Symbiont-specific responses of the lung lichen holobiont to excess light and nitrogen

Lobaria pulmonaria (L.) Hoffm.,a good bioindicator of forest continuity, as it mainly occurs in old-growth forests. This lichen is highly sensitive to increased light intensity and air pollution, particularly external nitrogen enrichment. The aim of our study is to investigate how the L. pulmonaria holobiont responds to nitrogen enrichment and varying light conditions, hypothesizing that these stressors affect the different lichen components; fungal, photobiont and cyanobiont symbiotic partners, as well as the associated bacterial microbiome, in distinct ways. Using a combination of amplicon sequencing and metaproteomic analyses, we examined changes in taxonomic composition and function of the holobiont, including both eukaryotic and prokaryotic components of the lichen, in response to experimental manipulation of light conditions via transplantation, as well as of external nitrogen loads simulating precipitation-driven nitrogen deposition. Our results demonstratedthatincreased light exposure enhanced both bacterial (Shannon index: p < 0.01) and eukaryotic alpha diversity. Although nitrogen enrichment did not significantly alter community composition, it affected eukaryotic protein expression (48.31% variance explained; p = 1e-04). ....In addition, our findings revealed the complexity of the L. pulmonaria holobiont beyond the known symbiotic partners. Protisttaxa such as Gregarinamorphea and microfauna within Rotifera and Tardigrada, suggest multi-trophic interactions within the holobiont. Functional redundancy within the microbiome likely contributed to holobiont functional stability despite environmental stressors. These findings highlight L. pulmonaria as a dynamic symbiotic hub, where abiotic factors distinctly shape microbial community structure and function, with implications for lichen resilience in changing ecosystems.