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.

Floral nectar of some animal-pollinated plants usually harbours highly adapted yeast communities which can profoundly alter nectar characteristics and, therefore, potentially have significant impacts on plant reproduction through their effects on insect foraging behaviour. Bacteria have also been occasionally observed in floral nectar, but their prevalence, phylogenetic diversity and ecological role within plant-pollinator-yeast systems remains unclear. Here we present the first reported survey of bacteria in floral nectar from a natural plant community. Culturable bacteria occurring in a total of 71 nectar samples collected from 27 South African plant species were isolated and identified by 16S rRNA gene sequencing. Rarefaction-based analyses were used to assess operational taxonomic units (OTUs) richness at the plant community level using nectar drops as sampling units. Our results showed that bacteria are common inhabitants of floral nectar of South African plants (53.5% of samples yielded growth), and their communities are characterized by low species richness (18 OTUs at a 16S rRNA gene sequence dissimilarity cut-off of 3%) and moderate phylogenetic diversity, with most isolates belonging to the Gammaproteobacteria. Furthermore, isolates showed osmotolerance, catalase activity and the ability to grow under microaerobiosis, three traits that might help bacteria to overcome important factors limiting their survival and/or growth in nectar.

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Mutualistic services by ant are essential for the pollination of Cytinus hypocistis. Although this parasite does not exhibit typical features of the 'ant-pollination syndrome', many other characteristics indicate that it is evolving to a more specialized ant-pollination system. The striking interspecific differences in the pollination systems of Mediterranean Cytinus (ant-pollinated) and some South African Cytinus (mammal-pollinated) make this genus an excellent model to investigate the divergent evolution of pollination systems in broadly disjunct areas.

Ants and their associated yeasts induce changes in nectar sugar traits, reducing the chemical control of the plant over this important floral trait. The potential relevance of this new role for ants as indirect nectar modifiers is a rich topic for future research into the ecology of ant-flower interactions.

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.

Chemical signals emitted by plants are crucial to understanding the ecology and evolution of plant-animal interactions. Scent is an important component of floral phenotype and represents a decisive communication channel between plants and floral visitors. Floral volatiles promote attraction of mutualistic pollinators and, in some cases, serve to prevent 5 flower visitation by antagonists such as ants. Despite ant visits to flowers have been suggested to be detrimental to plant fitness, in recent years there has been a growing recognition of the positive role of ants in pollination. Nevertheless, the question of whether floral volatiles mediate mutualisms between ants and ant-pollinated plants still remains largely unexplored. Here we review the documented cases of ant pollination and investigate the chemical 10 composition of the floral scent in the ant-pollinated plant Cytinus hypocistis. By using chemical-electrophysiological analyses and field behavioural assays, we examine the importance of olfactory cues for ants, identify compounds that stimulate antennal responses, and evaluate whether these compounds elicit behavioural responses. Our findings reveal that floral scent plays a crucial role in this mutualistic ant-flower interaction, and that only ant 15 species that provide pollination services and not others occurring in the habitat are efficiently attracted by floral volatiles. 4-oxoisophorone, (E)-cinnamaldehyde, and (E)-cinnamyl alcohol were the most abundant compounds in Cytinus flowers, and ant antennae responded to all of them. Four ant pollinator species were significantly attracted to volatiles emitted by Cytinus inflorescences as well as to synthetic mixtures and single antennal-active compounds. The 20 small amount of available data so far suggests that there is broad interspecific variation in floral scent composition among ant-pollinated plants, which could reflect differential responses and olfactory preferences among different ant species. Many exciting discoveries will be made as we enter into further research on chemical communication between ants and plants.25 3

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