightly constrained to an Ophrys orchids and their pollinators. Chem Scr 25:283–294 J Chem Ecol (2016) 42:17–23 23

Borg-Karlson A-K (1987) Chemical basis for the relationship between Peakall R, Smouse PE (2012) GenAlEx 6.5: genetic analysis in Excel. Ophrys orchids and their pollinators. Chem Scr 27:313–325 Population genetic software for teaching and research—an update. Borg-Karlson A-K (1990) Chemical and ethological studies of pol- Bioinformatics 28:2537–2539 lination in the genus Ophrys (Orchidaceae). Phytochemistry Peakall R, Ebert D, Poldy J, Barrow RA, Francke W, Bower CC, Schiestl 29:1359–1387 FP (2010) Pollinator specificity, floral odour chemistry and the phy- Cardé RT. 1990. Principles of mating disruption. Behavior-modifying logeny of Australian sexually deceptive Chiloglottis orchids: impli- chemicals for pest management: Applications of pheromones and cations for pollinator-driven speciation. New Phytol 188:437–450 other attractants. Marcel Dekker, New York:47–71 PhillipsRD,FaastR,BowerCC,BrownGR,PeakallR(2009) Coleman E (1929) Pollination of an Australian orchid by the male Implications of pollination by food and sexual deception for polli- ichneumonid Lissopimpla semipunctata, Kirby. Trans R Entomol nator specificity, fruit set, population genetics and conservation of Soc Lond 76:533–539 Caladenia (Orchidaceae). Aust J Bot 57:287–306 Curtiss LA, Redfern PC, Raghavachari K (2007) Gaussian-4 theory using Phillips RD, Scaccabarozzi D, Retter BA, Hayes C, Brown GR, reduced order perturbation theory. The J Chem Phys 127:124105 Dixon KW, Peakall R (2014) Caught in the act: pollination Francke W, Schulz S (2010) 4.04 - Pheromones of terrestrial inverte- of sexually deceptive trap-flowers by fungus gnats in Pterostylis – brates. In: Liu H-W, Mander L (eds) Comprehensive natural prod- (Orchidaceae). Ann Bot 113:629 641 ucts II. Elsevier, Oxford, pp. 153–223 Renou M, Guerrero A (2000) Insect parapheromones in olfaction re- Franke S, Ibarra F, Schulz CM, Twele R, Poldy J, Barrow RA, Peakall R, search and semiochemical-based pest control strategies. Annu Rev – Schiestl FP, Francke W (2009) The discovery of 2,5- Entomol 45:605 630 dialkylcyclohexan-1,3-diones as a new class of natural products. Roelofs WL, Comeau A (1968) Sex pheromone perception. Nature 220: – Proc Natl Acad Sci U S A 106:8877–8882 600 601 Roelofs WL, Comeau A (1969) Sex pheromone specificity: taxonomic Frisch M, Trucks G, Schlegel H, Scuseria G, Robb M, Cheeseman J, and evolutionary aspects in Lepidoptera. Science 165:398–400 Scalmani G, Barone V, Mennucci B, Petersson G (2009) Gaussian Schiestl FP, Cozzolino S (2008) Evolution of sexual mimicry in the or- 09, revision D. 01. Gaussian, Wallingford, CT, USA chid subtribe Orchidinae: the role of preadaptations in the attraction Gaskett AC (2011) Orchid pollination by sexual deception: pollinator of male bees as pollinators. BMC Evol Biol 8:27 perspectives. Biol Rev 86:33–75 Schiestl FP, Ayasse M, Paulus HF, Löfstedt C, Hansson BS, Ibarra F, Griffiths KE, Trueman JWH, Brown GR, Peakall R (2011) Molecular Francke W (1999) Orchid pollination by sexual swindle. Nature genetic analysis and ecological evidence reveals multiple cryptic 399:421–422 species among thynnine wasp pollinators of sexually deceptive or- Schiestl FP, Ayasse M, Paulus HF, Löfstedt C, Hansson BS, Ibarra F, chids. Mol Phylogenet Evol 59:195–205 Francke W (2000) Sex pheromone mimicry in the early spider Kullenberg B (1950) Investigations on the pollination of Ophrys species. orchid (Ophrys sphegodes): patterns of hydrocarbons as the – Oikos 2:1 19 key mechanism for pollination by sexual deception. J Comp Menz MHM, Phillips RD, Anthony JM, Bohman B, Dixon KW, Peakall Phys A 186:567–574 R (2015) Ecological and genetic evidence for cryptic ecotypes in a Schiestl FP, Peakall R, Mant JG, Ibarra F, Schulz C, Franke S, Francke W rare sexually deceptive orchid, . Bot J Linn Soc (2003) The chemistry of sexual deception in an orchid-wasp polli- – 177:124 140 nation system. Science 302:437–438 Niehuis O, Buellesbach J, Gibson JD, Pothmann D, Hanner C, Mutti NS, Schlüter PM, Schiestl FP (2008) Molecular mechanisms of floral mimicry Judson AK, Gadau J, Ruther J, Schmitt T (2013) Behavioural and in orchids. Trends Plant Sci 13:228–235 genetic analyses of Nasonia shed light on the evolution of sex pher- Schneider D, Lange R, Schwarz F, Beroza M, Bierl BA (1974) Attraction – omones. Nature 494:345 348 of male gypsy and nun moths to Disparlure and some of its chemical Ono M, Sasaki M (1987) Sex pheromones and their cross-activities in six analogues. Oecologia 14:19–36 Japanese sympatri species of the genus Vespa. Insect Soc 34: Stökl J, Dandekar A-T, Ruther J (2014) High chemical diversity in a wasp 252–260 pheromone: a blend of methyl 6-methylsalicylate, fatty alcohol ac- Paulus H, Gack C (1990) Pollinators as prepollinating isolation factors: etates and cuticular hydrocarbons releases courtship behavior in the evolution and speciation in Ophrys (Orchidaceae). Israel J Bot 39: Drosophila parasitoid Asobara tabida. J Chem Ecol 40:159–168 43–79 Symonds MR, Elgar MA (2008) The evolution of pheromone diversity. Peakall R (1990) Responses of male Zaspilothynnus trilobatus wasps to Trends Ecol Evol 23:220–228 females and the sexually deceptive orchid it pollinates. Funct Ecol 4: Vereecken NJ, Schiestl FP (2008) The evolution of imperfect floral mim- 159–167 icry. Proc Natl Acad Sci U S A 105:7484–7488 Peakall R, Smouse PE (2006) GENALEX 6: genetic analysis in Excel. Whitehead MR, Peakall R (2014) Pollinator specificity drives strong Population genetic software for teaching and research. Mol Ecol prepollination reproductive isolation in sympatric sexually decep- Notes 6:288–295 tive orchids. Evolution 68:1561–1575