Nectar Yeasts in the Tall Larkspur Delphinium barbeyi (Ranunculaceae) and Effects on Components of Pollinator Foraging Behavior

Microorganisms frequently colonize the nectar of angiosperm species. Though capable of altering a suite of traits important for pollinator attraction, few studies exist that test the degree to which they mediate pollinator foraging behavior. The objective of our study was to fill this gap by assessing the abundance and diversity of yeasts associated with the perennial larkspur Delphinium barbeyi (Ranunculaceae) and testing whether their presence affected components of pollinator foraging behavior. Yeasts frequently colonized D. barbeyi nectar, populating 54–77% of flowers examined depending on site. Though common, the yeast community was species-poor, represented by a single species, Metschnikowia reukaufii. Female-phase flowers of D. barbeyi were more likely to have higher densities of yeasts in comparison to male-phase flowers. Pollinators were likely vectors of yeasts, as virgin (unvisited) flowers rarely contained yeasts compared to flowers open to pollinator visitation, which were frequently colonized. Finally, pollinators responded positively to the presence of yeasts. Bombus foragers both visited and probed more flowers inoculated with yeasts in comparison to uninoculated controls. Taken together, our results suggest that variation in the occurrence and density of nectar-inhabiting yeasts have the potential to alter components of pollinator foraging behavior linked to pollen transfer and plant fitness.     

Relationships among nectar‐dwelling yeasts,flowers and ants: patterns and incidence on nectar traits

Nectar‐dwelling yeasts are emerging as widely distributed organisms playing a potentially significant and barely unexplored ecological role in plant pollinator mutualisms. Previous efforts at understanding nectar–pollinator–yeast interactions have focused on bee‐pollinated plants, while the importance of nectarivorous ants as vectors for yeast dispersal remains unexplored so far. Here we assess the abundance and composition of the nectar fungal microbiota of the ant‐pollinated plant Cytinus hypocistis, study whether yeast transmission is coupled with ant visitation, and discern whether ant‐ transported yeasts promote changes in nectar characteristics. Our results show that a high percentage of flowers (77%) and plants (94%) contained yeasts, with yeast cell density in nectar reaching up to 6.2 × 10⁴ cells mm^?3, being the highest densities associated with the presence of the nectar‐specialist yeast Metschnikowia reukaufii. The establishment of fungal microbiota in nectar required flower visitation by ants, with 70% of yeast species transported by them being also detected in nectar. Ant‐vectored yeasts diminished the nutritional quality of nectar, with flowers exposed to pollinators and yeasts containing significantly lower nectar sugar concentration than virgin flowers (13.4% and 22.8%, respectively). Nectar of flowers that harbored M. reukaufii showed the lowest quality, with nectar concentration declining significantly with increasing yeast density. Additionally, yeasts modified patterns of interpopulation variation in nectar traits, homo genizing differences between populations in some nectar attributes. We show for the first time that the outcome of the tripartite pollinator–flower–yeast interaction is highly dependent on the identity and inherent properties of the participants, even to the extent of influencing the species composition of this ternary system, and can be mediated by ecological characteristics of plant populations. Through their influence on plant functional traits, yeasts have the potential to alter nectar consumption, pollinator foraging behavior and ultimately plant reproduction.     

Nectar robbers deter legitimate pollinators by mutilating flowers

Nectar robbing – harvesting nectar illegitimately – can have a variety of outcomes for plant sexual reproduction and for the pollinator community. Nectar robbers can damage flowers while robbing nectar, which could affect the behavior of subsequent flower visitors and, consequently, plant reproduction. However, only nectar manipulation by nectar robbers has so far received attention. We found a short-tongued bee, Hoplonomia sp. (Halictidae), mutilating the conspicuous lower petal of the zygomorphic flowers of Leucas aspera (Lamiaceae) while robbing nectar. We hypothesized that the mutilation of the conspicuous lower petal deters legitimate pollinators on L. aspera flowers, which, in turn, might affect plant reproduction. We first assessed the proportion of naturally-robbed flowers in plant populations for three years to confirm that it was not a purely local phenomenon due to a few individual bees. We then studied diversity, community and visitation characteristics of pollinators, nectar dynamics and fruit set in unrobbed and robbed open flowers in naturally-robbed populations. The proportion of robbed flowers varied significantly across sites and years. Robbing did not affect nectar dynamics in flowers, but it did alter flower morphology, so much so that it reduced pollinator visitation and altered the pollinator community on robbed flowers. However, the maternal function of plant reproduction was not affected by nectar robbing. This study for the first time shows that a nectar robber can have an ecologically significant impact on floral morphology.     

Nectar secretion dynamic links pollinator behavior to consequences for plant reproductive success in the ornithophilous mistletoe Psittacanthus robustus

The mistletoe Psittacanthus robustus was studied as a model to link flower phenology and nectar secretion strategy to pollinator behaviour and the reproductive consequences for the plant. The bright‐coloured flowers presented diurnal anthesis, opened asynchronously throughout the rainy season and produced copious dilute nectar as the main reward for pollinators. Most nectar was secreted just after flower opening, with little sugar replenishment after experimental removals. During the second day of anthesis in bagged flowers, the flowers quickly reabsorbed the offered nectar. Low values of nectar standing crop recorded in open flowers can be linked with high visitation rates by bird pollinators. Eight hummingbirds and two passerines were observed as potential pollinators. The most frequent flower visitors were the hummingbirds Eupetomena macroura and Colibri serrirostris, which actively defended flowering mistletoes. The spatial separation between anthers, stigma and nectar chamber promotes pollen deposition on flapping wings of hovering hummingbirds that usually probe many flowers per visit. Seed set did not differ between hand‐, self‐ and cross‐pollinated flowers, but these treatments set significantly more seeds than flowers naturally exposed to flower visitors. We suggest that the limitation observed in the reproductive success of this plant is not related to pollinator scarcity, but probably to the extreme frequency of visitation by territorial hummingbirds. We conclude that the costs and benefits of plant reproduction depend on the interaction strength between flowers and pollinators, and the assessment of nectar secretion dynamics, pollinator behaviour and plant breeding system allows clarification of the complexity of such associations.     

Bouncy versus idles: On the different role of pollinators in the generalist Gentiana lutea L.

Generalist flowers are visited by a broad variety of insects that function as pollinators, occasional visitors and as pollen and/or nectar robbers. Moreover, among legitimate pollinators the pollination efficiency can be different. Nectar greatly affects visitor behaviour and fidelity to a certain species, influencing plant reproductive effort. In this study we have investigated a generalist system (Gentiana lutea L.), examining the role of flower visitors and quantifying the contribution of each pollinating taxon in three natural populations. In order to verify the level of generalization, we introduce an index of Pollinator Performance (PoP), based on insect visitation rate and cross-pollen transport efficiency. Our results confirm the high degree of pollinator-generalization of the study species. Nevertheless, flower visitors show various degree of pollinating performance, mainly defined by their sedentary versus dynamic behaviour. Sedentary insects enhance geitonogamous pollen transfer, which results in reduced seed set and pollen limitation. In particular, an unusual sluggish behaviour was observed in bumblebees feeding on nectar. The hexose-rich abundant nectar offered by G. lutea flowers is remarkably rich in proline and β-alanine amino acids: this composition presumably influences feeding choice and insect dynamism, likely exerting a narcotic effect on pollinators. The consequences on plant fitness are discussed in an evolutionary perspective.     

Visitation rate of pollinators and nectar robbers to the flowers and inflorescences of Tabebuia aurea (Bignoniaceae): effects of floral display size and habitat fragmentation

Large floral displays favour pollinator attraction and the import and export of pollen. However, large floral displays also have negative effects, such as increased geitonogamy, pollen discounting and nectar/pollen robber attraction. The size of the floral display can be measured at different scales (e.g. the flower, inflorescence or entire plant) and variations in one of these scales may affect the behaviour of flower visitors in different ways. Moreover, the fragmentation of natural forests may affect flower visitation rates and flower visitor behaviour. In the present study, video recordings of the inflorescences of a tree species (Tabebuia aurea) from the tropical savannah of central Brazil were used to examine the effect of floral display size at the inflorescence and tree scales on the visitation rate of pollinators and nectar robbers to the inflorescence, the number of flowers approached per visit, the number of visits per flower of potential pollinators and nectar robbers, and the interaction of these variables with the degree of landscape disturbance. Nectar production was quantified with respect to flower age. Although large bees are responsible for most of the pollination, a great diversity of flower insects visit the inflorescences of T. aurea. Other bee and hummingbird species are highly active nectar robbers. Increases in inflorescence size increase the visitation rate of pollinators to inflorescences, whereas increases in the number of inflorescences on the tree decrease visitation rates to inflorescences and flowers. This effect has been strongly correlated with urban environments in which trees with the largest floral displays are observed. Pollinating bees (and nectar robbers) visit few flowers per inflorescence and concentrate visits to a fraction of available flowers, generating an overdispersed distribution of the number of visits per inflorescence and per flower. This behaviour reflects preferential visits to young flowers (including flower buds) with a greater nectar supply.     

Nectar ecology of Datura ferox (Solanaceae): an invasive weed with nocturnal flowers in agro-ecosystems from central Argentina

Plant–pollinator interactions provide highly important ecological functions, and are influenced by floral nectar characteristics. The night blooming Datura ferox is an excellent model to test general hypotheses on the relationship between nectar traits (e.g., nectar secretion patterns, nectar chemical composition), pollinators and reproductive success for invasive, weedy species in highly modified ecosystems as crop fields. We hypothesized an adjustment between nectar composition and secretion dynamics through flower anthesis and the activity and requirements of nocturnal pollinators. Nectar chemical analyses showed low quantities of amino acids and lipids, phenolics, and alkaloids were not detected. D. ferox showed sucrose-dominant nectar with comparable amount of hexoses. Sugar proportions did not vary between populations or during flowering season. Most nectar is secreted before flower opening. Nectar resorption was detected at the end of anthesis. Experimentally drained flowers of both populations increased nectar production up to 50 % in the total amount of sugar per flower compared to control flowers. Nectar standing crop was relatively constant during the flowering season, but differences were detected between populations. Nectar traits of D. ferox would be favoring cross-pollination and maintaining seed production of this weed, since recently open flowers display a higher amount of nectar and they can renew nectar after a pollinator visit or reabsorb it at the end of anthesis. This nectar source may be important for native pollinators considering that human-induced forest fragmentation is related with the impoverishment of native flora from agro-ecosystems.     

Nectar Sugar Production across Floral Phases in the Gynodioecious Protandrous Plant Geranium sylvaticum

Many zoophilous plants attract their pollinators by offering nectar as a reward. In gynodioecious plants (i.e. populations are composed of female and hermaphrodite individuals) nectar production has been repeatedly reported to be larger in hermaphrodite compared to female flowers even though nectar production across the different floral phases in dichogamous plants (i.e. plants with time separation of pollen dispersal and stigma receptivity) has rarely been examined. In this study, sugar production in nectar standing crop and secretion rate were investigated in Geranium sylvaticum, a gynodioecious plant species with protandry (i.e. with hermaphrodite flowers releasing their pollen before the stigma is receptive). We found that flowers from hermaphrodites produced more nectar than female flowers in terms of total nectar sugar content. In addition, differences in nectar production among floral phases were found in hermaphrodite flowers but not in female flowers. In hermaphrodite flowers, maximum sugar content coincided with pollen presentation and declined slightly towards the female phase, indicating nectar reabsorption, whereas in female flowers sugar content did not differ between the floral phases. These differences in floral reward are discussed in relation to visitation patterns by pollinators and seed production in this species.     

Nectar robbing of a carpenter bee and its effects on the reproductive fitness of Glechoma longituba (Lamiaceae)

The floral biology and pollination process of Glechoma longituba (Nakai) Kuprian, a clonal gynodioecious, perennial herb that is widely distributed in China was investigated in natural populations. During visits to the flowers of G. longituba, the carpenter bee—Xylocopa sinensis mainly displayed nectar-robbing behavior with minimal pollination. Nectar robbing behavior began at the onset of flowering and continued for about 3 weeks ending at about the middle of the flowering period. A total of 18.6% floral visits in this period were by nectar robbers, with about 90% of the flowers in the study populations being subjected to two or two nectar-robbing episodes. During controlled experiments, lower pollination efficiency was recorded for X. sinensis compared to legitimate pollinators. Pollination by the robber-like pollinator X. sinensis during the early-mid phase of the flowering season yielded seeds of 16.16%. Although nectar robbing by the carpenter bee seemed to have a slight enhancing effect on seed set in G. longituba, this effect was effectively masked by the more pronounced detrimental effect of nectar robbing. Experiments indicated that nectar robbing by the carpenter bee reduced seed production by more than 26%, largely owing to the consequent shortening of the life span of robbed flowers. Furthermore, 10.43% of the ovules and 13.04% of the nectaries in the robbed flowers were damaged, thus causing a decrease or entire loss of reproductive opportunity in the robbed flowers. In addition, a higher number of pollen grains remained on the androecia of robbed flowers indicating that nectar robbing may have a lowering effect on male fitness in G. longituba.     

Variability in nectar production and standing crop, and their relation to pollinator visits in a Mediterranean shrub

Nectar standing crops in flowers within an individual plant are often highly variable. This variability may be a by-product of the foraging activity of insect pollinators. Alternatively, plants may be selected to produce highly variable rewards to reduce consecutive visitation by risk-averse pollinators, thus diminishing within-plant pollen transfer. This study evaluated the roles of pollinator control vs. plant control over nectar variability in the bee-pollinated shrub Rosmarinus officinalis L. (Lamiaceae). We sampled nectar production, standing crop and pollinator visits in three shrubs of one population over 17 days during one blooming season. Nectar production rates were highly variable (CV = 1.48), and increased after rainy days. Nectar standing crops were even more variable (CV = 2.16), decreased with increasing temperatures, and increased with time since the last rain. Pollinator visit rates decreased with variability in nectar standing crops, increased with flower number per shrub, and were unaffected by variability in nectar production rates. Repeated sampling of marked flowers revealed no correlation between their nectar standing crops and production rates. These findings support the role of reward variance in reducing pollinator visits, but suggest that plants are not in complete control of this variability. Rather, plant-generated variability can be modified by intensive foraging activity of pollinators. Such pollinator control over nectar variability is likely to reduce the selective advantage of plant-generated reward variation. Handling Editor: Neal Williams.     

Nectar Production of Calliandra longipedicellata (Fabaceae: Mimosoideae), an Endemic Mexican Shrub with Multiple Potential Pollinators

The nectar resource environment across which nectarivores forage may be patchy and variable. To understand the sources and consequences of such a variation, nectar production was investigated in Calliandra longipedicellata . Nectar was measured once a month throughout a 3-mo winter season in two successive years at three sites. We also conducted diurnal and nocturnal field observations to describe visitation rates of floral visitors, and a pollinator exclusion experiment to evaluate diurnal and nocturnal pollination at the three sites. In all populations, nectar secretion was primarily nocturnal, although flowers produced some nectar during the day. Sugar production per flower varied significantly at both the seasonal and population levels, although nectar production rates and a well-defined afternoon to morning production pattern were consistent across months, populations, and years. Average nectar production rates were high compared to other Calliandra species, and to most hummingbird- or hawkmoth-pollinated plants in the region. Flowers were regularly visited by hawkmoths, bats, hummingbirds and various diurnal insects, and all populations had similar rates of visitation. Nocturnal insects had the highest overall visitation rates (three times as high as those by diurnal insects). Fruit production varied among pollination treatments and populations, and significant differences were found in fruit production when flowers exposed to both diurnal and nocturnal visitation were compared to flowers exposed only to diurnal visitation. Our results and the bright-red staminal filaments of C. longipedicellata indicate lack of specialization for particular pollinators.     

Long corollas as nectar barriers in <Emphasis Type="Italic">Lonicera implexa</Emphasis>: interactions between corolla tube length and nectar volume

Long corollas are a classical example of nectar barriers, because they prevent undesired visitors from consuming the reward intended for more effective pollinators. As the investment in nectar barriers increases, flower attractiveness and nectar rewards may also increase to maintain loyal visitation of most effective pollinators; and flowers may become more prone to nectar robbing. We evaluated the effect of nectar barriers (corolla tube length), two related traits (nectar volume and upper lip size) and the associated risk of nectar robbing, on the fecundity of Lonicera implexa plants from three populations differing in the abundance of its most efficient pollinator, the hummingbird hawkmoth Macroglossum stellatarum. Corolla tube length varied most among individuals within populations (45–46 % of total variance) and inflorescences within individuals (23–32 %), and showed little variation among populations (0.2–11 %). Longer corolla tubes were always associated with larger nectar volumes and larger upper lips, although the strength of the relationships varied across populations and years. Robbing frequency increased with corolla tube length, decreased with nectar volume and upper lip size, and its weak effects on fecundity were predominantly positive. Plant fecundity peaked at two different optima: long corollas with little nectar and short corollas with abundant nectar. However, the exact shape of the interaction between corolla length and nectar volume, as well as the combination of traits showing the highest fecundity, differed between populations and years. This variation could be explained by among-population differences in pollinator assemblages, and inter-annual changes in resources dedicated to reproduction. Our study shows that large nectar volumes can modulate the effect of corolla length as a nectar barrier, and that the combination of these two traits that maximises fecundity may be related to the identity of pollinators within each population.     

Pollination by passerine birds: why are the nectars so dilute?

Bird-pollinated flowers are known to secrete relatively dilute nectars (with concentrations averaging 20–25% w/w). Many southern African plants that are pollinated by passerine birds produce nectars with little or no sucrose. Moreover, these hexose nectars are extremely dilute (10–15%). This suggests a link between sugar composition and nectar concentration. Nectar originates from sucrose-rich phloem sap, and the proportion of monosaccharides depends on the presence and activity of invertase in the nectary. Hydrolysis of sucrose increases nectar osmolality and the resulting water influx can potentially convert a 30% sucrose nectar into a 20% hexose nectar, with a 1.56 times increase in volume. Hydrolysis may also increase the gradient for sucrose transport and thus the rate of sugar secretion. When sucrose content and refractometer data were compared, some significant correlations were seen, but the occurrence of sucrose-rich or hexose-rich nectars can also be explained on phylogenetic grounds (e.g. Erythrina and Protea). Hexose nectars may be abundant enough to drip from open flowers, but evaporation leads to much variability in nectar concentration and increases the choices available to pollinators.     

贠建全:广东荷包岛维管束植物名录

以分布于秦岭的金花忍冬（Lonicera chrysantha Turcz.）、忍冬（L.japonica Thunb.）、葱皮忍冬（L.ferdinandii Franch.）和金银忍冬（L.maackii（Rupr.）Maxim.）为对象,通过定位观察、人工授粉实验、人为设置实验斑块的方法对忍冬属4种植物的开花生物学特性、繁育系统、花色变化现象、传粉过程进行了研究。结果表明,4种植物的单花花期、花部特征存在差异。人工授粉实验显示,4种植物均存在一定的花粉限制,自交不亲和。除葱皮忍冬外,其余3种植物随着花色由白变黄,花粉和花蜜报酬减少、雌雄生殖能力逐渐降低;葱皮忍冬花变色后花蜜量变化不显著,且仍保留较强的雌性生殖能力。变色花的保留被认为是植物的一种生殖策略,通过增大植物的花展示来扩大自身的吸引力,以吸引更多远距传粉者访花。人为控制白、黄花不同数量比的实验结果表明,大多数传粉者偏向访问白花（变色前的花）,且白花提供的报酬量和黄花（变色后的花）数量显著影响传粉者的访花频率,即当花蜜量减少或黄花数量增多时,传粉者访花频率随之降低。因此,我们认为忍冬属4种植物的花色变化可能除了增大植物对远距传粉者的吸引力外,对近距传粉者的访花行为也可能具有一定的影响。当传粉者接近植株时,变色后的花可能暗示其花蜜（花粉）报酬已经发生变化,并驱使昆虫离开并飞向同株或异株植物新开放的报酬丰富的白花,这既有利于提高传粉者的觅食效率,又能降低植物同株异花授粉的几率,对忍冬属植物及传粉者都具有重要意义,是植物长期与授粉昆虫相互适应的反映。     

Variation in nectar volume and composition of Impatiens capensis at the individual,plant, and population levels

Although the volume and chemical composition of nectars are known to vary among plant species and to affect pollinator response to plants, relatively little is known of the variation in volume, and sugar and amino acid composition within species. We collected nectar from Impatiens capensis in a nested design: three flowers from each of three plants from each of three populations. This design enabled us to quantify variation within individual plants, among plants within populations, and among populations. Using high performance liquid chromatography, we analyzed the sugar and amino composition of the 27 flowers. Analysis of variance showed that none of the parameters (volume, concentrations of three sugars and 24 amino compounds) varied within individuals. Variation in nectar volume was not significant among plants but was nearly significant among populations. Of the three sugars detected (sucrose, glucose, and fructose), the only significant variation was that of sucrose among populations. Concentrations of 12 amino compounds varied significantly at the plant level while 7 amino compounds varied among populations. The results indicate that: (1) pooling of nectar samples from flowers of individual plants can be an acceptable methodology for those seeking to understand within species variation; (2) amino compounds appear to vary more than either volumes or sugar concentrations; (3) future studies should assess how much of the observed variation is due to genetic versus environmental differences; (4) additional studies should examine the geographic variation in nectar parameters and pollinators of I. capensis in order to assess the role different pollinators play in shaping nectar composition.     

Pollinator foraging modifies nectar sugar composition in Helleborus foetidus (Ranunculaceae):An experimental test

We experimentally tested the hypothesis that the extensive within-plant variation of nectar sugar composition in Helleborus foetidus (Ranunculaceae) and other species results from differences between flowers and nectaries in pollinator visitation history. Experiments were conducted to mimic single-nectary visits by wild-caught individuals of the main bee pollinators of H. foetidus, which were assayed for their capacity to modify the sugar composition of natural and artificial nectar. Experimental nectar probing with bee mouthparts induced extensive changes in proportional sugar composition 48 h after treatment, and bee taxa differed widely in their effects. Nectar probing by Andrena, medium-sized Anthophoridae, Apis mellifera, and Lasioglossum had no subsequent effects on nectar sugar composition, while probing by Bombus terrestris and B. pratorum induced an extensive reduction in percentage sucrose, a marked increase in percentage fructose, and a slight increase in percentage glucose. Results support the hypothesis that stochastic variations among flowers or nectaries in the taxonomic identity of recent visitors and their relative visitation frequencies may eventually generate very small-scale mosaics in nectar sugar composition. Changes in nectar sugar composition following bumblebee probing may be the consequence of nectar contamination with pollinator-borne nectarivorous yeasts.     

Presence of yeasts in floral nectar is consistent with the hypothesis of microbial-mediated signaling in plant-pollinator interactions

Olfactory floral signals are significant factors in plant-pollinator mutualisms. Recently, unusual fermentation odors have been described in the nectar and flowers of some species. Since yeasts are common inhabitants of many angiosperms nectars, this raises the possibility that nectar yeasts may act as causal agents of fermentation odors in flowers and, therefore, as possible intermediate agents in plant signaling to pollinators. A recent field study has reported that nectar yeasts were quite frequent in floral nectar across three different regions in Europe and America, where they reached high densities (up to 10⁵ cells/mm³). Yeast incidence in floral nectar differed widely across plant host species in all sampling sites. A detailed study currently in progress on one of the species surveyed in that study (Helleborus foetidus, Ranunculaceae) has detected that, in addition to interespecific differences in yeast incidence, there is also a strong component of variance in yeast abundance that takes place at the subindividual level (among flowers of the same plant, among nectaries of the same flower). If yeast metabolism is eventually proved to contribute significantly to floral scent, then multilevel patchiness in the distribution of nectar yeasts (among species, among individuals within species, and among flowers and nectaries of the same individual) might contribute to concomitant multilevel variation in plant signaling and, eventually, also in pollination success, pollen flow and plant fitness.Key words: nectar, yeast, scent, plant-animal interaction, plant signalingPollinators forage on a wide range of flowers that differ in morphology, color, scent and quality and quantity of reward. The majority of these floral features are important visual and olfactory cues that are directly related to plant-pollinators signaling and the pollination process.^1–12 Recently, the intriguing possibility has been raised that microbial communities (especially nectarivorous yeasts) inhabiting flowers could explain better than, or in addition to, plant physiology itself, certain floral features that participate in plant-pollinators signaling, like yeasty nectar or floral scent.^13,14 However, some of these suggestions are based on circumstantial or indirect evidence indicative of the presence of microbes in flowers. For example, fermentation odors have been described in a number of Angiosperms,^14–16 in which different compounds found in nectar were not shared with any other floral parts.¹³ In addition, yeasty odors (ketones and shortchain alcohols) have only been observed in mature flowers that were already visited by pollinators and thus potentially contaminated with microbes, in contrast, for example, to the sesquiterpenes isolated in immature flowers that are also common in the foliage of many plants.¹⁴ Yeasty odors were found in species whose flowers are long-lived, produce large amounts of nectar, and are visited by flies and beetles, which are known to act as yeast vectors to flowers.^17–19 In spite of these plausible suggestions, studies indicating a potential role of microbes in the origin of floral scents generally have not looked directly for their presence or abundance in floral nectar, which clearly would provide critical empirical evidence in support of the hypothesis of microbial-mediated signaling in plant-pollinator interactions.That yeasts are common inhabitants of floral nectars was well known to microbiologists more than a century ago^20,21 and has been recently corroborated by Herrera et al.²² This study was conducted at three widely separated areas, which differed greatly in ecological features and biogeographical affinities: two study sites were located in the Southern Iberian Peninsula, about 350 km apart, and one in Yucatán Peninsula, eastern Mexico. Floral nectar samples from 40, 63 and 37 species, belonging to 21, 23 and 21 families, were examined microscopically for yeast cells at these three areas. Yeasts occurred very frequently in floral nectar at all areas, as revealed by the high proportion of nectar samples that contained them (31.8%, 42.3% and 54.4%; samples from all species at each site combined). In addition to being quite frequent in nectar samples, yeast cells often reached extraordinarily high densities in floral nectar at the three areas, which reached roughly 4 × 10⁵ cells/mm³. When plant species, rather than individual nectar samples, were considered as the units for analyses, Herrera et al.²² found wide variation among species in both the frequency of occurrence and the density of yeasts in nectar samples. A significant fraction of such variation was found to be correlated with differences in pollinator composition, a link between pollination ecology and floral nectar microbiology that has remained unexplored until now. Similar results showing high densities and frequency of occurrence of yeasts in nectar, and interespecific differences in these magnitudes related to variation in pollinator composition, have been also reported by de Vega et al.²³ for 40 South African plant species, which further supports the generality of the phenomenon. In addition to interespecific differences in the prevalence of nectar yeasts, the data examined by Herrera et al.²² and de Vega et al.²³ revealed also considerable intraespecific variability (i.e., among individuals plants of the same species), although this aspect of results was not explicitly considered in their studies.A study currently in progress has documented patterns of intraespecific variability in yeast occurrence in the nectaries of Helleborus foetidus (Ranunculaceae), a winter-flowering, bumble bee-pollinated perennial herb whose long-lived flowers last for roughly two weeks. Frequency of occurrence and cell density of yeasts in nectar were studied at six populations of this species from Sierra de Cazorla (SE Spain). Helleborus foetidus flowers have five separated horn-shaped nectaries hidden at the corolla base, each of which produces up to 5 µl of nectar. This enabled us to study patterns of yeast occurrence also at the within-flower level. At each population, total variance in yeast cell density on a per-nectary basis was partitioned into components due to differences between individual plants, flowers within plants and nectaries within flowers. We found extreme differences concerning the abundance and frequency of yeasts in H. foetidus nectar, the magnitude of intraespecific variation being similar or even greater than variation found in interespecific comparisons in the same study area (Pozo MI, et al. unpublished results). Our data suggest that temporal and spatial factors may explain differences regarding yeast abundance in H. foetidus nectar, and possibly other species as well. The largest component of intraespecific variance in yeast abundance occurred at the subindividual level, and was mainly accounted for by the variance between nectaries in the same flower (Fig. 1). This intraespecific variation in nectarivorous yeast incidence can have some important implications related to plant-pollinators interactions and, more specifically, to plant signaling, as outlined below.Open in a separate windowFigure 1Hierarchical dissection of variance in yeast abundance in single-nectary nectar samples of Helleborus foetidus. (A) Temporal patterns. Collection dates and plant, flower within plant and nectary within flower as hierarchical levels of variance analyzed. (B) Spatial patterns. Population, plant within populations and flower within plants as hierarchical levels of variance analyzed.Nectar-inhabiting yeasts modify certain flower characteristics linked to pollinator foraging behavior, such as nectar sugar composition and energetic value, by reducing total sugar concentration and altering the relative proportions of constituent sugars (sucrose, glucose and fructose) and the sucrose:hexose ratio.^23–26 Furthermore, as noted above, yeasts could be also implicated in floral volatiles emission.^13,14 Consequently, yeast incidence (measured both by frequency and abundance of yeast cells in nectar samples) may have been modifying signaling cues which have been postulated to be intrinsic plant species-specific. Although an empirical connection between yeast presence and fermentation nectar odor is needed, the fact that nectarivorous yeast presence would be as variable as described by our studies could imply the same variability for plant species signaling aspects, along with potential consequences for pollinators, since variance was mainly accounted for by variation below individual plant level. For example, in H. foetidus study variance in yeast abundance occurs mainly at the single nectary, which matches with the smallest scale that is perceived by a foraging insect. The fact that nectar is an important floral reward that plays a decisive role in the establishment of plant-pollinator mutualisms, together with the recently confirmed ubiquity of nectarivorous yeasts which could be acting as parasites of such mutualisms, open up new and exciting avenues to explore their effect on pollination success and pollen flow^27–30 and finally on plant fitness.^31–35     

Visual cues and foraging choices: bee visits to floral colour phases in Alkanna orientalis (Boraginaceae)

When a pollination vector is required, any mechanism that contributes to floral visitation will potentially benefit the reproductive fitness of a plant. We studied the effect of floral colour change in the desert perennial Alkanna orientalis on the foraging behaviour of the solitary bee Anthophora pauperata . Flowers changed colour over time from bright yellow (with moderate nectar reward) to pale yellow/white (with significantly lower nectar reward). Bee visitation was non-random with respect to colour phase availability within the flower population and was biased towards the more rewarding flowers. At plants where the availability of colour phases had been manipulated experimentally to produce 'bright' or 'pale' plants, bees visited significantly more flowers (and for longer periods) on the bright plants. The change of flower colour was not simply age-related; we observed variation in the temporal course of colour change and our data suggest that visitation, leading to deposition of cross-pollen, can accelerate the process. In subpopulations with limited pollinators, Alkanna can influence bees by using their colour-related foraging preferences to alter visitation patterns.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 87 , 427–435.     

Ecological context influences pollinator deterrence by alkaloids in floral nectar

Secondary compounds may benefit plants by deterring herbivores, but the presence of these defensive chemicals in floral nectar may also deter beneficial pollinators. This trade-off between sexual reproduction and defense has received minimal study. We determined whether the pollinator-deterring effects of a nectar alkaloid found in the perennial vine Gelsemium sempervirens depend on ecological context (i.e. the availability of alternative nectar sources) by monitoring the behavioural response of captive bumblebees (Bombus impatiens, an important pollinator of G. sempervirens in nature) to nectar alkaloids in several ecologically relevant scenarios. Although alkaloids in floral nectar tended to deter visitation by bumblebees, the magnitude of that effect depended greatly on the availability and nectar properties of alternative flowers. Ecological context should thus be considered when assessing ecological costs of plant defense in terms of pollination services. We consider adaptive strategies that would enable plants to minimize pollinator deterrence because of defensive compounds in flowers.     

Flowers as islands: spatial distribution of nectar-inhabiting microfungi among plants of Mimulus aurantiacus, a hummingbird-pollinated shrub

Microfungi that inhabit floral nectar offer unique opportunities for the study of microbial distribution and the role that dispersal limitation may play in generating distribution patterns. Flowers are well-replicated habitat islands, among which the microbes disperse via pollinators. This metapopulation system allows for investigation of microbial distribution at multiple spatial scales. We examined the distribution of the yeast, Metschnikowia reukaufii, and other fungal species found in the floral nectar of the sticky monkey flower, Mimulus aurantiacus, a hummingbird-pollinated shrub, at a California site. We found that the frequency of nectar-inhabiting microfungi on a given host plant was not significantly correlated with light availability, nectar volume, or the percent cover of M. aurantiacus around the plant, but was significantly correlated with the location of the host plant and loosely correlated with the density of flowers on the plant. These results suggest that dispersal limitation caused by spatially nonrandom foraging by pollinators may be a primary factor driving the observed distribution pattern.