## 1 Introduction
The exclusive use of culture-dependent methods is not enough to fully characterize microbial communities, detecting only a small fraction of the total diversity existing in a sample. This is verified not only because the growth conditions of many species are unknown, or difficult to reproduce, but also because of the existence of microorganisms in inactive (viable, but not cultivable) states [30; 31; 32; 33]. To bypass this limitation, culture-independent methodologies, namely molecular biology techniques, have been extensively used in the profiling of microbiological communities in biodeterioration studies. These techniques, based on DNA analysis, allow the profiling of complex communities. Due to their implementation, some studies have shown that the microbiological diversity in this type of substrate is much greater than was previously believed [30; 31; 32; 33; 34]. High-throughput next-generation sequencing (HTS) has revolutionized the study of genomics and molecular biology by allowing, in a much faster way, the sequencing and analysis of billions of DNA fragments, providing a much more detailed representation of the populations present in a sample [35; 36]. Nevertheless, only a small number of studies have been performed in which high-throughput sequencing was applied to limestone fungal populations in biodeterioration scenarios, particularly in Portugal [14; 16; 37]. Nonetheless, with advantages and disadvantages associated with both approaches, the classic microbiological methods complement molecular techniques in the identification, characterization, and description of microorganisms, allowing a better understanding of the real biodiversity within stone-colonizing communities [38]. The biological deterioration of the historical heritage is one of the greatest concerns of conservations and restorers worldwide. Knowing and understanding the factors that lead to the alteration of rocks is, without a doubt, the first and most important step in the development of more appropriate and effective guidelines and strategies, in order that preventive conservation and viable maintenance of our cultural heritage can be carried out. However, given the numerous intrinsic and extrinsic factors associated with stone alteration, a definitive and universal solution is unlikely to be found. Located in the central region of Portugal, the Church of Trofa houses the only element of the municipality of Agueda classified as a National Monument, officially integrated in the Portuguese Cultural Heritage. This classification arises from the fact that inside it has an emblematic sculptural-funerary ensemble of great architectural and artistic value--the Lemos Pantheon [39], that is barely known and rarely visited by tourists. After several years bearing a forgotten and degraded appearance, with biological colonization markedly affecting the limestone of this site, in August this year, restoration and requalification interventions were started, which included the cleaning of the interior and exterior facades [40], without a previous analysis or extensive knowledge of the biodeterioration phenomena present there. This posed a risk to the conservation of this monument that could lead to a future recolonization with potentially worse effects [41; 42]. The objective of this work was to thoroughly characterize the fungal diversity associated with different biodeterioration phenomena present in this monument, using high-throughput next-generation sequencing and cultivation-dependent methodologies. This information will ultimately contribute to science-supported restoration plans in future interventions.