## 4 Discussion  
The multi-analytical approach used in this study allowed us to characterize the microbiome of stones associated with petroglyph sites and, at the same time, to characterize the lithtic substrate, thus obtaining a deeper knowledge of possible mineral-microbial interactions. The microbiomes of the samples were generated using nanopore sequencing technology, which has been employed in different studies in the cultural heritage field [37; 43; 44; 45], showing some advantages compared to next-generation sequencing methods. In fact, this technology, which offers an easier workflow and much more affordable prices, when combined with whole genome amplification (WGA) protocol allows simultaneous obtainment of the real proportions of different groups of microorganisms (fungi, bacteria and archaea) as a whole in a given sample, which was one of the first aims of this study. The microorganism communities associated with rock varnish have been previously studied to determine whether or not there is a connection between the varnish and the  
Figure 9: (**a**) Normalized Raman spectra collected from the black varnish on two different spots (A, B), on the orange layer emerging from the black varnish (C) and on the orange crust of sample EZ4 (D) compared with reference spectra of burnt Sienna (E) (Pigments Checker Raman Database [39]), hematite (F) and magnetite (G) (RRUFF [38]) in the spectral range between 150 and 1700 cm\({}^{-1}\). (**b**) Normalized Raman spectra collected on the orange layer beneath the black crust (a) and on a spot colonized by cyanobacteria (b) in the range from 900 cm\({}^{-1}\) to 1700 cm\({}^{-1}\), showing the characteristic peaks of carotenoids. microorganisms inhabiting the stone. The results of our metagenomic study are consistent with the findings reported in the literature [25,33], revealing a microbiome dominated by bacteria, with lower proportions of eukaryotes, and archaea representing less than 1% of the total assigned reads. Within the bacteria, Actinobacteria and Cyanobacteria were the most abundant phyla, followed by Proteobacteria and Bacteroidetes, as found also by other authors in metagenomics studies on microbial communities of stones in desert environments [16,27,30]. Notable is that sample EZ4, which does not present the superficial black varnish, showed a different microbiome, in which the genus _Rubrobacter_ (_Actinobacteria_ phylum) together with the genus Acinetobacter (_Proteobacteria_ phylum) and the genus _Pontibacter_ (_Bacteroidetes_ phylum) made up almost 90% of the total reads, while Cyanobacteria represented less than 1%. In the other samples (AV1, AV3 and EZ1), Cyanobacteria represented between 26 and 39% of the bacterial communities, with a rather high biodiversity. Cyanobacteria are common photoautotroph bacteria inhabiting rocks in desert environments [29,46,47]; they are considered primary producers with the ability to withstand stressful environmental conditions due to their thick cell wall and the production of protective pigments such as scytonemin and carotenoids, which protect them against photooxidative damage [16,48]. Among the cyanobacteria, the order _Nostocales_, a nitrogen-fixing family that contains several pigment-secreting taxa, was dominant, with _Nostoc_ spp. being the most abundant. The order _Nostocales_ was previously reported to be desiccation resistant [49] and radiation tolerant and a dominant taxa in hypolith communities of the Atacama desert [50]. The genus _Criinalium_ (order _Oscillatoriales_) and the genus _Gloecapas_ (order _Chroococales_) had relatively high abundance in our samples and are also known to be well adapted to harsh environments [51,52]. Within the order _Chroococales_, the extremely desiccation and radiation resistant genus _Chroococidopsis_ is often indicated as dominant in cyanobacterial communities on stone in deserts [27,53,54]; however, in our study it represented only around 1% of total reads.