The Impact of Visible Symptoms of Thallus Damage on the Phycobiota of Mediterranean Epiphytic Lichens

Microbial Ecology https://doi.org/10.1007/s00248-026-02794-3 Article in Press The Impact of Visible Symptoms of Thallus Damage on the Phycobiota of Mediterranean Epiphytic Lichens Tamara Pazos, Patricia Moya, Salvador Chiva, Pavel Škaloud, Veronika Kantnerová, Eva Barreno & Isaac Garrido-Benavent Received: 16 January 2026 Accepted: 17 May 2026 Cite this article as: Pazos T., Moya P., Chiva S. et al. The Impact of Visible Symptoms of Thallus Damage on the Phycobiota of Mediterranean Epiphytic Lichens. Microb Ecol (2026). https://doi. org/10.1007/s00248-026-02794-3 A S S We are providing an unedited version of this manuscript to give early access to its findings. Before final publication, the manuscript will undergo further editing. Please note there may be errors present which affect the content, and all legal disclaimers apply. E R P If this paper is publishing under a Transparent Peer Review model then Peer Review reports will publish with the final article. 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To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. ACCEPTED ARTICLEMANUSCRIPT IN PRESS The impact of visible symptoms of thallus damage on the phycobiota of Mediterranean epiphytic lichens Tamara Pazos1, Patricia Moya1, Salvador Chiva2, Pavel Škaloud3, Veronika Kantnerová3, Eva Barreno1 & Isaac Garrido-Benavent4 1. Instituto Cavanilles de Biodiversidad y Biología Evolutiva (ICBiBE) – Botánica, Universitat de València, C/ Dr. Moliner 50, 46100 Burjassot, València, Spain; *Corresponding author e–mail: 2. Institut Universitari de Recerca en Biotecnologia i Biomedicina (BIOTECMED), Universitat de València, Av. Vicent Andrés Estellés 19, 46100, Burjassot, Spain 3. Department of Botany, Faculty of Science, Charles University, Benátská 2, 12800 Prague, Czech Republic S S E R P 4. Departament de Botànica i Geologia, Universitat de València, C/Dr. Moliner 50, 46100 Burjassot, València, Spain Abstract E L C I T R A IN Lichens are excellent bioindicators of overall ecosystem health. The symbiotic nature of their thalli enables tracking changes in humidity, temperature, habitat disturbance and air pollution, often before larger plants do. Sensitive species usually show visible thallus damage, such as bleaching or changes in colour (including total or partial necrosis, and death of the photosynthetic component of the symbiosis), slow growth, and/or biases in reproductive strategies. Particularly, the extent to which these damages is associated with changes in the microscopic photosynthetic community inhabiting lichen thalli (phycobiota) remains poorly understood. Here, we combined Sanger and Illumina sequencing techniques to characterize the diversity and community structure of the eukaryotic phycobiome in selected epiphytic macrolichens showing different levels of thallus damage. Phylogenetic analyses revealed a high microalgal diversity, largely dominated by a few Trebouxia species, ACCEPTED ARTICLEMANUSCRIPT IN PRESS which are the most prevalent lichenized microalgae, accompanied by several low-abundance co-occurring genera. Notably, microalgal diversity peaked at intermediate levels of thallus damage. This pattern is consistent with disturbance-mediated modulation of microalgal community evenness rather than a categorical shift in symbiotic composition. These findings reveal previously unrecognized variability within the lichen phycobiota, providing new insights into the ecological dynamics and stress responses of these communities. In conclusion, our work offers a new perspective on the potential of lichens as sensitive bioindicators of air quality and ecosystem health. Key words: diversity indices, Illumina, Intermediate Disturbance Hypothesis (IDH), metabarcoding, photobiont, symbiosis. 1. Introduction S S E R P Lichen thalli represent the structural and functional outcome of complex mutualistic IN interactions between filamentous heterotrophic fungi —referred to as mycobionts— and one E L C I T R A or more autotrophic partners, or photobionts, typically green microalgae and/or cyanobacteria [1-3]. Among phycobionts (i.e., lichen-symbiotic green microalgae), most belong to the chlorophyte genus Trebouxia Puymaly (e.g., [4-8]. This genus stands as one of the best-studied symbiotic microalgal lineages from taxonomic [9-11], phylogenetic [12, 13], physiological [14-19], genomic [20-22] and geographical perspectives [13, 23, 24]. However, lichenological research has traditionally prioritized the fungal partner, leaving photobiont diversity and evolution comparatively understudied. Current knowledge of the factors shaping myco-phycobiont interactions is largely based on Sanger sequencing of a single genetic locus [25]. So far, evidence has shown that associations with diverse phycobiont lineages in lichen-forming fungal species can be influenced by both symbiont co-dispersal history [26-29], and environmental conditions, including micro- and macroclimatic gradients [30-33]. At a community level, advances in molecular techniques have propelled the study of myco-photobiont interactions in a variety of ecological contexts, including metal-rich substrata [34, 35], soils [36, 37], rocks [38-40], ACCEPTED ARTICLEMANUSCRIPT IN PRESS and forest ecosystems [41-43]. Although these studies have improved our understanding of fungal–photobiont interactions, a new paradigm of lichen symbioses has emerged. Recent evidence revealed that lichens harbor a much greater microalgal diversity than previously recognized [25, 44-46]. These additional algal associates coexist alongside the dominant photobiont in the thallus [45, 47, 48] and from a pure taxonomical perspective, these microalgae are collectively referred to as the phycobiota. Moreover, lichens host diverse bacterial communities (“bacteriota”; [49-53]) and non-lichenized fungi together with the main mycobiont [the mycobiota; 54-59]. Metagenomic and metatranscriptomic analyses have further confirmed that these microbial communities, along with the mycobiont and the primary photobiont, constitute the lichen holobiome [60-64], reinforcing the view of lichens as self-sustaining microecosystems [3, 65]. The development of high-thr (...truncated)