Effects of pollen and nectar inoculation by yeasts, bacteria or both on bumblebee colony development.

It is increasingly recognized that gut microbiota have a major effect on the physiology, biology, ecology and evolution of their animal hosts. Because in social insects, the gut microbiota is acquired through the diet and by contact with nest provisions, it can be hypothesized that regular supplementation of microorganisms to the diet will have an impact on the fitness of the consumer and on the development of the whole colony. To test this hypothesis, we investigated how supplementation of bacteria, yeasts, and combinations of the two to either pollen or nectar affected colony development in the social bumblebee Bombus terrestris. Three yeasts and three bacterial species that live at the flower-insect interface were used in the experiments and the development of bumblebee colonies was monitored over a period of 10 weeks. The results showed that administration of microbes via pollen had a stronger positive impact on colony development than when provided via sugar water. Supplementation of bacteria led, in general, to a faster egg laying, higher brood size and increased production of workers during the first weeks, whereas yeasts or a combination of yeasts and bacteria had less impact on colony development. However, the results differed between microbial species, with Wickerhamiella bombiphila and Rosenbergiella nectarea showing the strongest increase in colony development. Torulaspora delbrueckii induced early male production, which is likely a fitness cost. We conclude that the tested bacteria-yeast consortia did not result in better colony development than the interacting species alone.

The effect of DNA methylation on bumblebee colony development.

BACKGROUND: Although around 1% of cytosines in bees' genomes are known to be methylated, less is known about methylation's effect on bee behavior and fitness. Chemically altered DNA methylation levels have shown clear changes in the dominance and reproductive behavior of workers in queen-less colonies, but the global effect of DNA methylation on caste determination and colony development remains unclear, mainly because of difficulties in controlling for genetic differences among experimental subjects in the parental line. Here, we investigated the effect of the methylation altering agent decitabine on the developmental rate of full bumblebee colonies. Whole genome bisulfite sequencing was used to assess differences in methylation status. RESULTS: Our results showed fewer methylated loci in the control group. A total of 22 CpG loci were identified as significantly differentially methylated between treated and control workers with a change in methylation levels of 10% or more. Loci that were methylated differentially between groups participated in pathways including neuron function, oocyte regulation and metabolic processes. Treated colonies tended to develop faster, and therefore more workers were found at a given developmental stage. However, male production followed the opposite trend and it tended to be higher in control colonies. CONCLUSION: Overall, our results indicate that altered methylation patterns resulted in an improved cooperation between workers, while there were no signs of abnormal worker dominance or caste determination.

Yeast-nectar interactions: metacommunities and effects on pollinators.

About 90% of all flowering plant species are pollinated by animals. Animals are attracted to flowers because they often provide food in the form of nectar and pollen. While floral nectar is assumed to be initially sterile, it commonly becomes colonized by yeasts after animals have visited the flowers. Although yeast communities in floral nectar appear simple, community assembly depends on a complex interaction between multiple factors. Yeast colonization has a significant effect on the scent of floral nectar, foraging behavior of insects and nectar consumption. Consumption of nectar colonized by yeasts has been shown to improve bee fitness, but effects largely depended on yeast species. Altogether, these results indicate that dispersal, colonization history and nectar chemistry strongly interact and have pronounced effects on yeast metacommunities and, as a result, on bee foraging behavior and fitness. Future research directions to better understand the dynamics of plant-microbe-pollinator interactions are discussed.

Addition of pollen increases growth of nectar-living yeasts.

Nectar is frequently inhabited by a limited number of microorganisms. Nonetheless, these species can quickly attain relatively high cell densities. This is quite surprising because of the limited availability of nutrients and unbalanced Carbon/Nitrogen ratios. Because nectar yeasts commonly aggregate around pollen and pollen grains are particularly rich in proteins, it has been suggested that the presence of pollen in nectar contributes to enhanced growth of yeasts in nectar, but compelling experimental evidence is still lacking. In this study, we conducted in vitro growth experiments to investigate whether the addition of pollen to sugar water increased growth of yeasts that naturally occur in nectar and honey provisions: Metschnikowia reukaufii, Starmerella orientalis and Torulaspora delbueckii. Our results indicate that yeasts benefit from the addition of pollen to a sugar-dominated medium, but the effects depended on type of pollen used. Overall, these results demonstrate that pollen plays an important role in the population dynamics of nectar-inhabiting yeasts and supports the idea that the chemical composition and the concentration of dehisced pollen may be more important factors determining the population growth of nectar yeasts than the chemistry of the nectar itself.

Hibernation Leads to Altered Gut Communities in Bumblebee Queens (Bombus terrestris).

Many reptiles, amphibians, mammals, and insects practice some form of hibernation during which their metabolic rate is drastically reduced. This allows them to conserve energy and survive the harsh winter conditions with little or no food. While it can be expected that a reduction in host metabolism has a substantial influence on the gut microbial community, little is known about the effects of hibernation on the composition of the microbial gut community, especially for insects. In this study, we assessed and compared the bacterial gut community composition within the midgut and ileum of indoor-reared queens of Bombus terrestris before and after an artificial hibernation period of 16 weeks. Deep sequencing of 16S ribosomal RNA gene amplicons and clustering of sequence reads into operational taxonomic units (OTUs) at a similarity threshold of 97% revealed several bacterial taxa that are known to be strongly associated with corbiculate bees. Bacterial community composition after hibernation compared to before hibernation was characterized by higher OTU richness and evenness, with decreased levels of the core bacteria Gilliamella (Proteobacteria, Orbaceae) and Snodgrassella (Proteobacteria, Neisseriaceae), and increased relative abundance of non-core bacteria, including several psychrophilic and psychrotrophic taxa.

Surviving in the absence of flowers: do nectar yeasts rely on overwintering bumblebee queens to complete their annual life cycle?

Floral nectar represents an ephemeral habitat that is restricted in time and space to zoophilous flowering vegetation. To survive in these habitats, nectar-inhabiting microorganisms rely on animal vectors to disperse from one flower to the next. However, it remains unclear how nectar yeasts persist when flowers and nectar cease to be present. Here, we tested the hypothesis that hibernating bumblebee queens function as a reservoir for nectar yeasts in the absence of plants or pollinators during winter. Our results show that the nectar yeast, Metschnikowia reukaufii, was present in the gastrointestinal tract of wild bumblebee queens that emerged from hibernation and that it could persist inside the gut of hibernating queens under experimental conditions. However, no evidence for such persistence was found in the case of the second most frequent nectar yeast, M. gruessii. Furthermore, a phylloplane yeast that occasionally inhabits nectar, Rhodotorula mucilaginosa, was able to colonize the gut under experimental conditions. Two bumblebee-associated yeasts, Candida bombi and C. bombiphila, were successfully passed down generations after administration in commercial lab-reared bumblebees. Overall, these results demonstrate that bumblebees could act as a reservoir for nectar yeasts during winter when floral nectar is absent.

Effects of agricultural fungicides on microorganisms associated with floral nectar: susceptibility assays and field experiments.

Pesticides have become an inseparable element of agricultural intensification. While the direct impact of pesticides on non-target organisms, such as pollinators, has recently received much attention, less consideration has been given to the microorganisms that are associated with them. Specialist yeasts and bacteria are known to commonly inhabit floral nectar and change its chemical characteristics in numerous ways, possibly influencing pollinator attraction. In this study, we investigated the in vitro susceptibility of nectar yeasts Metschnikowia gruessi, Metschnikowia reukaufii, and Candida bombi to six widely used agricultural fungicides (prothioconazole, tebuconazole, azoxystrobin, fenamidone, boscalid, and fluopyram). Next, a commercial antifungal mixture containing tebuconazole and trifloxystrobin was applied to natural populations of the plant Linaria vulgaris and the occurrence, abundance, and diversity of nectar-inhabiting yeasts and bacteria was compared between treated and untreated plants. The results showed that prothioconazole and tebuconazole were highly toxic to nectar yeasts, inhibiting their growth at concentrations varying between 0.06 and 0.5 mg/L. Azoxystrobin, fenamidone, boscalid, and fluopyram on the other hand exhibited considerably lower toxicity, inhibiting yeast growth at concentrations between 1 and 32 mg/L or in many cases not inhibiting microbial growth at all. The application of the antifungal mixture in natural plant populations resulted in a significant decrease in the occurrence and abundance of yeasts in individual flowers, but this did not translate into noticeable changes in bacterial incidence and abundance. Yeast and bacterial species richness and distribution did not also differ between treated and untreated plants. We conclude that the application of fungicides may have negative effects on the abundance of nectar yeasts in floral nectar. The consequences of these effects on plant pollination processes in agricultural systems warrant further investigation.

Nectar yeasts of the Metschnikowia clade are highly susceptible to azole antifungals widely used in medicine and agriculture.

The widespread use of azole antifungals in medicine and agriculture and the resulting long-persistent residues could potentially affect beneficial fungi. However, there is very little information on the tolerance of non-target environmental fungi to azoles. In this study, we assessed the susceptibility of diverse plant- and insect-associated yeasts from the Metschnikowia clade, including several ecologically important species, to widely used medical and agricultural azoles (epoxiconazole, imazalil, ketoconazole and voriconazole). A total of 120 strains from six species were tested. Minimum inhibitory concentrations (MICs) were determined by the EUCAST broth microdilution procedure after some necessary modifications were made. The majority of species tested were highly susceptible to epoxiconazole, ketoconazole and voriconazole (>95% of strains showed MICs ≤ 0.125 mg l(-1)). Most strains were also very susceptible to imazalil, although MIC values were generally higher than for the other azoles. Furthermore, certain Metschnikowia reukaufii strains displayed a 'trailing' phenotype (i.e. showed reduced but persistent growth at antifungal concentrations above the MIC), but this characteristic was dependent on test conditions. It was concluded that exposure to azoles may pose a risk for ecologically relevant yeasts from the Metschnikowia clade, and thus could potentially impinge on the tripartite interaction linking these fungi with plants and their insect pollinators.

Species coexistence in simple microbial communities: unravelling the phenotypic landscape of co-occurring Metschnikowia species in floral nectar.

Identifying the ecological processes that underlie the distribution and abundance of species in microbial communities is a central issue in microbial ecology and evolution. Classical trade-off based niche theories of resource competition predict that co-occurrence in microbial communities is more likely when the residing species show trait divergence and complementary resource use. We tested the prediction that niche differentiation explained the co-occurrence of two yeast species (Metschnikowia reukaufii and M. gruessii) in floral nectar. Assessment of the phenotypic landscape showed that both species displayed a significantly different physiological profile. Comparison of utilization profiles in single versus mixed cultures indicated that these two species did not compete for most carbon and nitrogen sources. In mixed cultures, M. reukaufii grew better in sucrose solutions and in the presence of the antimicrobial compound digitonin than when grown as pure culture. M. gruessii, on the other hand, grew better in mixed cultures in glucose and fructose solutions. Overall, these results provide clear evidence that M. reukaufii and M. gruessii frequently co-occur in nectar and that they differ in their phenotypic response to variation in environmental conditions, suggesting that niche differentiation and resource partitioning are important mechanisms contributing to species co-occurrence in nectar yeast communities.

The impact of nectar chemical features on phenotypic variation in two related nectar yeasts.

Floral nectars become easily colonized by microbes, most often species of the ascomycetous yeast genus Metschnikowia. Although it is known that nectar composition can vary tremendously among plant species, most probably corresponding to the nutritional requirements of their main pollinators, far less is known about how variation in nectar chemistry affects intraspecific variation in nectarivorous yeasts. Because variation in nectar traits probably affects growth and abundance of nectar yeasts, nectar yeasts can be expected to display large phenotypic variation in order to cope with varying nectar conditions. To test this hypothesis, we related variation in the phenotypic landscape of a vast collection of nectar-living yeast isolates from two Metschnikowia species (M. reukaufii and M. gruessii) to nectar chemical traits using non-linear redundancy analyses. Nectar yeasts were collected from 19 plant species from different plant families to include as much variation in nectar chemical traits as possible. As expected, nectar yeasts displayed large variation in phenotypic traits, particularly in traits related to growth performance in carbon sources and inhibitors, which was significantly related to the host plant from which they were isolated. Total sugar concentration and relative fructose content significantly explained the observed variation in the phenotypic profile of the investigated yeast species, indicating that sugar concentration and composition are the key traits that affect phenotypic variation in nectarivorous yeasts.

Microbiology of sugar-rich environments: diversity, ecology and system constraints.

Microbial habitats that contain an excess of carbohydrate in the form of sugar are widespread in the microbial biosphere. Depending on the type of sugar, prevailing water activity and other substances present, sugar-rich environments can be highly dynamic or relatively stable, osmotically stressful, and/or destabilizing for macromolecular systems, and can thereby strongly impact the microbial ecology. Here, we review the microbiology of different high-sugar habitats, including their microbial diversity and physicochemical parameters, which act to impact microbial community assembly and constrain the ecosystem. Saturated sugar beet juice and floral nectar are used as case studies to explore the differences between the microbial ecologies of low and higher water-activity habitats respectively. Nectar is a paradigm of an open, dynamic and biodiverse habitat populated by many microbial taxa, often yeasts and bacteria such as, amongst many others, Metschnikowia spp. and Acinetobacter spp., respectively. By contrast, thick juice is a relatively stable, species-poor habitat and is typically dominated by a single, xerotolerant bacterium (Tetragenococcus halophilus). A number of high-sugar habitats contain chaotropic solutes (e.g. ethyl acetate, phenols, ethanol, fructose and glycerol) and hydrophobic stressors (e.g. ethyl octanoate, hexane, octanol and isoamyl acetate), all of which can induce chaotropicity-mediated stresses that inhibit or prevent multiplication of microbes. Additionally, temperature, pH, nutrition, microbial dispersion and habitat history can determine or constrain the microbiology of high-sugar milieux. Findings are discussed in relation to a number of unanswered scientific questions.

Nonrandom genotype distribution among floral hosts contributes to local and regional genetic diversity in the nectar-living yeast Metschnikowia reukaufii.

Environmental heterogeneity has been often suggested as a major driving force preserving genetic variation in clonal microorganisms. This study examines this hypothesis for the specialized nectar-dwelling, clonal yeast Metschnikowia reukaufii (Ascomycota, Saccharomycetales). We examined whether M. reukaufii subpopulations associated with flowers of different host plant species, and different individuals of the same host species, differed in genetic characteristics. Amplified fragment length polymorphisms (AFLP) fingerprints of M. reukaufii strains isolated from floral nectar of different host species and individuals sampled at different spatial scales revealed a strong host-mediated component of genetic and genotypic diversity at all scales considered. Genotypes were nonrandomly distributed among flowers of different species and, in the case of the single host species studied in detail (Helleborus foetidus), also among flowers of conspecific individuals coexisting locally. These host-mediated patterns of genetic structuring are compatible with those expected under the diversifying selection hypothesis for the maintenance of local and regional genetic diversity in clonal organisms. It is proposed that a combination of intrafloral selection and biased pollinator-mediated migration may ultimately account for observed host-mediated genetic structuring in populations of M. reukaufii.

Yeasts in nectar of an early-blooming herb: sought by bumble bees, detrimental to plant fecundity.

Through their effects on physicochemical features of floral nectar, nectar-dwelling yeasts can alter pollinator behavior, but the effect of such changes on pollination success and plant reproduction is unknown. We present results of experiments testing the effects of nectar yeasts on foraging patterns of captive and free-ranging bumble bees, and also on pollination success and fecundity of the early-blooming, bumble bee-pollinated Helleborus foetidus (Ranunculaceae). Under controlled experimental conditions, inexperienced Bombus terrestris workers responded positively to the presence of yeasts in artificial sugar solutions mimicking floral nectar by visiting proportionally more yeast-containing artificial flowers. Free-ranging bumble bees also preferred yeast-containing nectar in the field. Experiments conducted in two different years consistently showed that natural and artificial nectars containing yeasts were more thoroughly removed than nectars without yeasts. Experimental yeast inoculation of the nectar of H. foetidus flowers was significantly associated with reductions in number of pollen tubes in the style, fruit set, seed set, and mass of individual seeds produced. These results provide the first direct evidence to date that nectar yeasts can modify pollinator foraging patterns, pollination success, and the quantity and quality of seeds produced by insect-pollinated plants.

Permanent genetic resources added to Molecular Ecology Resources Database 1 February 2013-31 March 2013.

This article documents the addition of 142 microsatellite marker loci to the Molecular Ecology Resources database. Loci were developed for the following species: Agriophyllum squarrosum, Amazilia cyanocephala, Batillaria attramentaria, Fungal strain CTeY1 (Ascomycota), Gadopsis marmoratus, Juniperus phoenicea subsp. turbinata, Liriomyza sativae, Lupinus polyphyllus, Metschnikowia reukaufii, Puccinia striiformis and Xylocopa grisescens. These loci were cross-tested on the following species: Amazilia beryllina, Amazilia candida, Amazilia rutila, Amazilia tzacatl, Amazilia violiceps, Amazilia yucatanensis, Campylopterus curvipennis, Cynanthus sordidus, Hylocharis leucotis, Juniperus brevifolia, Juniperus cedrus, Juniperus osteosperma, Juniperus oxycedrus, Juniperus thurifera, Liriomyza bryoniae, Liriomyza chinensis, Liriomyza huidobrensis and Liriomyza trifolii.

Nectar yeasts of two southern Spanish plants: the roles of immigration and physiological traits in community assembly.

Recent studies have shown that dense yeast populations often occurring in floral nectar are numerically dominated by a few species from the flower-insect interface specialized genus Metschnikowia, while generalist yeast species commonly occurring on leaf surfaces, soil, freshwater, and air were rarely isolated from nectar samples. This study was designed to understand the main factors responsible for the assembly of nectar yeast communities, by combining field experiments with laboratory tests characterizing the physiological abilities of all yeast species forming the pool of potential colonizers for two Spanish flowering plants (Digitalis obscura and Atropa baetica). Yeast frequency and species richness were assessed in external sources (bee glossae, air, plant phylloplane) as well as in pollinator rewards (pollen, nectar). Yeasts were most frequent in external sources (air, flower-visiting insects), less so in the proximate floral environment (phylloplane), and least in pollen and nectar. Nectar communities appeared to be considerably impoverished versions of those in insect glossae and phylloplane. Nectar, pollen, and insect yeast assemblages differed in physiological characteristics from those in other substrates. Nectarivorous Metschnikowia were not more resistant than other yeast species to plant secondary compounds and high sugar concentrations typical of nectar, but their higher growth rates may be decisive for their dominance in ephemeral nectar communities.

Species richness of yeast communities in floral nectar of southern Spanish plants.

Floral nectar of insect-pollinated plants often contains dense yeast populations, yet little quantitative information exists on patterns and magnitude of species richness of nectar-dwelling yeasts in natural plant communities. This study evaluates yeast species richness at both the plant community and plant species levels in a montane forest area in southern Spain, and also explores possible correlations between the incidence of different yeast species in nectar and their reported tolerance to high sugar concentrations, and between yeast diversity and pollinator composition. Yeast species occurring in a total of 128 field-collected nectar samples from 24 plant species were identified by sequencing the D1/D2 domain of the large subunit rDNA, and rarefaction-based analyses were used to estimate yeast species richness at the plant community and plant species levels, using nectar drops as elemental sampling units. Individual nectar samples were generally characterized by very low species richness (1.2 yeast species/sample, on average), with the ascomycetous Metschnikowia reukaufii and Metschnikowia gruessii accounting altogether for 84.7% of the 216 isolates identified. Other yeasts recorded included species in the genera Aureobasidium, Rhodotorula, Cryptococcus, Sporobolomyces, and Lecythophora. The shapes and slopes of observed richness accumulation curves were quite similar for the nectar drop and plant species approaches, but the two approaches yielded different expected richness estimates. Expected richness was higher for plant species-based than for nectar drop-based analyses, showing that the coverage of nectar yeast species occurring in the region would be improved by sampling additional host plant species. A significant correlation was found between incidence of yeast species in nectar and their reported ability to grow in a medium containing 50% glucose. Neither diversity nor incidence of yeasts was correlated with pollinator composition across plant species.

Nectar yeasts warm the flowers of a winter-blooming plant.

Yeasts are ubiquitous in terrestrial and aquatic microbiota, yet their ecological functionality remains relatively unexplored in comparison with other micro-organisms. This paper formulates and tests the novel hypothesis that heat produced by the sugar catabolism of yeast populations inhabiting floral nectar can increase the temperature of floral nectar and, more generally, modify the within-flower thermal microenvironment. Two field experiments were designed to test this hypothesis for the winter-blooming herb Helleborus foetidus (Ranunculaceae). In experiment 1, the effect of yeasts on the within-flower thermal environment was tested by excluding them from flowers, while in experiment 2 the test involved artificial inoculation of virgin flowers with yeasts. Nectary temperature (T(nect)), within-flower air temperature (T(flow)) and external air temperature (T(air)) were measured on experimental and control flowers in both experiments. Experimental exclusion of yeasts from the nectaries significantly reduced, and experimental addition of yeasts significantly increased, the temperature excess of nectaries (DeltaT(nect) = T(nect) - T(air)) and the air space inside flowers in relation to the air just outside the flowers. In non-experimental flowers exposed to natural pollinator visitation, DeltaT(nect) was linearly related to log yeast cell density in nectar, and reached +6 degrees C in nectaries with the densest yeast populations. The warming effect of nectar-dwelling yeasts documented in this study suggests novel ecological mechanisms potentially linking nectarivorous microbes with winter-blooming plants and their insect pollinators.

Inhospitable sweetness: nectar filtering of pollinator-borne inocula leads to impoverished, phylogenetically clustered yeast communities.

Identifying the rules and mechanisms that determine the composition and diversity of naturally co-occurring species assemblages is a central topic in community ecology. Although micro-organisms represent the 'unseen majority' of species, individuals and biomass in many ecosystems and play pivotal roles in community development and function, the study of the factors influencing the assembly of microbial communities has lagged behind that of plant and animal communities. In this paper, we investigate experimentally the mechanisms accounting for the low species richness of yeast communities inhabiting the nectar of the bumble-bee-pollinated Helleborus foetidus (Ranunculaceae), and explore the relationships between community assembly rules and phylogenetic relatedness. By comparing yeast communities on the glossae of foraging bumble-bees (the potential species pool) with those eventually establishing in virgin nectar probed with bee glossae (the realized community), we address the questions: (i) does nectar filter yeast inocula, so that the communities eventually established there are not random subsamples of species on bumble-bee glossae? and (ii) do yeast communities establishing in H. foetidus nectar exhibit some phylogenetic bias relative to the species pool on bumble-bee glossae? Results show that nectar filtering leads to species-poor, phylogenetically clustered yeast communities that are a predictable subset of pollinator-borne inocula. Such strong habitat filtering is probably due to H. foetidus nectar representing a harsh environment for most yeasts, where only a few phylogenetically related nectar specialists physiologically endowed to tolerate a combination of high osmotic pressure and fungicidal compounds are able to develop.

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 10(5) cells/mm(3)). 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.

Yeasts in floral nectar: a quantitative survey.

BACKGROUND AND AIMS: One peculiarity of floral nectar that remains relatively unexplored from an ecological perspective is its role as a natural habitat for micro-organisms. This study assesses the frequency of occurrence and abundance of yeast cells in floral nectar of insect-pollinated plants from three contrasting plant communities on two continents. Possible correlations between interspecific differences in yeast incidence and pollinator composition are also explored. METHODS: The study was conducted at three widely separated areas, two in the Iberian Peninsula (Spain) and one in the Yucatán Peninsula (Mexico). Floral nectar samples from 130 species (37-63 species per region) in 44 families were examined microscopically for the presence of yeast cells. For one of the Spanish sites, the relationship across species between incidence of yeasts in nectar and the proportion of flowers visited by each of five major pollinator categories was also investigated. KEY RESULTS: Yeasts occurred regularly in the floral nectar of many species, where they sometimes reached extraordinary densities (up to 4 x 10(5) cells mm(-3)). Depending on the region, between 32 and 44 % of all nectar samples contained yeasts. Yeast cell densities in the order of 10(4) cells mm(-3) were commonplace, and densities >10(5) cells mm(-3) were not rare. About one-fifth of species at each site had mean yeast cell densities >10(4) cells mm(-3). Across species, yeast frequency and abundance were directly correlated with the proportion of floral visits by bumble-bees, and inversely with the proportion of visits by solitary bees. CONCLUSIONS: Incorporating nectar yeasts into the scenario of plant-pollinator interactions opens up a number of intriguing avenues for research. In addition, with yeasts being as ubiquitous and abundant in floral nectars as revealed by this study, and given their astounding metabolic versatility, studies focusing on nectar chemical features should carefully control for the presence of yeasts in nectar samples.