## 3 Results and Discussion
### Metabarcoding vs. Culture-Dependent Diversity and Biodeterioration-Related Organisms
As a result, molecular profiling can be expected to provide different results from those obtained from culture isolation techniques. Combining molecular techniques with a more classical approach may allow a much greater understanding of fungal communities. Concerning the cultivable diversity, differences between the fungal profiles obtained by isolation and metabarcoding were noted. With the exception of _Alternaria_, _Aspergillus_, _Cladosporium_, _Cypellophora_, _Parengyodonium_, _Penicillium_, _Talaronyces_ and _Toxicoladosporium_, some of the most abundant taxa in the metabarcoding approach were absent from the fungi identified by culture-based methods. This was the case for _Fusarium_, _Mortierella_ and _Botrytis_, for example, all of which were previously isolated from the stone and are fast-growing and easy to culture. _Neodevriesa_, despite being particularly abundant in samples L2 and L3, is a slow-growing and weak competitive black fungi, therefore being difficult to isolate. Nonetheless, a very large number of strains from different genera (e.g., _Neophysalospora_, _Epicoccum_, _Stachybortys_, _Acremonium_ and _Phelbiopsis_) were isolated, despite their low proportions in the metabarcoding analysis. Additionally, _Arthrinium_, _Microascus_, _Microscynha_, _Utstilago_, _Aeminium_ and _Circumfusicillium_ were not detected at all through the metabarcoding analysis. The detection by cultivation of numerous taxa that go undetected in metagenomic studies is far from insignificant, even if these microorganisms are in low abundance. This may be verified because these species go under unidentified groups in HTS analysis or are present below the detection thresholds of large-scale molecular studies, demonstrating a major \"depth bias\" of the metagenomic approaches. Metabarcoding proved insufficient on its own, and isolation provided an added value for the accurate characterization of the complex fungal community found on limestone. Therefore, for a more complete and thorough characterization, the use of both approaches is probably the best solution. In fact, one of the main advantages of culture isolation is that the different organisms can be preserved for further testing, to understand their ecological functions and identify specific interactions with materials and other organisms. Furthermore,only with isolation can new strains be obtained for further characterization and description, enhancing our current knowledge of the true diversity of fungi. Similar conclusions have been recently reported by [PERSON] et al. [120] in a study that compared culture-dependent and amplicon sequencing approaches of black fungi in Antarctic cryptoendolithic communities, where their results also sustain the theory that the application of both approaches is desirable to discern microbial diversity and community structure in deeper detail, since the respective limits are complementary. Furthermore, in a new publication, [PERSON] and [PERSON] [121] also suggest that the analysis of the microbial community by HTS should be further improved using RNA-based cDNA, to verify the metabolically active population present in a sample, revealing a more meaningful community composition and structure. Fungal mediated biodeterioration is related to: discoloration caused by pigments released from, or contained within, the microorganism (aesthetic action); biocorrosion of the mineral support by acidosis and oxidoreductase processes generated by metabolism (biochemical action); and physical damage caused by the mechanical action of the biomass colonizing the material during its growth (physical action) that leads to the disruption of the material or to its distortion [32]. From the isolated species, _Aspergillus fumigatus, Epicoccum nigrum, Talaromyces purpuggenus, Alternaria_ spp. and _Cladosporium_ spp., and highly melanized black fungi such as _Aeminium ludgeri_ and _Cyphellophora olivaceae_ are known pigment producers, and therefore their growth contributes to aesthetic alterations [24; 26; 29; 122].