Uncovering the Fungal Diversity Colonizing Limestone Walls of a Forgotten Monument in the Central Region of Portugal by High-Throughput Sequencing and Culture-Based Methods

Featured Application This work expands the current knowledge of fungal agents and their role in the biodeterioration of limestone rocks, while providing tools for the development of better and more adequate strategies for the conservation, restoration, and rehabilitation of the heritage. Although the information presented here is related to a single monument in Portugal, the data can be extrapolated to other monuments of similar lithology. Fungal organisms are considered one of the most relevant stone colonizers, and biodeteriogens. They are ubiquitous heterotrophs, metabolically versatile, ranging from generalist to extremophiles. Limestone, a sedimentary rock characterized by high levels of calcium carbonate, has low compressive strength and hardness and high porosity. These features make it highly susceptible to fungal colonization and an exceptional target for biodeterioration. Understanding the mycobiome composition associated with different biodeterioration scenarios is key for the development of effective guidelines and strategies for preventive conservation and viable maintenance of our cultural heritage. In this work, a thorough analysis of the fungal community composition on the Lemos Pantheon, a limestone-built artwork located in Portugal, was performed using high-throughput sequencing complemented with culture-based methods. The combined results allowed a detailed characterization of the fungal communities of each analyzed spot, revealing highly diverse and dissimilar communities according to the type of biodeterioration observed. In addition, we verified that both cultivation and metagenomics methodologies should be employed synergistically to tackle inherent limitations.