## 3 Results and Discussion
### Fungal Community  
As for the _16S_ data, the Beta diversity index (Figure 4a) shows a difference between the fungal communities of Naples and Pisa samples. The median Shannon values, indicating Alpha diversity, are similar for the two communities (Figure 4b). Figure 4: Diversity measures of ITS2 sequencing data. (**a**) Beta diversity represented by Principle Coordinate Analysis Emperor plot on a Bray–Curtis distance matrix. Green dots: Naples, orange dots: Pisa. (**b**) Boxplots of Alpha-diversity Index (calculated as Shannon index) in the two archaeological sites. The sequencing results showed that the fungal communities of the wood samples coming from Naples were dominated by the phylum Ascomycota, accounting on average for more than 50% and reaching in some cases more than 90% of the total reads (Figure 5a). In Pisa samples, a neat prevalence of the phylum Basidiomycota was registered. Sequences attributed to this phylum represented more than 65% of the identified taxa in almost all analyzed samples. It is interesting to note the presence of sequences belonging to the fungal phyla Chytridiomycota and Rozellomycota in some of the P samples and of the phylum Cercozoa (supergroup Rhizaria) in both N and P samples (reaching more than 67% in sample PO). Again, the extraction method does not seem to have modulated the composition of the fungal community (Figure 5a). Figure 5: ITS2. Stacked barcharts of the taxonomic profile at phylum (**a**) and genus (**b**) level. At class level (Figure S3), the communities of N samples were mainly composed of Sordariomycetes (on average 55% of the total reads), Leotiomycetes (on average 39%), and Dacrymycetes (5% of the N88 community). Agaromycetes dominated the P samples, accounting for more than 70% of the total reads on average. The class Dothideomycetes was identified in almost all analyzed samples and reached more than 5% of the relative frequency in most of them. The class Eurotiomycetes was present only in sample PF accounting for 14% (Protocol 2) and 19% (Protocol 1) of total reads. The most represented families in N samples are Pleurotheciaceae (genus _Pleurotheciella_), Lasiosphaeriaceae (g. _Podospora_), Coniochataceae (g. _Coniochatea_), Herpotrichiellaceae (g. _Coniosporium_, _Philophora_ and _Exophilata_), Dacrymycetaceae (g. _Cerinosterus_, _Calocera_) and Reticulascaceae (g. _Reticulascues_). Pleurotheciaceae accounted for more than 50% of total reads in all analyzed samples except for N88. The genera of the families Serendipitaceae (unidentified genus) and Herpotrichiellaceae (g. _Cladophilophora_), were enriched in P samples. In particular, the family Serendipitaceae accounted for a percentage of reads ranging from 2% to 88% in the different samples (Figure S4 and Figure 5b). Differently from what has been observed in the bacterial communities, most of fungal genera were exclusively identified in one or two of the analyzed samples (Figure 5b). Usually, the relative frequencies obtained for each taxon with the two extraction protocols do not coincide; therefore, in the results, both values are reported. Fungal identification at species level through ITS barcoding is not very reliable due to problems linked to the insufficient hypervariability and/or amplicon length (especially for species-rich ascomycete genera), insufficient annotations in public DNA repositories, and the unreliable sequences deposited in the reference databases [96; 97; 98]. However, in the discussion of the results some of the identified species (Figure S5) will be mentioned due to the interest they have for the present work. The results obtained for Naples samples will be analyzed first. _Pleurotheciella_ is the most abundant genus in all analyzed N samples except for N88 where it is absent. Species of the genus _Pleurotheciella_ are usually isolated from freshwater habitats, the species _P. rivularia_ (identified in the N samples) was collected on decaying wood submerged in freshwater [99; 100]. The literature has not reported a possible ligninolytic or cellulolytic activity of this species.