Mixed-species interactions constrain diversification and shape biofilm evolution

Experimental evolution provides a powerful framework for dissecting how ecological interactions shape adaptive trajectories. Here, we evolved Klebsiella pneumoniae, Pseudomonas protegens, and P. aeruginosa in single- and mixed-species biofilm communities for 24 weeks and tracked changes in population dynamics, phenotypes, and genomes. In mono-species evolution, all three species exhibited similar dynamics of adaptation, with steadily increasing biofilm-associated populations. In contrast, mixed-species communities displayed striking compositional shifts, with P. protegens emerging as the dominant biofilm former and K. pneumoniae dominating the supernatant. Phenotypic assays revealed that all three species showed enhanced biofilm formation, but this increase was consistently greater in isolates from mono-species than mixed species communities, with P. protegens showing the largest gains. Beyond biofilm production, biofilm-associated isolates exhibited greater phenotypic diversification than planktonic isolates, whereas mixed-species interactions constrained diversification. Whole-genome sequencing identified species-specific putative adaptations such as csrD in K. pneumoniae, yfiBNR in P. protegens, and cheA in P. aeruginosa that arose early, persisted, and were enriched in mixed-species isolates. Functional assays support the contribution of these mutations to enhanced biofilm formation, with yfiBNR mutations in P. protegens increasing cyclic-di-GMP production and producing a competitive advantage that recapitulated its dominance in evolved biofilms. Our findings show that biofilm evolution fosters phenotypic diversification, whereas interspecies interactions shape adaptive trajectories, with specific mutations associated with keystone drivers of ecological dynamics in multi-species communities.