Interspecies interactions in bacterial biofilms have important impacts on the composition and function of communities in natural and engineered systems. To investigate these interactions, synthetic communities provide experimentally tractable systems. Colony biofilms are one such system that have been used for understanding the eco-evolutionary and biophysical forces that determine community composition and spatial distribution in biofilms. Prior work has focused on intraspecies interactions, using differently fluorescent tagged but identical or genetically modified strains of the same species. Here, we investigated how physiological differences determine the community composition and spatial distribution in synthetic biofilm communities of Pseudomonas aeruginosa, Pseudomonas protegens and Klebsiella pneumoniae. Visualizing this biofilm 'community morphology' microscopically revealed that the outcomes of interspecies interactions in multispecies biofilms are influenced by type IV pilus mediated motility, extracellular matrix secretion, environmental parameters and the specific species involved. These results indicate that the patterns observable in mixed species colony biofilms can be used to understand the mechanisms that drive interspecies interactions, which are dependent on the interplay between specific species' physiology and environmental conditions.### Competing Interest StatementThe authors have declared no competing interest.

Interspecies interactions in bacterial colonies are determined by physiological traits and the environment