Thynnine Wasp Research Articles

Pollination by sexual deception promotes outcrossing and mate diversity in self-compatible clonal orchids.

The majority of flowering plants rely on animals as pollen vectors. Thus, plant mating systems and pollen dispersal are strongly influenced by pollinator behaviour. In Australian sexually deceptive orchids pollinated by male thynnine wasps, outcrossing and extensive pollen flow is predicted due to floral deception, which minimizes multiple flower visitations within patches, and the movement of pollinators under mate-search rather than foraging behaviours. This hypothesis was tested using microsatellite markers to reconstruct and infer paternity in two clonal, self-compatible orchids. Offspring from naturally pollinated Chiloglottis valida and C.aff. jeanesii were acquired through symbiotic culture of seeds collected over three seasons. In both species, outcrossing was extensive (tm =0.924-1.00) despite clone sizes up to 11m wide. The median pollen flow distance based on paternity for both taxa combined was 14.5m (n=18, range 0-69m), being larger than typically found by paternity analyses in other herbaceous plants. Unexpectedly for orchids, some capsules were sired by more than one father, with an average of 1.35 pollen donors per fruit. This is the first genetic confirmation of polyandry in orchid capsules. Further, we report a possible link between multiple paternity and increased seed fitness. Together, these results demonstrate that deceptive pollination by mate-searching wasps enhances offspring fitness by promoting both outcrossing and within-fruit paternal diversity.

Identification of hydroxymethylpyrazines using mass spectrometry.

Pyrazines are well-known natural products that are present in bacterial odours and food flavouring agents and are used as insect pheromones. Recently, a number of hydroxymethylpyrazines have been identified as thynnine wasp pheromones and orchid semiochemicals that are essential for pollination in sexually deceptive plants. These compounds are present in low amounts in complex blends, making GC-MS (including high-resolution techniques) the method of choice for their structure elucidation. We report the EI mass spectra for 14 representative compounds and have found that based on characteristic fragmentations, it is possible to distinguish between different positional isomers of hydroxymethylpyrazines. The presence or absence of either [M - 17]+ , [M - 18]+· or [M - 19]+ fragment species provides characteristic information to allow the distinction between the different isomers. Considering the importance of pyrazines as a group of bioactive natural products, and the recent findings of biological activity for hydroxymethylpyrazines, our results presented here will aid the identification of these compounds in other biological systems. Copyright © 2015 John Wiley & Sons, Ltd.

Differences in metabolic rate and evaporative water loss associated with sexual dimorphism in thynnine wasps

Species with sexual dimorphism provide powerful study systems for understanding adaptation to different lifestyles as it removes the potentially confounding effects of phylogeny. Thynnine wasps have a stark sexual dimorphism where males fly patrols in search of the flightless, predominantly fossorial females with which to mate. Using flow-through respirometry, we tested the prediction that the highly active males of the thynnine wasp Zaspilothynnus nigripes would have high metabolic rates (VCO2) relative to females. Further, the females, which spend more time underground, were predicted to exhibit lower evaporative water loss (EWL) than males. Metabolic rate of both sexes increased exponentially between 12 and 28°C. As predicted, males had higher mass-corrected VCO2 at identical temperatures than females. Alternatively, there were no differences in the EWL at identical temperatures between sexes, suggesting that experiencing the same environmental conditions during mating may favour similar EWL. Interestingly, Z. nigripes were estimated to undergo a decrease in metabolism at approximately 30°C. It is proposed that Z. nigripes persist despite sensitivity to high temperatures using a combination of behavioural strategies and emergence during a period of relatively benign climate that ameliorates the impacts of high temperatures.

The role of preadaptations or evolutionary novelties for the evolution of sexually deceptive orchids.

The role of preadaptations or evolutionary novelties for the evolution of sexually deceptive orchids.

Pyrazines Attract Catocheilus Thynnine Wasps.

Five previously identified semiochemicals from the sexually deceptive Western Australian hammer orchid Drakaea livida, all showing electrophysiological activity in gas chromatography–electroantennogram detection (EAD) studies, were tested in field bioassays as attractants for a Catocheilus thynnine wasp. Two of these compounds, (3,5,6-trimethylpyrazin-2-yl)methyl 3-methylbutanoate and 2-(3-methylbutyl)-3,5,6-trimethylpyrazine, were attractive to male wasps. Additionally, the semiochemical 3-(3-methylbutyl)-2,5-dimethylpyrazine, a close analogue to 2-(3-methylbutyl)-3,5,6-trimethylpyrazine, identified in five other species of thynnine wasps, was equally active. The three remaining compounds from D. livida, which were EAD-active against Catocheilus, did not attract the insects in field trials. It is interesting that two structurally similar compounds induce similar behaviours in field experiments, yet only one of these compounds is present in the orchid flower. Our findings suggest the possibility that despite the high specificity normally characterising sex pheromone systems, the evolution of sexual deception may not be entirely constrained by the need to precisely match the sex pheromone constituents and blends. Such evolutionary flexibility may be particularly important during the early stages of speciation.

Discovery of pyrazines as pollinator sex pheromones and orchid semiochemicals: implications for the evolution of sexual deception

Sexually deceptive orchids employ floral volatiles to sexually lure their specific pollinators. How and why this pollination system has evolved independently on multiple continents remains unknown, although preadaptation is considered to have been important. Understanding the chemistry of sexual deception is a crucial first step towards solving this mystery. The combination of gas chromatography-electroantennographic detection (GC-EAD), GC-MS, synthesis and field bioassays allowed us to identify the volatiles involved in the interaction between the orchid Drakaea glyptodon and its sexually attracted male thynnine wasp pollinator, Zaspilothynnus trilobatus. Three alkylpyrazines and one novel hydroxymethyl pyrazine were identified as the sex pheromone of Z.trilobatus and are also used by D.glyptodon for pollinator attraction. Given that our findings revealed a new chemical system for plants, we surveyed widely across representative orchid taxa for the presence of these compounds. With one exception, our chemical survey failed to detect pyrazines in related genera. Collectively, no evidence for preadaptation was found. The chemistry of sexual deception is more diverse than previously known. Our results suggest that evolutionary novelty may have played a key role in the evolution of sexual deception and highlight the value of investigating unusual pollination systems for advancing our understanding of the role of chemistry in evolution.

Specialized ecological interactions and plant species rarity: The role of pollinators and mycorrhizal fungi across multiple spatial scales

Understanding the limitations to persistence and formation of populations is critical to the conservation of rare species. Engaging in specialized ecological interactions may constrain a species’ distribution and abundance through limiting the availability of suitable habitat or reducing fecundity. In a generic-wide study of Drakaea (Orchidaceae), containing three common and six rare species, we tested if mycorrhizal specificity and pollinator availability is associated with intrinsic rarity across multiple spatial scales and if the ecology of common and rare species consistently differs. All Drakaea formed a mycorrhizal association with the same widely distributed species of Tulasnella, demonstrating that rarity does not arise from the mycorrhizal interaction. Drakaea are pollinated by sexual deception of thynnine wasps. Experiments at >300 sites across the geographic range of the genus revealed extreme pollinator specialization, with each species primarily pollinated by a single orchid-specific wasp species. In only two of the six rare species was the pollinator detected beyond the range of the orchid. At the scale of habitat patches, the pollinator of rare orchids occurred in fewer patches of otherwise suitable orchid habitat. Pollination rate was not significantly different between common and rare species. However, pollination rate was highest in small populations suggesting that pollinator-mediated rarity is likely to arise through patchy distribution of pollinators rather than infrequent pollen transfer in small populations. In five of the six rare species there was evidence that a specialized pollination system is contributing to rarity, demonstrating that conservation programs must prioritize the ecological requirements of pollinators.

Sharing of Pyrazine Semiochemicals between Genera of Sexually Deceptive Orchids

It has recently been discovered that novel di-, tri- and tetra- substituted pyrazines are semiochemicals in Drakaea, an orchid genus that secures pollination by the sexual deception of male thynnine wasps. We examined if similar pyrazines were also present in the distantly related Caladenia barbarossa, a sexually deceptive orchid that is also pollinated by a thynnine wasp. Here we report for the first time the occurrence of two pyrazines, (3,5,6-trimethylpyrazin-2-yl)methyl 3-methylbutanoate (1) and 3-(3-methylbutyl)-2,5-dimethylpyrazine (2) in the orchid genus Caladenia. The former is known as a semiochemical involved in pollinator attraction in Drakaea livida. This convergence of floral odour between distantly related plants provides an exciting opportunity to understand the evolution and molecular basis of this sophisticated chemical mimicry.

Convergent specialization – the sharing of pollinators by sympatric genera of sexually deceptive orchids

Summary Pollinator sharing can offer powerful insights into the floral traits associated with the evolution of a pollination system and the consequences of floral differences for pollinator behaviour. Here, we investigate the first known case of pollinator sharing between two sexually deceptive plant genera. Floral manipulations were used to test the importance of floral traits for pollinator behaviour and pollination efficiency. We also explored the ecological differences enabling species co‐occurrence. Drakaea livida and Caladenia pectinata (Orchidaceae) exhibit dramatic differences in floral display and the insectiform appearance of the labellum, yet both are pollinated by sexually attracted males of the thynnine wasp Zaspilothynnis nigripes. Because of the prevalence of cryptic species in some genera of thynnine wasps, we confirmed pollinator sharing by a mark–recapture study and sequencing of the mtDNA CO1 region. Floral dissections revealed that semiochemicals used to attract the pollinator are released from the labellum in D. livida and sepaline clubs in C. pectinata. Drakaea livida was more efficient at converting pollinator attraction into potential pollen deposition leading to higher fruit set. Floral manipulations showed that pollinator contact with the labellum increases when it is the point of semiochemical release. However, sexual attraction to the labellum remained infrequent in C. pectinata in all experimental treatments. While their distribution and climatic range show extensive overlap, the differences in edaphic requirements of the two orchid species suggest that they rarely co‐occur. Therefore, the potential cost of sharing the same pollinator species is not realized. Synthesis. This case of pollinator sharing confirms that morphological traits do not place a strong constraint on the evolution of sexual deception. However, interspecific differences in floral traits have important consequences for converting attraction into pollination, suggesting that selection can act to increase efficiency at multiple steps of the pollination process. This system provides a novel opportunity to elucidate the chemical, visual and morphological adaptations underpinning the evolution of sexual mimicry.

Mate-Searching Behaviour of Common and Rare Wasps and the Implications for Pollen Movement of the Sexually Deceptive Orchids They Pollinate

Pollinator behaviour directly affects patterns of pollen movement and outcrossing rates in plants. In orchids pollinated by sexual deception of insects, patterns of pollen movement are primarily determined by the mate-searching behaviour of the deceived males. Here, using a capture-mark-recapture study (CMR) and dietary analysis, we compare mate-searching behaviour in relation to local abundance of two pollinator species and explore the implications for pollen movement in sexually deceptive Drakaea (Orchidaceae). Drakaea are pollinated solely by the sexual deception of male thynnine wasps. The rare Drakaea elastica and widespread D. livida occur sympatrically and are pollinated by the rare but locally common Zaspilothynnus gilesi, and the widespread and abundant Z. nigripes, respectively. Local abundance was significantly different with Z. nigripes twice as abundant as Z. gilesi. For the 653 marked wasps, there was no significant difference in median movement distance between Z. gilesi and Z. nigripes. However, the maximum movement distance was twice as high for Z. gilesi (556 m) compared with Z. nigripes (267 m). This is up to three times greater than previously reported for thynnines in CMR studies. Recapture rates were six times higher in Z. gilesi (57%) compared to Z. nigripes (9%). Pollen loads and wasp longevity were similar, suggesting that this difference in recapture rate arises due to differences in the number of males moving at a scale >500 m rather than through diet or mortality. Differences in the frequency of longer movements may arise due to variation in the spatial distribution of the wingless females. We predict that pollen movement will largely be restricted to within populations of Drakaea (<500 m), with few movements between populations (>500 m).

Discovery of Tetrasubstituted Pyrazines As Semiochemicals in a Sexually Deceptive Orchid

Sexually deceptive orchids employ mimicry of insect sex pheromones to exploit a diverse group of pollinators. The chemical structures of five semiochemicals (1-3, 7, 8) produced by populations of the warty hammer orchid, Drakaea livida, pollinated by a thynnine wasp in the genus Catocheilus were elucidated. With the exception of (2,5-dimethylpyrazin-3-yl)methyl 3-methylbutanoate (7), all active compounds were tetrasubstituted pyrazines, including hydroxymethyl (1) and ester (2 and 3) trimethylpyrazine derivatives. Male Catocheilus wasps were responsive to all of these compounds in GC-EAD experiments.

Identification of the First Alkenyl Chiloglottone Congener

AbstractOrchids of the genus Chiloglottis rely on sexual deception to attract a male thynnine wasp, whereupon a courtship routine results in the deposition and transfer of pollinia and subsequent pollination of the orchid. The chemical cues responsible for this behavior were recognized as a new class of natural products and efforts to synthetically prepare them, to assist in structural identification and to aid in ecological studies, have been ongoing. Most recently the first alkenylated member of this class was proposed based on GC–MS analysis of a physiologically active compound present in an orchid extract. The current work reveals synthetic efforts that provide access to diastereomerically pure unsaturated chiloglottone analogues and in combination with laboratory‐based electrophysiological assays and field‐based studies have unambiguously allowed identification of 5‐allyl‐2‐ethyl‐1,3‐cyclohexanedione (1d, chiloglottone 4) as the first alkenyl congener in the chiloglottone family. Convergent synthetic approaches, involving copper(I)‐mediated conjugate addition to a glutaconic ester converging on cadmium‐mediated desymmetrization of cyclic anhydrides or a Johnson–Claisen rearrangement of a mixed ketene acetal provided the appropriately substituted δ‐keto acids suitable for carbocyclization to furnish the required chiloglottone analogues.

Molecular genetic analysis and ecological evidence reveals multiple cryptic species among thynnine wasp pollinators of sexually deceptive orchids

Sexually deceptive Chiloglottis orchids lure their male thynnine wasp pollinators to the flower by emitting semiochemicals that mimic the specific sex pheromone of the wasp. Sexual deception is possible because chemical rather than visual cues play the key role in wasp mate search, suggesting that cryptic wasp species may be frequent. We investigated this prospect among Neozeleboria wasp pollinators of Chiloglottis orchids, drawing on evidence from molecular phylogenetic analysis at three genes (CO1, rhodopsin and wingless), population genetic and statistical parsimony analysis at CO1, orchid associations and their semiochemicals, and geographic ranges. We found a compelling relationship between genetically defined wasp groups, orchid associations, semiochemicals and geographic range, despite a frequent lack of detectable morphological differences. Our findings reveal multiple cryptic species among orchid pollinators and indicate that chemical changes are important for wasp reproductive isolation and speciation. The diversity of Neozeleboria may have enabled, rather than constrained, pollinator-driven speciation in these orchids.

Description of Two New Pseudaposematic Species with a Review of Defensive Adaptations in the Subfamily Thynninae (Hymenoptera: Thynnidae)

Two new species of thynnine wasps, 'Lestricothynnus abispoides' and 'Zaspilothynnus scolioides', are described, the males of which are mimics in size and colour pattern of 'Abispa' spp. (hymenoptera: Vespidae) and 'Scolia' spp. (hymenoptera: Scoliidae) respectively. Defensive mechanisms found in the Australian Thynninae are reviewed, including newly discovered defensive pheromones and stridulation.

Effect of pollen load, self-pollination and plant size on seeds and germination in the endangered pink-lipped spider orchid, Caladenia behrii

Caladenia behrii Schltdl. (Orchidaceae) (syn. Arachnorchis behrii) is a sexually deceptive, endangered orchid that produces aggregated pollen as pollinia. It is pollinated by a thynnine wasp, and may also be pollinated incidentally by other insects. Pollinator effectiveness may depend on the number of pollinia that pollinators carry and deposit, and on whether they mediate cross-pollination or self-pollination. To understand the role of pollinators and guide conservation programs, we determined the effect of pollen load (one pollinium v. two pollinia) and self-pollination on seed number, seed (embryo) size and germination at 35 days. We also examined the effect of plant size on seed size and seed number. By using partial correlations with leaf width, seed size, seed number, capsule volume and stem length, we found that leaf width was a good predictor for seed number, and that seed size was not correlated with any of the variables examined. Flowers pollinated with one pollinium and two pollinia did not produce seeds that differed in size or number. Cross-pollinated flowers produced fewer but larger seeds, which germinated faster than did seeds from self-pollinated flowers. We conclude that seed production in the field may be estimated from leaf size, that pollinators carrying one pollinium are as effective as those carrying two pollinia and that selfing affects germination negatively, partly because of the smaller size of selfed seeds. Conservation programs aiming to perform hand-pollination of this species should use crossing with a single pollinium.

Synthesis of chiloglottones – semiochemicals from sexually deceptive orchids and their pollinators

A five-step synthesis of monoalkyl- and 2,5-dialkyl-1,3-cyclohexanediones (1) is described via a sequence involving sequential Birch reductions and alkylations from the readily accessible and inexpensive starting material, 3,5-dimethoxybenzoic acid. Two approaches were considered in which alkylation at C-2 occurs either prior or subsequent to the proposed reduction. The successful route, in which Birch reduction of a 3-alkyl resorcinol derivative (3) precedes alkylation was applied in the synthesis of chiloglottone 1 (1dc), in 58% overall yield. Chiloglottone 1 is a member of a new class of natural products, representing a known sex pheromone of the thynnine wasp Neozeleboria cryptoides and pollinator attractant in the Australian sexually deceptive orchid genus Chiloglottis. The synthetic homologues were assessed for their biological activity via electroantennographic detection.

Implications of pollination by food and sexual deception for pollinator specificity, fruit set, population genetics and conservation of Caladenia (Orchidaceae)

Caladenia is very unusual in that it contains species that attract pollinators by two different strategies, food and sexual deception. Among the sexually deceptive species, baiting for pollinators has shown that within populations orchid species are typically pollinated by a single species of thynnine wasp. However, some wasp species can be pollinators of more than one species of orchid usually when their ranges do not overlap. There is a trend for closely related orchids to exploit wasps from the same genus, with different lineages of orchids often pollinated by different genera. Very little is known about pollination of food-deceptive Caladenia species, although it is evident they attract a suite of generalist food-seeking insects. Food-deceptive species have a higher pollination rate than do sexually deceptive species. Studies of population genetics and pollen movements are few, although they suggest a pattern of fine-scale genetic structuring within populations, owing to predominantly restricted seed dispersal and low genetic differentiation among populations as a consequence of rare long-distance seed-dispersal events. Both evolutionary and ecological research of Caladenia will greatly benefit from a better understanding of the insect species involved in pollination, their ecological requirements and the ecological and genetic consequences of food and sexual deception.

Bees and white spiders: unravelling the pollination syndrome of Caladenia rigida (Orchidaceae)

Orchids of the genus Caladenia have been shown to utilise two quite different pollination strategies, namely species-specific sexual deception of thynnine wasps and a more generalist strategy attracting a larger spectrum of foraging insects. While baiting techniques have enabled the identification of numerous pollinators of sexually deceptive Caladenia, insects that pollinate food-advertising species have received little attention. The present study employed a multidisciplinary approach to better evaluate the pollination syndrome of the white spider orchid, Caladenia rigida R.S.Rogers, a species previously reported to utilise both food and sexual deception. This included the observation and capture of potential pollinators of C. rigida through direct observation, pantraps, Malaise traps and pollinator-baiting experiments, and the use of molecular techniques to identify orchid pollinia isolated from carrier insects. We describe a suite of generalist insects visiting and bearing pollinia from C. rigida. In addition, samples collected from the labellum and column of C. rigida contained sugars at levels comparable to those of a known nectar-producing orchid, Microtis parviflora R.Br. Potential osmophores in the clubs and calli stained positively with neutral red and although this character is often associated with sexual deception, we found no evidence for this secondary pollination syndrome in C. rigida. The present study is the first one to provide a detailed description of the pollinators and pollination syndrome of a non-sexually deceptive species within the genus Caladenia and the first report to provide evidence of nectar production by a species within this genus.

An enthusiasm for orchids: sex and deception in plant evolution

The male thynnine wasp's extreme sexual enthusiasm is crucial to reproduction of hammer orchids in the wild. Hammer orchids have co-evolved to produce odors identical to those manufactured by female thynnine wasps. The male wasp's superb sensitivity to the scent of his female mate is the basis the hammer orchid's deceit--in effect, orchids exploit the male insect's highly adaptive sense of smell their own propogation. While pollinating orchids is a waste of time, and thus a maladaptive activity a wasp, his mistake comes about because he must react quickly whenever he senses a possible mate nearby. Alcock suggests that, for insects, he who hesitates is lost, although perhaps it would be better to say that he who hesitates often loses a chance to pass on his genes. This book abounds with clever explanations how these exceptionally complex flowers came to be shaped as they are. The reader can explore many aspects of orchid biology and history ranging from how some species avoid inbreeding, to the origins of orchids from an ancestor that belonged to the asparagus family. Examining each component of an orchid's flower, Alcock explains how the various parts work together to produce the plant's minute offspring. Each element of an orchid, as quirky as it may seem, is biologically significant, bearing the imprint of natural selection. Readers can share in the delight that Darwin and all other orchid enthusiasts have felt in making sense of even the smallest of details of these most wonderful plants.

Opportunistic pollinator shifts among sexually deceptive orchids indicated by a phylogeny of pollinating and non-pollinating thynnine wasps (Tiphiidae)

The thynnine wasp genus Neozeleboria Rohwer is the main pollinating group of the sexually deceptive Australian orchid genus, Chiloglottis R.Br. In a highly specialized interaction, Chiloglottis species attract males from a single or very few Neozeleboria species through the chemical mimicry of the female wasp's sex pheromone. An earlier study examining the historical association among Chiloglottis and Neozeleboria using DNA sequence data found matching phylogenetic patterns suggestive of cospeciation between orchids and pollinators. However, patterns of constraint in Neozeleboria emergence phenology and sex pheromones suggested that the close association among orchid and wasp clades may be due to pollinator switching among closely related wasp taxa that have similar traits. In this study, we further examine the association by incorporating a morphological phylogenetic analysis of non-pollinating as well as pollinating Neozeleboria. The morphological analysis is then compared with DNA sequence data from one nuclear and one mitochondrial gene for an increased sample of outgroup genera. The combined molecular data set finds a monophyletic Neozeleboria, although support for this was not strong in the individual data sets. A high congruence between molecular and morphological analyses was found among higher groupings of Neozeleboria. Neozeleboria species that pollinate Chiloglottis species are not found as a monophyletic group but, rather, are scattered throughout a phylogeny comprising pollinators and non-pollinators. Under the cospeciation model, the presence of related Neozeleboria non-pollinators carries the unlikely implication that the association between plant and pollinator has been repeatedly lost. Instead, we favour the alternative ‘preferential pollinator switching’ model that accounts for the specialization among orchid and wasp lineages in terms of similarities in traits among related Neozeleboria. © 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 86, 381–395.