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Obligate pollination mutualisms, where seed predators and their host plants trade pollination services for the rearing of larvae, are among the most specialized insect–plant interactions known. Classical examples of such ‘nursery’ pollination systems (sensuDufaÿ & Ansett, 2003) are those involving yucca moths (Pellmyr, 2003) and fig wasps (Weiblen, 2002), and several analogous associations have been discovered more recently, including Chiastocheta flies on globeflower Trollius europaeus (Pellmyr, 1989), the moth Upiga virescens on the senita cactus Lophocereus schottii (Fleming & Holland, 1998) and Epicephala moths on Phyllanthaceae plants (Kato et al., 2003; Kawakita & Kato, 2004a,b). Although these interactions have served as important focal systems for studies on the origin, stability, and reversal of mutualisms (Pellmyr & Huth, 1994; Pellmyr et al., 1996; Pellmyr & Leebens-Mack, 1999; Holland et al., 2002; Rønsted et al., 2005; Kawakita & Kato, 2009), as well as analyses of reciprocal diversification among interacting lineages within mutualisms (Kawakita et al., 2004; Machado et al., 2005), data on the specific sensory cues guiding obligate pollinators to their hosts are still very scarce. The floral scent has been chemically characterized for several taxa of Ficus (Grison et al., 1999; Grison-Pigéet al., 2002b), Yucca (Svensson et al., 2005, 2006), and Glochidion (Okamoto et al., 2007), and olfactory-based host attraction has been confirmed for both fig wasps (Hossaert-McKey et al., 1994; Ware & Compton, 1994; Gibernau et al., 1998; Song et al., 2001; Grison-Pigéet al., 2002a) and Epicephala moths (Okamoto et al., 2007), but the pollinator attractant has only been chemically identified for one such association (Chen et al., 2009).
Highly species-specific mutualistic or antagonistic interactions between plants and pollinators, or between fungi and spore dispersers, have been suggested to be mediated by a few, system-specific compounds through ‘private channels’ (e.g. Raguso, 2003; Steinebrunner et al., 2008), but to date few data are available with which to test this prediction. Unique compounds have indeed been documented as pollinator attractants in sexually deceptive orchids, for example one Ophrys species (Ayasse et al., 2003) and several Chiloglottis species (Schiestl et al., 2003; Franke et al., 2009), as well as a spore disperser attractant in Epichloë fungi (Steinebrunner et al., 2008). However, most other Ophrys and insect-attracting fungi use blends of ‘conventional’ compounds frequently occurring in flowering plants to attract respective pollinators and spore dispersers (Raguso & Roy, 1998; Schiestl et al., 1999). Thus, more studies are needed on the chemical ecology of plant–pollinator interactions to test whether a higher degree of specialization in such interactions is correlated with a more specific floral scent chemistry of the pollinator-attracting signal.
We tested the hypothesis that a ‘private channel’ mediates an obligate insect–plant mutualism, using the recently described mutualism between Breynia vitis-idaea and its species-specific Epicephala moth pollinator as a model (Kawakita & Kato, 2004b). To date, it is estimated that 500 Phyllanthaceae species are each pollinated by a host-specific Epicephala pollinator, which in turn lays eggs in female flowers, from which develop larvae that consume a fraction of the seeds (Kawakita & Kato, 2009). The ovipositing female moths actively pollinate host flowers to ensure that larval food (i.e. seeds) is produced for their offspring. At night, a female collects pollen from host male flowers using the proboscis, and then visits a female flower, on which she deposits pollen and subsequently lays an egg. Whether moths visit male and female flowers on the same plant, or fly to a new plant after collecting pollen, is as yet unknown. The pollen collection and deposition behaviours are highly distinct and stereotypic, and are accompanied by a morphological specialization: the female proboscis is equipped with numerous hairs that are absent in the males, which probably facilitate effective handling of pollen. Because pollination takes place only at night, the female moth is predicted to use olfactory cues for host location. Also, as male Epicephala moths are commonly found on host leaves, male moths may rely on floral cues for mate location, although they have not been observed on flowers and apparently do not pollinate the host. Furthermore, insects other than the pollinator Epicephala females have rarely been observed on flowers, which led us to hypothesize that a private signal mediating these intimate associations is present.
In the present study: we chemically characterized the odour bouquets of male and female flowers of B. vitis-idaea; we tested whether floral scent alone is sufficient for attraction of the host-specific Epicephala pollinator; and we identified the blend of floral volatiles functioning as the pollinator attractant in this mutualism.
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Contrary to our expectation of a ‘private signal’ guiding the exclusive pollinator to host flowers in an obligate pollination seed-consuming mutualism, the volatiles functioning as the attractant in the Epicephala–Breynia interaction (2-PEA and 2-PAN) should be regarded as general floral compounds, as they occur in the floral scent of a large number of angiosperms. Knudsen et al. (2006) reported 2-PEA and 2-PAN as constituents of the floral fragrance in 234 species from 49 families, and in 64 species from 18 families, respectively, showing the widespread occurrence of these compounds as floral volatiles among flowering plants. These two compounds were also found to occur together in the floral scent of a number of species (Knudsen et al., 2006). Although there are still limited data on whether and to what extent 2-PEA and 2-PAN function as attractants, or possibly repellents, in insect–plant interactions, the frequent occurrence of the two compounds as floral volatiles may indicate that they are used as attractants by many insect species during, for instance, nectar foraging. For example, 2-PEA alone has been shown to be an attractant for the generalist moth Trichoplusia ni (Haynes et al., 1991).
The first identification of a floral scent signal mediating an obligate pollination seed-consuming mutualism was reported very recently for Ficus semicorda (Chen et al., 2009). A single compound, 4-methylanisole, constitutes the attractive signal for the obligate pollinator in this association, and thus may represent a typical ‘private channel’. This is in contrast to scent data from other associations between figs and their host-specific pollinators, where general floral volatiles have mainly been identified (Grison-Pigéet al., 2002a,b). Interestingly, floral scent data from five species of Glochidion (Okamoto et al., 2007), a close relative of Breynia, which is also exclusively pollinated by host-specific Epicephala moths, suggest that these interactions are mediated by conventional floral scent compounds, similar to findings for B. vitis-idaea. Chemical and electrophysiological analyses of the floral scent in the yucca–yucca-moth mutualism strongly indicate that pollinator attraction in this association is mediated by specific chemistry (Svensson et al., 2005, 2006; G. P. Svensson, O. Pellmyr & R. A. Raguso, unpublished data). No data are yet available on the chemical ecology of the other two documented cases of obligate pollination mutualism, involving the globeflower and senita cactus and their respective obligate pollinators.
Until recently, the only nursery pollination system for which the pollinator attractant had been chemically characterized was the association between Silene latifolia and its pollinating seed predator Hadena bicruris (Jürgens et al., 2002; Dötterl et al., 2006). Electrophysiological analyses revealed almost 20 compounds in the floral headspace of S. latifolia eliciting an antennal response in H. bicruris. When the behavioural response to such compounds was tested in a flight tunnel, moths were found to be highly attracted to lilac aldehydes alone, and no difference in attraction was observed between these compounds and the scent from single S. latifoila flowers, showing that few key compounds constitute the active signal in this pollinator–plant interaction (Dötterl et al., 2006). Interestingly, nursery pollination systems involving Silene species and moths of the genera Hadena (Pettersson, 1992) and Perizoma (Westerbergh, 2004) do not constitute obligate associations, do not involve active pollination, and range from antagonistic to potentially mutualistic interactions (Kephart et al., 2006). Thus, additional data from these less specialized interactions, as well as other obligate associations, should greatly improve our understanding of whether floral chemistry is in fact more specialized in obligate plant–pollinator interactions than in less specialized mutualisms (Raguso, 2003).
We found that a blend of volatiles rather than a single volatile constitutes the attractive signal in our model system. This is in contrast to the study by Chen et al. (2009), who reported that a single compound was responsible for the attraction of a fig wasp pollinator to its host. Similar variation has been observed for sexually deceptive orchids, where Ophrys species mimic the multi-component blend of the female-produced sex pheromone of the pollinator (Schiestl et al., 1999; Ayasse et al., 2003), whereas Chiloglottis species rely on individual compounds for pollinator attraction (Schiestl et al., 2003; Schiestl & Peakall, 2005). Available data from more generalized pollinator–plant interactions suggest that blends of compounds rather than unique volatiles function as pollinator attractants. Plepys et al. (2002) used GC-EAD analyses and flight tunnel assays to identify which floral compounds from Platanthera bifolia, an orchid predominantly pollinated by sphingid and noctuid moths, constitute the attractive signal for the generalist moth Autographa gamma. Although 13 floral compounds elicited consistent EAD responses in the moths, only a small subset of the physiologically active compounds (the lilac aldehydes) were found to be critical for attraction, similar to findings in the Silene–Hadena association (Dötterl et al., 2006).
Our PCAs revealed a clear sexual dimorphism in floral scent production in the monoecious B. vitis-idaea, irrespective of whether all identified compounds or only EAD-active compounds were included in the analysis (Fig. 3). The two compounds making up the active pollinator signal (2-PEA and 2-PAN) showed a clear dominance in male flower samples but not in female flower samples (Table 1). In addition, male flowers emitted much higher amounts of the EAD-active compounds compared with female flowers (Fig. 2). Despite this clear difference in odour blends between flower types, the same EAD-active compounds were detected in both male and female flowers, and female moths were attracted to both odour stimuli in Y-tube bioassays (Fig. 5). Although elucidation of the role of sexual dimorphism in floral scent in this mutualism requires further study, the present results have some interesting implications as to how the evolutionary outcomes of floral scent may differ between floral sexes.
One potential cause of the quantitative difference in scent production between the sexes is the difference in their floral sizes (Ashman, 2009). However, although Breynia male flowers are larger than the female flowers (Fig. 1), the difference is probably no greater than twofold, which is far smaller than the observed difference in the amount of scent produced (Fig. 2). Alternatively, and more probably, the observed quantitative difference may be an outcome of sexual selection. As a consequence of asymmetry in reproductive interest between floral sexes, wherein fitness through male function linearly increases with increased access to female flowers, while female fitness is mostly limited by available resources, monoecious plants are expected to allocate resources differently through male and female function to maximize overall fitness. Waelti et al. (2009) showed that, in the S. latifolia–H. bicruris association, male flowers emit larger amounts of pollinator-attracting compounds compared with female flowers, and the pollinator prefers male flowers over female flowers in flight tunnel assays, indicating that the divergence in the floral signal in S. latifolia is caused by sexual selection. A similar pattern has been observed in the gynodioecious strawberry Fragaria virginiana, with hermaphroditic flowers emitting more scent, resulting in more pollinator visits compared with female flowers (Ashman et al., 2005).
However, there was also a qualitative difference between floral scents produced by male and female Breynia flowers. A nonadaptive explanation for the observed difference is that different floral structures, such as stamens and pistils, produce nonidentical compounds, resulting in floral bouquets that differ in chemical composition between the sexes (Ashman, 2009). However, it is also possible that the divergent scents in male and female flowers are the direct result of the highly specialized Epicephala pollination. Because Epicephala females exhibit distinctly different behaviours on male and female flowers, that is, pollen collection on male flowers and pollen deposition and oviposition on female flowers, host plants may be selected to produce different fragrance blends to induce compatible behaviours on flowers of each sex. Further analyses of sexual floral scent dimorphism in related plants without Epicephala pollination, as well as more detailed moth behavioural assays, are necessary to test this adaptive hypothesis.
Although we have identified 2-PEA and 2-PAN as critical components of the floral attractant in B. vitis-idaea, the actual concentrations of the compounds individual Epicephala moths are exposed to under field conditions are difficult to quantify. The doses of compounds used in our bioassays were 200 ng or c. 7000 male flower h−1 equivalents, which was arbitrarily chosen because of the difficulties in predicting such concentrations. The number of scent-producing flowers per plant in Epicephala-pollinated taxa of Breynia, Glochidion and Phyllanthus could be up to hundreds of thousands; thus, the moths are probably guided to their host by the scent signal produced by a large fraction of the flowers on a plant rather than by odour plumes derived from individual flowers. Moreover, because male and female flowers differ in their scent profiles (Fig. 3), the whole bouquet of a single plant may change depending on the relative numbers of male and female flowers produced at a given time. Therefore, the questions of whether the strength of the odour attractant or the relative proportions of male and female flowers affect patterns of moth attraction should be addressed in more detail in future studies.
Another important question is how Epicephala moths discriminate among hosts in areas where several Phyllanthaceae plants coexist. Floral scent has been shown to be an important isolation mechanism between closely related sympatric species in more generalized pollination systems (Waelti et al., 2008), and this trait may also play a key role in the tightly coevolved mutualisms involving pollinating seed predators. If heterospecific pollinations will not lead to seed maturation, an Epicephala female making such a mistake will have zero fitness, as her progeny will starve to death. Thus, to facilitate host specificity in these interactions, strong divergent selection should act on the floral scent profiles among sympatrically occurring species. Scent data are not yet available for additional Breynia species, but analyses on Glochidion plants show that sympatric species differ in the composition of the floral odour, and that female moths discriminate host from nonhost on the basis of floral scent (Okamoto et al., 2007), although the specific compounds responsible for this host specificity have not been identified. Similar odour-based host discrimination has been observed in fig wasps (Grison-Pigéet al., 2002a; Chen et al., 2009).
In summary, our study shows that B. vitis-idaea uses a blend of conventional floral scent compounds as an attractant for its exclusive Epicephala pollinator, demonstrating that system-specific chemistry is not a necessity for efficient host location by species-specific seed predators in a tightly coevolved pollination mutualism. The Phyllantheae–Epicephala association has recently emerged as a promising model system for studies on various aspects of mutualism, and this study and the previous study by Okamoto et al. (2007) show that this association is very suitable for research on how floral scent influences host location and host discrimination in herbivorous insects. Studies of floral scent in other Phyllantheae plants, including those with and without Epicephala pollination, should therefore provide new insights into how olfactory signal coevolution may have facilitated the origin of obligate mutualisms and shaped the subsequent co-diversification of the plants and pollinators.