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An integrative bioinformatics pipeline shows that honeybee-associated microbiomes are driven primarily by pollen composition.

The microbiomes associated with bee nests influence colony health through various mechanisms, although it is not yet clear how honeybee congeners differ in microbiome assembly processes, in particular the degrees to which floral visitations and the environment contribute to different aspects of diversity. We used DNA metabarcoding to sequence bacterial 16S rRNA from honey and stored pollen from nests of four honeybee species (Apis cerana, A. dorsata, A. florea, A. laboriosa) sampled throughout Yunnan, China, a global biodiversity hotspot. We developed a computational pipeline integrating multiple databases for quantifying key facets of diversity, including compositional, taxonomic, phylogenetic and functional ones. Further, we assessed candidate drivers of observed microbiome dissimilarity, particularly differences in floral visitations, habitat disturbance, and other key environmental variables. Analyses revealed that microbiome alpha diversity was broadly equivalent across the study sites and between bee species, apart from functional diversity which was very low in nests of the reclusive A. laboriosa. Turnover in microbiome composition across Yunnan was driven predominantly by pollen composition. Human disturbance negatively impacted both compositional and phylogenetic alpha diversity of nest microbiomes, but did not correlate with microbial turnover. We herein make progress in understanding microbiome diversity associated with key pollinators in a biodiversity hotspot, and provide a model for the use of a comprehensive informatics framework in assessing pattern and drivers of diversity, which enables the inclusion of explanatory variables both subtly and fundamentally different and enables elucidation of emergent or unexpected drivers. This article is protected by copyright. All rights reserved.