Biofilm organization and ECM remodeling in Trebouxia lynniae during colony growth and lichen symbiosis

Lichen-forming microalgae are traditionally studied within their symbiotic context, yet growing evidence shows they also persist as free-living organisms, revealing ecological flexibility, physiological autonomy, and potential contributions to ecosystem resilience. De novo lichen formation — the establishment of a new thallus from free-living fungal and algal partners — depends on traits of these free-living photobionts, yet the mechanisms that enable partner recognition, spatial organization, and functional integration remain poorly characterized. Understanding how microalgae organize themselves outside the symbiosis is therefore crucial to uncover how these traits contribute to thallus development and symbiotic reassembly. This study explores whether biofilm traits in the model microalga Trebouxia lynniae , including abundant extracellular matrix production and morphological and physiological stratification, are developmentally coordinated and retained or modified within the lichen thallus formed with the mycobiont Ramalina farinacea . Using confocal microscopy, flow cytometry, and ultrastructural analyses, we show that T. lynniae forms structured, biofilm-like colonies with spatially stratified photosynthetic activity. Within the lichen thallus, vegetative algal cells maintain layered organization in repeating undulating patterns, yet extracellular matrix is markedly reduced or absent, suggesting a functional repurposing of extracellular polymeric substances from structural cohesion to fungal nutrition. Flow cytometry further indicates that most lichenized algal cells remain in a vegetative, non-reproductive state, consistent with suppression of photobiont sexual reproduction within the thallus. Our findings reveal that extracellular matrix remodeling and biofilm architecture are central to coordinating symbiotic integration in lichens, highlighting biofilm-like growth as an evolutionary step toward stable mutualism. • Trebouxia lynniae microalga forms structured biofilm-like colonies in culture. • Biofilm architecture is partially retained within the lichen thallus. • The algal extracellular matrix is remodeled from structural to nutritional roles. • Lichenized algal cells remain in a vegetative, non-reproductive state.