Species boundaries structure competitive interactions among honeybee gut bacteria

Bacterial species often harbor extensive strain-level diversity, raising the question of whether strains, rather than species, are the relevant ecological units. We quantified pairwise interactions among 12 strains from four prevalent Lactobacillus species of the honeybee gut microbiota using gnotobiotic bees. Microbiota-depleted bees were colonized with individual strains and all 66 pairwise combinations, with strain abundances measured by colony counts and strain-resolved amplicon sequencing. Negative interactions predominated, with most significant interactions involving mutual inhibition. Within-species interactions were stronger and more asymmetric than between-species interactions, producing less even community compositions. Across repeated cycles of colonization in microbiota-depleted bees, three of four within-species pairs lost one strain, whereas all four between-species pairs persisted. These results indicate that closely related strains experience greater niche overlap and stronger competition than strains from different species, supporting bacterial species as ecologically differentiated units while highlighting the importance of strain-specific traits in shaping community assembly and stability.