Floral nectar is a specialized environment which supports a specific community of microbes (bacteria, microfungi, yeasts). Floral visitors and pollinators are believed to be the primary vectors for microbial inoculation of floral nectar, yet the influence of initial microbial deposition on downstream microbial composition is not well understood. Here we use a floral nectar model system to explore microbial dispersal dynamics, as well as microbial community succession along a temporal trajectory. Using both culture-dependent and culture-independent methods, we describe the microbial taxonomic and functional composition within the floral nectar environment of a hummingbird-pollinated shrub, Epilobium canum. We examine the effects of floral visitation (pollination versus nectar robbing) on microbial abundance, species composition and function in nectar, sampling across flowers that vary in development status. We sampled nectar from a total of 212 E. canum flowers within four visitation regimes: no visitation, pollinated, robbed, and both pollinated and robbed, across five floral stages of development. First, we quantified the abundance of culturable bacteria and fungi in nectar, then described the taxonomic composition of cultured isolates using targeted gene sequencing. We also performed shotgun metagenomic sequencing for a culture-independent description of taxonomic composition and metabolic potential of the nectar-associated microbial communities.
We found that floral visitors deposited microbes into floral nectar, as both bacterial and yeast abundance was higher in nectar of visited flowers compared to non-visited flowers. Furthermore, microbial abundance increased through time from the initial petal stage post-anthesis to senescence. We identified 13 bacterial and 12 fungal/yeast genera from cultured isolates. Common nectar inhabitants, Acinetobacter and Metschnikowia, were the most abundant microbial genera; yet were lacking from nectar of non-visited flowers. Metagenomic analysis revealed differences in the diversity and composition of nectar-associated microbes among visitation treatments. Further, metagenomics libraries from pollinated or robbed flowers had a greater number of genes associated with sugar degradation and utilization pathways compared to non-visited flowers. Overall, our results indicate that 1) floral visitors are a primary vector for microbial deposition to nectar, and 2) microbial abundance and effects on nectar sugars increases through flower development. Our results imply that hummingbird pollinators and nectar robbers deposit distinct suites of microbes to nectar, which may differ in metabolic potential and influence on nectar sugar composition. Our study adds to the understanding of the influence of dispersal on microbial community structure and function, and how the structure and function of microbial communities change through time.