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Transitions from wind pollination to animal pollination were milestones in the diversification of plants, yet floral trait evolution associated with such transitions remains poorly characterized. The fossil record has revealed glimpses of the evolution of inflorescence morphology and pollen traits associated with transitions to animal pollination in early plants (Hu et al., 2008), but other key floral traits, such as color and scent, cannot be determined from fossils. Extant basal flowering plants and cycads provide some insights into trait evolution associated with early transitions from wind pollination to animal pollination (Pellmyr, 1992; Endress & Doyle, 2009). However, functional and comparative studies of more recent transitions from wind pollination to animal pollination, within plant lineages that have reverted from animal pollination to wind pollination, can yield further insights into the roles of functional traits in this important evolutionary transition.
Animal- and wind-pollinated plants are characterized by distinct suites of floral traits that appear to be adaptive. Animal-pollinated flowers tend to have larger, more colorful perianths and produce fragrance and nectar that attract and reward pollinators, whereas wind-pollinated plants tend to have more flowers with fewer (often single) ovules and smoother, less sticky, and more consistently sized (17–58 μm) pollen that promotes pollen transport by wind (Friedman & Barrett, 2008, 2009b). Animal-pollinated plants tend to be cosexual, allowing each pollinator visit to both remove and deposit pollen, whereas wind-pollinated plants are often dioecious (Friedman & Barrett, 2008), which prevents selfing. However, not all of these trait shifts are required for a transition between pollination modes: some may instead occur after a transition, as an adaptation to enhance efficiency of a new pollination mode.
Transitions from animal pollination to wind pollination have occurred at least 65 times in the flowering plants (Linder, 1998) and phylogenetic analyses suggest that only a few traits – nonsticky pollen and small flowers (Linder, 1998), and perhaps unisexual flowers or plants (Friedman & Barrett, 2008) – are required for this transition. Transitions from wind pollination to animal pollination have been much rarer (Dodd et al., 1999), occurring in the families Caryophyllaceae (Weller et al., 1998) and Moraceae (Datwyler & Weiblen, 2004) and possibly Fagaceae (Manos et al., 2001), Salicaceae (Peeters & Totland, 1999), Joinvilleaceae and Flagellariaceae (Linder & Rudall, 2005). In the ancestrally and predominantly wind-pollinated sedge family Cyperaceae (Linder & Rudall, 2005), a number of transitions to insect pollination have been inferred from shifts in floral color and scent (Goetghebeur, 1998; Magalhães et al., 2005) and observations of insects carrying pollen (Stelleman, 1984; Thomas, 1984). Indeed, it appears that there may have been more transitions from wind pollination to insect pollination in the sedge family than in all other plants combined. However, there has been no experimental evidence that insects contribute substantially to pollination of any sedge lineage. Therefore, Friedman & Barrett (2008) did not include putative transitions from wind pollination to insect pollination in sedges, which lack nectar and often occur in open habitats, in their analysis that concluded that transitions from wind pollination to insect pollination are more likely in nectariferous lineages of closed habitats. Thus, sedges will be a key group for advancing our limited understanding of which trait shifts are required for this rare transition.
Anecdotal observations of insects visiting Cyperus obtusiflorus and Cyperus sphaerocephalus, sedges with showy bracts and slight floral scents (Fig. 1), led us to hypothesize that there has been a transition from wind pollination to exclusive insect pollination, associated with modifications of pollen and floral advertizing traits, in the clade containing these species. We tested the following predictions arising from this hypothesis: (1) excluding insects (but not wind) would reduce seed set in these putatively insect-pollinated species but not in wind-pollinated relatives; (2) pollen of these putatively insect-pollinated species would be less motile in the wind than pollen of wind-pollinated relatives; (3) inflorescences of these putatively insect-pollinated species would be convergent in color with those of other insect-pollinated species, and would be more apparent and attractive to pollinating insects than those of wind-pollinated relatives; and (4) inflorescences of these putatively insect-pollinated species would be convergent in emission rate and chemical composition of scent with those of other insect-pollinated species, and would attract pollinating insects, whereas inflorescences of wind-pollinated relatives would emit fewer compounds and be no more scented than their leaves.
Figure 1. Mean (± SE) seed set in three sedge species. Exclusion of insect-borne but not wind-borne pollen reduced seed set in Cyperus obtusiflorus (b) and Cyperus sphaerocephalus (c) but not in Pycreus oakfortensis (a). Conversely, the additional exclusion of wind-borne pollen reduced seed set in P. oakfortensis (a) but not in C. obtusiflorus (b) or in C. sphaerocephalus (c). Sample sizes are (florets/spikelets/inflorescences). Insect visitors pictured are (a) leaf beetle, Monolepta cruciata, Chrysomelidae; (b) honeybee, Apis mellifera, Apidae; and (c) monkey beetle, Eriesthis fallax, Scarabaeidae (foreground). Bars, 5 mm.
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Our hypothesis that the sedges C. obtusiflorus and C. sphaerocephalus are dependent on pollination by insects was strongly supported, as their inflorescences are visited frequently by insects, which are effective in depositing pollen, their pollen has relatively little motility in wind, and there was a dramatic reduction in seed set when insects were excluded. Given that sedges were ancestrally wind-pollinated, these results firmly establish at least one transition to insect pollination in the family. Cyperus sphaerocephalus was considered a variety of C. obtusiflorus in some earlier taxonomic treatments but relationships within the Cyperus clade remain unresolved (Gordon-Gray, 1995; Muasya et al., 2009), so we cannot determine whether these species derive from one or two transitions to insect pollination.
We can think of three alternative explanations for the dramatic reduction in seed set when insects were excluded from inflorescences of C. obtusiflorus and C. sphaerocephalus, but none are tenable. First, the insect-exclusion bags, which modestly reduced deposition of wind-borne pollen, may have hindered wind pollination of these putatively insect-pollinated species. Second, tethering the inflorescences to stakes to support the bags may have prevented them from oscillating in the wind, which could have inhibited wind pollination (Niklas, 1987). However, neither possibility can account for our results for C. obtusiflorus because identical insect-exclusion bags and tethering simultaneously had no effect on seed set of intermingled plants of wind- pollinated P. oakfortensis. These alternative explanations are also implausible for C. sphaerocephalus, given its similarity to C. obtusiflorus in inflorescence morphology, pollen motility and habitat. Third, the bags may have inhibited pollen tube growth or seed development by altering the inflorescence microclimate. This explanation can be excluded because hand-pollinated florets of C. obtusiflorus and C. sphaerocephalus inside identical bags set full complements of seeds. Thus, we can firmly conclude that C. obtusiflorus and C. sphaerocephalus depend on insects for pollination.
While seed set of the two insect-pollinated species was dramatically reduced by exclusion of insects, it was not zero (Fig. 1b,c). With insects excluded, seed set was slightly higher when wind was admitted than when it was excluded (Fig. 1b,c). These differences in seed set could have been caused by a very low level of wind pollination, or by tiny insects occasionally penetrating the insect-exclusion mesh. However, these differences were not statistically significant so the low but nonzero seed set when insects were excluded may well have resulted entirely from rare self-fertilizations, or contamination with cross-pollen before the exclusion treatments were applied to buds.
Visits to inflorescences of the insect-pollinated species were fairly evenly split between bees, flies and beetles (Table 1). These comprised at least 75 insect taxa (P.D. Wragg, unpublished). Except for bract-feeding weevils (Curculionidae), all were observed to feed on pollen. All visitor species carried sedge pollen and representatives of all three orders effected pollination during single visits (P.D. Wragg, unpublished), indicating a generalized pollination system. Solitary bees (Apidae and Halictidae) and certain beetles (Scarabaeidae and Chrysomelidae), which had the highest visitation rates and carried the largest and purest loads of sedge pollen, were likely the most effective pollinators (P.D. Wragg, unpublished). By contrast, the few recorded visits to wind-pollinated species were primarily by a few slow-moving beetle taxa: the insect fauna visiting the wind-pollinated species appeared to be similar to that visiting nonfloral vegetation (P.D. Wragg, pers. obs.). This is to be expected given that the three wind-pollinated species are visually indistinguishable from the green-leaf background, at least to the bees that are likely major pollinators, as indicated by their placement at the center of the color hexagon (Fig. 3) and confirmed by their perceptual distance from green-leaf samples of < 0.045 Euclidean hexagon units, which is near the threshold of discrimination (Dyer & Chittka, 2004). By contrast, both insect-pollinated species are > 0.25 Euclidean hexagon units from the center of the hexagon and from the green-leaf samples (Fig. 3), well above discrimination thresholds, and thus highly apparent to bees (Dyer & Chittka, 2004).
Floral scent composition in C. obtusiflorus and C. sphaerocephalus is chemically consistent with that of other species with generalized insect pollination systems (Dobson, 2006). The scents of these two species are more complex (mean of 28 compounds per sample, Fig. 4a) than that of Eleocharis elegans, the only other showy, potentially insect-pollinated sedge in which floral scent has been characterized: E. elegans yielded only 16 compounds, all aliphatics and sesquiterpenes (Magalhães et al., 2005). Floral scent appears to be associated with insect pollination in sedges as we recorded very few compounds in our wind-pollinated species (Fig. 5, Table S2) and Magalhães et al. (2005) were unable to detect any scent compounds emitted from the inconspicuous, presumably wind-pollinated, inflorescences of Eleocharis sellowiana. The scent emission rates of C. obtusiflorus and C. sphaerocephalus (Fig. 4b) are comparable with those of other insect-pollinated plants (Raguso et al., 2003; Shuttleworth & Johnson, 2009).
White and yellow colors were sufficient to attract all the major pollinators of the insect-pollinated species to model inflorescences, at visit rates comparable to those in natural populations (c. 0.3 visits h−1), even in the absence of scent (Fig. 6, Table 1). Choice experiments using a Y-maze olfactometer suggested that scent plays a role in attracting insects. However, we were unable to demonstrate this in the field, possibly because our blend of compounds was not similar enough to the floral bouquet or because additional replication would be required (Raguso, 2008a). A high proportion of sedge pollen in the pollen loads of solitary bees and monkey beetles suggests that they showed constancy to the insect-pollinated species (P.D. Wragg, unpublished); this may have been enhanced by floral scent (Raguso, 2008b), particularly given the presence of other similarly colored flowers. Nevertheless, our prediction that color and scent are key functional traits in this transition from wind pollination to insect pollination was supported.
Pollen of the insect-pollinated species is slightly larger than that of some wind-pollinated species (see the Results section) but pollen of all our species is within the size range typical of wind pollination (Friedman & Barrett, 2009b). Contrary to expectation, pollen production, and hence pollen:ovule ratios, tend to be higher in our insect-pollinated species than in our wind-pollinated species, but within the range of wind-pollinated Cyperaceae and Poaceae (Subba Reddi & Reddi, 1986). Some showy sedges such as Kyllinga spp. have micro-echinate pollen ornamentation that may be associated with insect pollination (Tanaka et al., 2004; Nagels et al., 2009; Sannier et al., 2009), but our insect-pollinated species did not (Fig. S1). Thus, the main difference in pollen traits was the presence of evident pollenkitt in our insect-pollinated species compared with its apparent absence in the wind-pollinated species. The amount and distribution of pollenkitt determines pollen stickiness and hence the ease with which pollen is removed from anthers by wind, the degree to which it clumps, and the degree to which it adheres to insects, among other functions (Pacini & Hesse, 2005). Thus, pollenkitt is strongly associated with animal pollination (Hesse, 1979; Hu et al., 2008). The presence of pollenkitt exclusively in our insect-pollinated species probably caused the lower pollen motility in the wind tunnel of these species compared with our wind-pollinated species, as well as lower pollen removal by wind in the field for insect-pollinated C. obtusiflorus than for wind-pollinated P. oakfortensis (Fig. S2).
The transition from insect pollination to wind pollination has been posited to be irreversible, given the loss of apparently complex traits such as scent and nectar (Cox & Grubb, 1991; Culley et al., 2002). However, the example of sedges shows that traits sufficient to mediate a transition from wind pollination to insect pollination can be regained. As shown by our experiments, color is a key functional trait for this transition. Showy white or yellow bracts appear to have evolved independently in the Cyperus clade containing our insect-pollinated species, at least three times within Rhynchospora section Dichromena (Thomas, 1984), and several more times in other sedge lineages (Goetghebeur, 1998; W.W. Thomas, pers. comm.), suggesting that they can evolve relatively easily. Floral scent also appears to have evolved independently in the sedges E. elegans (Magalhães et al., 2005) and Ficinia radiata (S.D. Johnson & P.D. Wragg, unpublished), which are distantly related to the Cyperus clade. Monoterpenes characteristic of C. obtusiflorus and C. sphaerocephalus floral scent occur in trace amounts in samples of the wind-pollinated sedges studied here (Table S2) and are present in defensive roles across the plant kingdom (Harrewijn et al., 1994), for example in essential oils of other wind-pollinated sedges (Kilani et al., 2008), so enhanced emission of these compounds in floral scent may also be a relatively easy evolutionary shift. This study shows that it is not necessary to evolve nectar to make the transition to insect-pollination, at least where sexual parts are close enough together for pollen-collecting insects to contact stigmas.
Another reason the transition from insect pollination to wind pollination has been considered irreversible is the tendency for wind-pollinated species to evolve separation of male and female functions in space (through unisexual flowers or plants) or time (dichogamy, especially protogyny) to avoid inbreeding (Cox & Grubb, 1991; Culley et al., 2002). Separation of the sexes makes pollination by pollen-collecting insects unlikely, because they have no reason to visit female flowers. Thus, unisexuality hinders the transition from wind pollination to animal pollination (Friedman & Barrett, 2008). In such lineages a reward provided by both sexes, such as nectar (Friedman & Barrett, 2008), or a mechanism of deception in one or both of the sexes (as in many cycads, Donaldson, 1997; Proches & Johnson, 2009), may be required to allow the transition from wind pollination to insect pollination. Although shifts from wind pollination to animal pollination are less likely in protogynous than protandrous lineages (Sargent & Otto, 2004), in sedges protogyny rarely appears complete enough at the inflorescence level to prevent the evolution of insect pollination. This is indicated by failure of protogyny to prevent geitonogamous self-pollination in our species (P.D. Wragg, unpublished; self-incompatibility prevented seed set) and in several Carex species (Friedman & Barrett, 2009a).
Our discovery of a transition from wind pollination to insect pollination in Cyperus sedges of open habitats appears to run counter to the tendency for this transition to occur in closed habitats with low wind speeds that inhibit wind pollination (Friedman & Barrett, 2008). While the lack of a detailed phylogeny prevents us from excluding the possibility that this transition took place in a closed habitat, this seems unlikely given that most species in this group occur in open wetlands and grasslands (Gordon-Gray, 1995). Hence, we need to explore other potential ecological drivers of this transition, such as higher efficiency of insect pollination than wind pollination at low density: in this study the insect-pollinated C. obtusiflorus occurred at lower density than co-occurring wind-pollinated species, and this appears to be a general trend (Friedman & Barrett, 2009b).
In conclusion, there do not appear to be any unsurpassable barriers to the transition from wind pollination to insect pollination: any wind-pollinated plant, but especially those with male and female functions proximal in space and time, can shift to insect pollination by evolving showy floral color, floral scent and pollen of low motility. It thus seems reasonable to infer that the first plants to evolve insect pollination possessed some combination of these traits.