Separating selection by diurnal and nocturnal pollinators on floral display and spur length in Gymnadenia conopsea

Most plants attract multiple flower visitors that may vary widely in their effectiveness as pollinators. Floral evolution is expected to reflect interactions with the most important pollinators, but few studies have quantified the contribution of different pollinators to current selection on floral traits. To compare selection mediated by diurnal and nocturnal pollinators on floral display and spur length in the rewarding orchid Gymnadenia conopsea, we manipulated the environment by conducting supplemental hand-pollinations and selective pollinator exclusions in two populations in central Norway. In both populations, the exclusion of diurnal pollinators significantly reduced seed production compared to open pollination, whereas the exclusion of nocturnal pollinators did not. There was significant selection on traits expected to influence pollinator attraction and pollination efficiency in both the diurnal and nocturnal pollination treatment. The relative strength of selection among plants exposed to diurnal and nocturnal visitors varied among traits and populations, but the direction of selection was consistent. The results suggest that diurnal pollinators are more important than nocturnal pollinators for seed production in the study populations, but that both categories contribute to selection on floral morphology. The study illustrates how experimental manipulations can link specific categories of pollinators to observed selection on floral traits, and thus improve our understanding of how species interactions shape patterns of selection.     

Geographic structure of pollinator communities, reproductive assurance, and the evolution of self-pollination

Reproductive assurance is often invoked as an explanation for the evolution of self-fertilization in plants. However, key aspects of this hypothesis have received little empirical support. In this study, I use geographic surveys of pollinator communities along with functional studies of floral trait variation to examine the role of pollination ecology in mating system differentiation among populations and subspecies of the annual plant Clarkia xantiana. A greenhouse experiment involving 30 populations from throughout the species' range indicated that variation in two floral traits, herkogamy and protandry, was closely related to levels of autofertility and that trait variation was partitioned mainly among populations. Emasculation experiments in the field showed that autonomous selfing confers reproductive assurance by elevating fruit and seed production. Surveys of pollinator communities across the geographic range of the species revealed that bee pollinator abundance and community composition differed dramatically between populations of the outcrossing subspecies xantiana and the selfing subspecies parviflora despite their close proximity. Specialist bee pollinators of Clarkia were absent from selfing populations, but they were the most frequent visitors to outcrossing populations. Moreover, within the outcrossing subspecies xantiana, there was a close correspondence between specialist abundance and population differentiation in herkogamy, a key mating system trait. This spatial covariation arose, in part, because geographically peripheral populations had reduced herkogamy, higher autofertility, and lower pollinator abundance compared to central populations of xantiana. Finally, I detected strong spatial structure to bee communities both across the range of the species and within the outcrossing subspecies. In both cases, spatial structure was stronger for specialist bees compared to generalist bees, and pollinator communities varied in parallel with population variation in herkogamy. These results provide evidence that mating system differentiation parallels spatial variation in pollinator abundance and community composition at both broad and more restricted spatial scales, consistent with the hypothesis that pollinator abundance and reproductive assurance are important drivers of plant mating system evolution.     

Trait-mediated effects on flowers: artificial spiders deceive pollinators and decrease plant fitness

Although predators can affect foraging behaviors of floral visitors, rarely is it known if these top-down effects of predators may cascade to plant fitness through trait-mediated interactions. In this study we manipulated artificial crab spiders on flowers of Rubus rosifolius to test the effects of predation risk on flower-visiting insects and strength of trait-mediated indirect effects to plant fitness. In addition, we tested which predator traits (e.g., forelimbs, abdomen) are recognized and avoided by pollinators. Total visitation rate was higher for control flowers than for flowers with an artificial crab spider. In addition, flowers with a sphere (simulating a spider abdomen) were more frequently visited than those with forelimbs or the entire spider model. Furthermore, the presence of artificial spiders decreased individual seed set by 42% and fruit biomass by 50%. Our findings indicate that pollinators, mostly bees, recognize and avoid flowers with predation risk; forelimbs seem to be the predator trait recognized and avoided by hymenopterans. Additionally, predator avoidance by pollinators resulted in pollen limitation, thereby affecting some components of plant fitness (fruit biomass and seed number). Because most pollinator species that recognized predation risk visited many other plant species, trait-mediated indirect effects of spiders cascading down to plant fitness may be a common phenomenon in the Atlantic rainforest ecosystem.     

Flower color influences insect visitation in alpine New Zealand

Despite a long-standing belief that insect pollinators can select for certain flower colors, there are few experimental demonstrations that free-flying insects choose between natural flowers based on color. We investigated responses of insect visitors to experimental manipulations of flower color in the New Zealand alpine. Native syrphid flies (Allograpta and Platycheirus) and solitary bees (Hylaeus and Leioproctus) showed distinct preferences for visiting certain flower species. These responses were determined, in part, by flower color, as insects also responded to experimental manipulations of visible petal color in 7 out of 11 tests with different combinations of flower species and insect type. When preferences were detected, syrphid flies chose yellow over white petals regardless of flower species, whereas Hylaeus chose white over yellow Ourisia glandulosa. In some cases, the strength and direction of color preference depended on the context of other floral traits, in which case the response usually favored the familiar, normal combination of traits. Syrphid flies also visited in response to floral morphological traits but did not show preference based on UV reflectance. The unusually high preponderance of white flowers in the New Zealand alpine is not explained by complete generalization of flower color choice. Instead, the insect visitors show preferences based on color, including colors other than white, along with other floral traits. Furthermore, they can respond in complex ways to combinations of floral cues, suggesting that traits may act in nonadditive ways in determining pollinator visitation.     

Advertising to the enemy: enhanced floral fragrance increases beetle attraction and reduces plant reproduction

Many organisms face challenges in avoiding predation while searching for mates. For plants, emitting floral fragrances to advertise reproductive structures could increase the attraction of detrimental insects along with pollinators. Very few studies have experimentally evaluated the costs and benefits of fragrance emission with explicit consideration of how plant fitness is affected by both pollinators and florivores. To determine the reproductive consequences of increasing the apparency of reproductive parts, we manipulated fragrance, pollination, and florivores in the wild Texas gourd, Cucurbita pepo var. texana. With enhanced fragrance we found an increase in the attraction of florivores, rather than pollinators, and a decrease in seed production. This study is the first to demonstrate that enhanced floral fragrance can increase the attraction of detrimental florivores and decrease plant reproduction, suggesting that florivory as well as pollination has shaped the evolution of floral scent.     

Disruption of a belowground mutualism alters interactions between plants and their floral visitors

Plants engage in diverse and intimate interactions with unrelated taxa. For example, aboveground floral visitors provide pollination services, while belowground arbuscular mycorrhizal fungi (AMF) enhance nutrient capture. Traditionally in ecology, these processes were studied in isolation, reinforcing the prevailing assumption that these above- and belowground processes were also functionally distinct. More recently, there has been a growing realization that the soil surface is not a barrier to many ecological interactions, particularly those involving plants (who live simultaneously above and below ground). Because of the potentially large impact that mycorrhizae and floral visitors can have on plant performance and community dynamics, we designed an experiment to test whether these multi-species mutualisms were interdependent under field conditions. Using benomyl, a widely used fungicide, we suppressed AMF in a native grassland, measuring plant, fungal, and floral-visitor responses after three years of fungal suppression. AMF suppression caused a shift in the community of floral visitors from large-bodied bees to small-bodied bees and flies, and reduced the total number of floral visits per flowering stem 67% across the 23 flowering species found in the plots. Fungal suppression has species-specific effects on floral visits for the six most common flowering plants in this experiment. Exploratory analyses suggest these results were due to changes in floral-visitor behavior due to altered patch-level floral display, rather than through direct effects of AMF suppression on floral morphology. Our findings indicate that AMF are an important, and overlooked, driver of floral-visitor community structure with the potential to affect pollination services. These results support the growing body of research indicating that interactions among ecological interactions can be of meaningful effect size under natural field conditions and may influence individual performance, population dynamics, and community structure.     

Imperfect Replacement of Native Species by Non‐Native Species as Pollinators of Endemic Hawaiian Plants

Native plant species that have lost their mutualist partners may require non‐native pollinators or seed dispersers to maintain reproduction. When natives are highly specialized, however, it appears doubtful that introduced generalists will partner effectively with them. We used visitation observations and pollination treatments (experimental manipulations of pollen transfer) to examine relationships between the introduced, generalist Japanese White‐eye (Zosterops japonicus) and 3 endemic Hawaiian plant species (Clermontia parviflora, C. montis‐loa, and C. hawaiiensis). These plants are characterized by curved, tubular flowers, apparently adapted for pollination by curve‐billed Hawaiian honeycreepers. Z. japonicus were responsible for over 80% of visits to flowers of the small‐flowered C. parviflora and the midsize‐flowered C. montis‐loa. Z. japonicus‐visited flowers set significantly more seed than did bagged flowers. Z. japonicus also demonstrated the potential to act as an occasional Clermontia seed disperser, although ground‐based frugivory by non‐native mammals likely dominates seed dispersal. The large‐flowered C. hawaiiensis received no visitation by any birds during observations. Unmanipulated and bagged C. hawaiiensis flowers set similar numbers of seeds. Direct examination of Z. japonicus and Clermontia morphologies suggests a mismatch between Z. japonicus bill morphology and C. hawaiiensis flower morphology. In combination, our results suggest that Z. japonicus has established an effective pollination relationship with C. parviflora and C. montis‐loa and that the large flowers of C. hawaiiensis preclude effective visitation by Z. japonicus. Remplazo Imperfecto de Especies Nativas por Especies No‐Nativas como Polinizadores de Plantas Endémicas de Hawaii     

Herbivory-mediated pollinator limitation: negative impacts of induced volatiles on plant-pollinator interactions

Although induced plant responses to herbivory are well studied as mechanisms of resistance, how induction shapes community interactions and ultimately plant fitness is still relatively unknown. Using a wild tomato, Solanum peruvianum, native to the Peruvian Andes, we evaluated the disruption of pollination as a potential ecological cost of induced responses. More specifically, we tested the hypothesis that metabolic changes in herbivore-attacked plants, such as the herbivore-induced emission of volatile organic compounds (VOCs), alter pollinator behavior and consequentially affect plant fitness. We conducted a series of manipulative field experiments to evaluate the role of herbivore-induced vegetative and floral VOC emissions as mechanisms by which herbivory affects pollinator behavior. In field surveys and bioassays in the plants' native habitat, we found that real and simulated herbivory (methyl jasmonate application) reduced attractiveness of S. peruvianum flowers to their native pollinators. We show that reduced pollinator preference, not resource limitation due to leaf tissue removal, resulted in reduced seed set. Solitary bee pollinators use floral plant volatiles, emitted in response to herbivory or methyl jasmonate treatment, as cues to avoid inflorescences on damaged plants. This herbivory-induced pollinator limitation can be viewed as a general cost of induced plant responses as well as a specific cost of herbivory-induced volatile emission.     

Nectar secondary compounds affect self-pollen transfer: implications for female and male reproduction

Pollen movement within and among plants affects inbreeding, plant fitness, and the spatial scale of genetic differentiation. Although a number of studies have assessed how plant and floral traits influence pollen movement via changes in pollinator behavior, few have explored how nectar chemical composition affects pollen transfer. As many as 55% of plants produce secondary compounds in their nectar, which is surprising given that nectar is typically thought to attract pollinators. We tested the hypothesis that nectar with secondary compounds may benefit plants by encouraging pollinators to leave plants after visiting only a few flowers, thus reducing self-pollen transfer. We used Gelsemium sempervirens, a plant whose nectar contains the alkaloid gelsemine, which has been shown to be a deterrent to foraging bee pollinators. We found that high nectar alkaloids reduced the total and proportion of self-pollen received by one-half and one-third, respectively. However, nectar alkaloids did not affect female reproduction when we removed the potential for self-pollination (by emasculating all flowers on plants). We then tested the assumption that self-pollen in combination with outcrossed pollen depresses seed set. We found that plants were weakly self-compatible, but self-pollen with outcrossed pollen did not reduce seed set relative to solely outcrossed flowers. Finally, an exponential model of pollen carryover suggests that high nectar alkaloids could benefit plants via increased pollen export (an estimate of male function), but only when pollinators were efficient and abundant and plants had large floral displays. Results suggest that high nectar alkaloids may benefit plants via increased pollen export under a restricted set of ecological conditions, but in general, the costs of high nectar alkaloids in reducing pollination balanced or outweighed the benefits of reducing self-pollen transfer for estimates of female and male reproduction.     

Facilitation in an insect-pollinated herb with a floral display dimorphism

Population context should influence pollination success and selection on floral display in animal-pollinated plants because attraction of pollinators depends not only on the characteristics of individual plants, but also on the attractiveness of co-occurring conspecifics. The insect-pollinated herb Primula farinosa is polymorphic for inflorescence height. Natural populations may include both long-scaped plants, which present their flowers well above the soil surface, and short-scaped plants, with their flowers positioned close to the ground. We experimentally tested whether seed production in short-scaped P. farinosa varied with local morph frequency and surrounding vegetation height. In tall vegetation, short-scaped plants in polymorphic populations produced more fruit and tended to produce more seeds than short-scaped plants did in monomorphic populations. In low vegetation, population composition did not significantly affect fruit and seed output of short-scaped plants. The results suggest that long-scaped plants facilitate short-scaped plants in terms of pollinator attraction and that the facilitation effect is contingent on the height of the surrounding vegetation. The documented facilitation should contribute to the maintenance of the scape length polymorphism in ungrazed areas where litter accumulates and vegetation grows tall.     

Daily temporal structure in African savanna flower visitation networks and consequences for network sampling

Ecological interaction networks are a valuable approach to understanding plant-pollinator interactions at the community level. Highly structured daily activity patterns are a feature of the biology of many flower visitors, particularly provisioning female bees, which often visit different floral sources at different times. Such temporal structure implies that presence/absence and relative abundance of specific flower-visitor interactions (links) in interaction networks may be highly sensitive to the daily timing of data collection. Further, relative timing of interactions is central to their possible role in competition or facilitation of seed set among coflowering plants sharing pollinators. To date, however, no study has examined the network impacts of daily temporal variation in visitor activity at a community scale. Here we use temporally structured sampling to examine the consequences of daily activity patterns upon network properties using fully quantified flower-visitor interaction data for a Kenyan savanna habitat. Interactions were sampled at four sequential three-hour time intervals between 06:00 and 18:00, across multiple seasonal time points for two sampling sites. In all data sets the richness and relative abundance of links depended critically on when during the day visitation was observed. Permutation-based null modeling revealed significant temporal structure across daily time intervals at three of the four seasonal time points, driven primarily by patterns in bee activity. This sensitivity of network structure shows the need to consider daily time in network sampling design, both to maximize the probability of sampling links relevant to plant reproductive success and to facilitate appropriate interpretation of interspecific relationships. Our data also suggest that daily structuring at a community level could reduce indirect competitive interactions when coflowering plants share pollinators, as is commonly observed during flowering in highly seasonal habitats.     

Fine-scale flower-visiting behavior revealed by using a high-speed camera

Most estimations of the pollination efficiency of insects have been based on observation by the naked human eye. However, insect behaviors are often too rapid to analyze sufficiently this way. Here we demonstrate the use of high-speed cameras to analyze the fine-scale behaviors of Macroglossum pyrrhosticta, Xylocopa appendiculata, and Papilio dehaanii when visiting Clerodendrum trichotomum. The fine-scale nectar drinking time, number of contacts with anthers and/or stigmas, and frequencies of body part contact with anthers and/or stigmas differed significantly among pollinator species. Pollination efficiency was not equal among pollinators. In addition, M. pyrrhosticta made the least number of contacts with anthers and/or stigmas even though it showed the highest visitation frequency. These results demonstrate that when examined from the viewpoint of rapid visitation behaviors, pollination dynamics differ among pollinator species, and flower visits and pollination rates are not equal.     

Effects of Pollinator Loss on Endemic New Zealand Mistletoes (Loranthaceae)

Abstract: The endemic mistletoes Peraxilla colensoi and P. tetrapetala (Loranthaceae) have declined considerably in New Zealand since 1840, reputedly because of introduced herbivores but coincident with a major decline in native bird densities. We show that at two South Island sites ( Craigieburn and Ohau) there are too few bird pollinators visiting the flowers to allow full fruit set. We studied pollination rates in P. colensoi at Wakefield and P. tetrapetala at Craigieburn over four flowering seasons and P. tetrapetala in one season at Ohau. Supplemental hand pollination increased fruit production 1.25–5.3 times at Craigieburn and Ohau but not at Wakefield. Excluding birds by covering mistletoes with mesh bags decreased fruit set significantly at Wakefield but had little effect at Craigieburn and Ohau. Bellbirds ( Anthornis melanura ) and Tuis (   Prosthemadera novaeseelandiae ) visited flowers significantly more often at Wakefield than at Craigieburn. A lack of pollen tubes in the style, not self-incompatibility or resource shortage, caused the low fruit production in unmanipulated flowers at Craigieburn. Thus, at the two P. tetrapetala sites (Craigieburn and Ohau) fruit set was chronically pollen limited, whereas P. colensoi at Wakefield was not pollen-limited. Data from other Peraxilla sites also suggest pollination failure. Our study suggests that the conservation of Peraxilla species will require maintenance of native bird populations. Tuis and Bellbirds are important pollinators and dispersers of many other New Zealand plants, and the breakdown of such mutualistic relationships may have widespread consequences.     

Analysis of bumblebee visitation sequences within single bouts: implication of the overstrike effect on short-term memory

Pollinators whose foraging habitats consist of several plant types (species or morph) may continue to choose the plant type last visited because information about the type of plant last visited dominates over all other memory contents, in particular of short-term memory. In this study, I extracted this overstrike effect on the plant choices of pollinators by analyzing patterns of visitation sequences within a single round-trip between the hive and foraging patch (bout). First, I simulated the visitation sequences within single bouts with a model to show how factors, including the bees plant-type preferences, the arrangement of plants and the effect of overstrike on short-term memory, affect visitation sequences. Here, bees are assumed to forage in a patch consisting of two plant types (H and L). The model predicts that only the effect of overstrike on short-term memory causes assorted visitation sequences according to plant type (within-bout flower constancy). That is, if the overstrike-effect on short-term memory is the primary determinant of plant choice, then bees will fly to a type-L plant after visiting a type-L plant even if they predominantly visit type-H plants and vice versa. Next, I investigated individual bumblebees visitation sequences at a patch of artificial inflorescences with a set-up similar to that assumed in the model. Two types of inflorescences were arranged on a Cartesian grid. Assorted visitation sequences according to inflorescence type were observed, depending on the distances among inflorescences. This result supports the hypotheses that bees fly to the same plant type as that last visited because short-term memory is displaced (overstruck) with information about the most recently visited plant type.Communicated by M. Giurfa     

Experimental Demonstration of an Allee Effect in American Ginseng

Abstract: Harvesting of wild American ginseng (   Panax quinquefolius ) for the herbal trade has lowered natural population sizes. We tested for reproductive limitation due to small population size (a form of the Allee effect) by experimentally planting "natural" populations numbering 4, 16, and 64 using 4-year-old cultivated plants. Plant size traits and reproductive traits ( bud, flower, green fruit, and mature fruit) were recorded through the ensuing summer. Fruit production per flower and per plant increased in proportion to flowering population size (  p = 0.0063 and p = 0.0017, respectively), strongly suggesting that an Allee effect occurs in very small populations. The increase in fruit production was not explained by either plant or inflorescence size differences. Although population size-dependent pollination, through insufficient pollinator visitation rate or pollen transfer rate, seems the most likely cause of the observed effects, our limited observations of pollinators were not sufficient to demonstrate a change in pollination rates as a function of population size. Knowledge of the presence as well as the mechanism underlying this Allee effect may be especially useful for management and determination of minimum viable population size of the species in the wild.     

Collapse of a pollination web in small conservation areas

A suspected global decline in pollinators has heightened interest in their ecological significance. In a worst-case scenario, the decline of generalist pollinators is predicted to trigger cascades of linked declines among the multiple specialist plant species to which they are linked, but this has not been documented. I studied a portion of a pollination web involving a generalist pollinator, the oil-collecting bee Rediviva peringueyi, and a community of oil-secreting plants. Across 27 established conservation areas located in the Cape Floral Region, I found substantial variation in the bees' occurrence in relation to soil type and the successional stage of the vegetation. Anthropogenic declines were detectable against this background of naturally occurring variation: R. peringueyi was absent from small conservation areas (< 385 ha) in an urban matrix. In the absence of the bee, seed set failed in six specialist plant species that are pollinated only by R. peringueyi but remained high in a pollination generalist, which had replacement pollinators. The findings are consistent with theoretical predictions of the importance of generalist pollinators in maintaining the structure of pollination webs.     

Anther-mimicking floral guides exploit a conflict between innate preference and learning in bumblebees (<Emphasis Type="Italic">Bombus terrestris</Emphasis>)

Bee-pollinated plants are frequently dichogamous: e.g. each flower has a discernable male and female phase, with only the male phase offering a pollen reward. Pollen-collecting bees should therefore discriminate against female-phase flowers to maximise their rate of pollen harvest, but this behaviour would reduce plant fitness due to inferior pollination. Here, we test the hypothesis that flowers use pollen-mimicking floral guides to prevent flower-phase discrimination. Such floral guides resemble pollen in spectral reflection properties and are widespread among angiosperm flowers. In an array of artificial flowers, bumblebees learned less well to discriminate between flower variants simulating different flowering phases when both flower variants carried an additional pollen-yellow guide mark. This effect depended crucially on the pollen-yellow colour of the guide mark and on its spatial position within the artificial flower. We suggest that floral guides evolved to inhibit flower-phase learning in bees by exploiting the innate colour preferences of their pollinators.     

Mimics and magnets: the importance of color and ecological facilitation in floral deception

Plants that lack floral rewards can attract pollinators if they share attractive floral signals with rewarding plants. These deceptive plants should benefit from flowering in close proximity to such rewarding plants, because pollinators are locally conditioned on floral signals of the rewarding plants (mimic effect) and because pollinators are more abundant close to rewarding plants (magnet effect). We tested these ideas using the non-rewarding South African plant Eulophia zeyheriana (Orchidaceae) as a study system. Field observations revealed that E. zeyheriana is pollinated solely by solitary bees belonging to a single species of Lipotriches (Halictidae) that appears to be closely associated with the flowers of Wahlenbergia cuspidata (Campanulaceae), a rewarding plant with which the orchid is often sympatric. The pale blue color of the flowers of E. zeyheriana differs strongly from flowers of its congeners, but is very similar to that of flowers of W. cuspidata. Analysis of spectral reflectance patterns using a bee vision model showed that bees are unlikely to be able to distinguish the two species in terms of flower color. A UV-absorbing sunscreen was applied to the flowers of the orchid in order to alter their color, and this resulted in a significant decline in pollinator visits, thus indicating the importance of flower color for attraction of Lipotriches bees. Pollination success in the orchid was strongly affected by proximity to patches of W. cuspidata. This was evident from one of two surveys of natural populations of the orchid, as well as experiments in which we translocated inflorescences of the orchid either into patches of W. cuspidata or 40 m outside such patches. Flower color and location of E. zeyheriana plants relative to rewarding magnet patches are therefore key components of the exploitation by this orchid of the relationship between W. cuspidata and Lipotriches bee pollinators.     

Predispersal seed herbivores, not pollinators, exert selection on floral traits via female fitness

Herbivores that oviposit in flowers of animal-pollinated plants depend on pollinators for seed production and are therefore expected to choose flowers that attract pollinators. This provides a mechanism by which seed herbivores and pollinators could impose conflicting selection on floral traits. We measured phenotypic selection on floral traits of Lobelia siphilitica (Lobeliaceae) via female fitness to determine the relative strength of selection by pollinators and a specialist predispersal seed herbivore. We were able to attribute selection on flowering phenology to the herbivores. However, no selection could be attributed to pollinators, resulting in no conflicting selection on floral traits. Unlike pollinators, whose preference for certain floral traits does not always translate into higher fitness, any discrimination by seed herbivores is likely to decrease fitness of the preferred floral phenotype. Thus predispersal seed herbivores may be a significant agent of selection on floral traits.     

A Molecular Genetic Assessment of Mating-System Variation in a Naturally Bird-Pollinated Shrub: Contributions from Birds and Introduced Honeybees

Abstract: Introductions of European honeybees have occurred globally, potentially affecting the natural pollination ecology of many plants. Introduced honeybees are now the most frequent visitors to the inflorescences of the self-compatible, bird-pollinated Australian shrub Grevillea macleayana and may therefore be expected to have altered the mating system. To examine the degree to which birds continue to play a role in determining the mating system of G. macleayana in this disturbed system, we compared outcrossing rates in open-pollinated inflorescences with inflorescences from which birds had been selectively excluded. Outcrossing rates were estimated from the microsatellite genotypes of over 100 seeds per population in three populations. Outcrossing rates (  t ) in open-pollinated seeds were surprisingly low (0.062–0.225) and did not vary significantly among the three populations. Nevertheless, outcrossing was significantly lower when birds were excluded (data pooled from all populations). Two lines of evidence suggest that there are temporal fluctuations in outcrossing rate and hence that birds usually have a major effect on the mating system of G. macleayana . First, at one site, t was substantially lower than estimates from an earlier study (0.06 in 1995 cf. 0.85 in 1990). Second, fixation indices based on seeds were high in all populations (  >0.68), whereas values for the established plants ( parental generation) were much lower in two of the three populations (0.06–0.32). Our findings suggest that honeybee activity is so high that the contribution of birds to pollination in G. macleayana is sometimes relatively trivial.