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In 2013, UNESCO classified the 'University of Coimbra – Alta and Sofia ' (Portugal) as World Heritage site. Some of the limestone monuments that compose the site, such as the Old Cathedral of Coimbra, show clear visual effects of biological colonization by cyanobacteria and microalgae. The aim of this study was to thoroughly characterize the structural diversity of the photoautotrophic community at this site, combining isolation and molecular identification with next-generation sequencing techniques. Culture-dependent approaches resulted on the identification of green microalgae belonging to Prasiolales , Chlorellales , Watanabea , Chlamydomonadales and Sphaeropleales clades, and cyanobacteria belonging to Nostocales and Synechococcales clades. Culture-independent methods using Illumina Platform resulted in the identification of 610 Operational Taxonomic Units (OTUs), revealing the presence of populations within phyla Chlorophyta , Cyanobacteria , Rhodophyta and Stramenopiles / Bacillariophyta. In addition, principal component analysis (PCA) and a network analysis were generated and successfully related the different populations to specific biodeterioration scenarios observed at this monument. The use of culture-dependent and -independent techniques together with PCA and network analysis allowed us to understand the distribution patterns of the photoautotrophic community among the different biofilms observed at this monument, as well as their ecological specifications. This study highlights that both cultivation and metagenomics methodologies should be employed synergistically in similar biodeterioration studies. • Culture-dependent and NGS Illumina sequencing of phototrophic populations at the Old Cathedral of Coimbra. • Predominance of microalgae over cyanobacteria among collected samples. • Distinct phototrophic populations associated with different biofilm patterns. • Cultivation and metagenomics approaches revealed to be complementary. [ABSTRACT FROM AUTHOR]

During the ongoing characterization of limestone biodeterioration in the UNESCO World Heritage site of "University of Coimbra – Alta and Sofia" (Coimbra, Portugal), it was brought to our attention that the limestone relieves adorning the tomb of D. Afonso I (First Portuguese King), displayed several signs of distinctive biodeterioration patterns. According to the ICOMOS Illustrated glossary on stone deterioration patterns, these could be characterized as visual mould proliferation, presence of black crusts, stone erosion and disintegration. Due to the invaluable nature of this monument, a multi-disciplinary approach (biological and analytical) to fully characterize these phenomena was applied. The results obtained allowed the characterization of the fungal diversity colonizing this monument, as well as the identification of various deterioration products. The results highlighted that the detected indoor black crusts displayed various characteristics from polluted environments, although lacking the typical association with gypsum. The black crusts were also characterized by the presence of lead oxides, damaging salts and carbonaceous particles. In addition, it could be verified that the erosion phenomena found are a result from fungal mediated calcium carbonate dissolution and calcium oxalate formation. The establishment of different fungal populations coupled with the deposition of environmental particles contributed to the development of the distinct phenomena detected through differential biodeterioration mechanisms. • Application of a multi analytical approach to study D. Afonso I tomb pathologies. • High fungal contamination associated with all biodeteriorated areas. • Black crusts holding lead oxides, anhydrite, syngenite and carbonaceous particles. • Stone erosions with calcium oxalates. • Biodeterioration by chromatic changes, stone dissolution and calcium oxalate formation. [ABSTRACT FROM AUTHOR]