Spatial segregation and cross-kingdom interactions drive stingless bee hive microbiome assembly

Studying host-associated microbiome assembly is key to understanding microbial and host evolution and health. While honey bee microbiomes have been central models for such investigations among pollinators, they overlook the diversity of eusocial dynamics and multi- kingdom interactions. Stingless bees, highly eusocial managed bees that rely on yeast for larval development, offer a valuable complementary system to study microbiome assembly, and within an eco-evolutionary framework. Using amplicon sequencing, metagenomics, and microbial experiments, we investigate the drivers of stingless bee microbiome assembly. We reveal a spatially structured, site-adapted microbiome, where high microbial influx hive components are segregated from the brood, which harbors a stable, multi-kingdom community. We show that the brood microbiome is not only physically protected but also actively maintained through highly selective bacterial-fungal interactions. Our findings uncover multi-layered mechanisms shaping an eusocial insect microbiome, from host biology to cross-kingdom interactions, while providing critical insights into microbiome maintenance of important pollinators.