Population density constrains coexistence of cooperative and cheating Kluyveromyces lactis through frequency-dependent physical interactions

Coexistence between cooperative and cheating individuals remains a central puzzle in social evolution. Here, we investigate how population density modulates coexistence using Kluyveromyces lactis strains that interact via physical attachment. Snowflake (cooperative) strains flocculate into multicellular clusters that settle efficiently, while Co-Uni (cheater) strains remain planktonic unless attached to Snowflakes, enabling them to exploit settling benefits without contributing to cluster formation. We manipulated settling density over 15 transfers (~60 generations) to alter encounter rates between strains while holding initial frequencies constant. At high densities, Co-Uni persistence was stabilized by frequent co-settling with Snowflakes; at low densities, reduced interaction frequency drove more rapid exclusion. Model-based projections revealed that both the rate of exclusion and the predicted equilibrium frequency of Co-Uni depend on settling density. Direct measurements of settling fitness confirmed that Co-Uni strains were less enriched in the settled fraction at low densities. These results demonstrate that interaction frequency, as modified by population density, can shape the ecological stability of social traits even in the absence of evolved enforcement or metabolic exchange. Our findings support the idea that density acts as a generalizable structural constraint on coexistence in microbial systems.