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The bacterial communities dominated over the eukaryotic communities in all the drawings. The most dominant phylum showed to be the Proteobacteria with dominance of the Gammaproteobacteria class in drawings 2–4 (Figure 4B). Inside this class, members of the order Pseudomonadales were detected, such as the genus Acinetobacter, present in drawings L2, L3, and L7, the genus Moraxella, which was highly abundant in L2 (representing over 51% of total reads) but also relative abundant in L3–L5 (1–5.25% total reads), the genus Psychrobacter being representative solely in L5 and the genus Pseudomonas, which was relative abundant in drawings L7 and L8 (Figure 6). Members of the Enterobacteriales were detected in drawings L2–L6 but absent in drawings L7–L8. The genus Salmonella was detected in L2 and L5 while Escherichia was identified in L4–L6. The genera Proteus, Xenorhabdus and Buchnera were present exclusively in L3 (Figure 6). Interestingly, the genus Alcanivorax (order Oceanospirillales) was present in drawings L7 and L8 and Stenotrophomonas (order Xanthomonadales) was only present in L8 with a high relative abundance (over 10% of total reads, Figure 6).

In contrast, members of the Betaproteobacteria class were shown to be more abundant in drawings L5–L8 (Figure 4B) with Ralstonia as the dominant genus in all drawings (∼3–40% of total reads) (Figure 6). The genera Burkholderia and Cupriavidus were significative only in drawings L7 and L8 and Paraburkholderia exclusively in drawing L7. Members of the Alphaproteobacteria class were identified in all drawing but in relative lower proportions (Figure 4B). The genus Caulobacter showed to be exclusively present and with a relative high abundance (over 5% of total reads) in drawing L5. The genus Rhizobium was identified in L5 and L8 while Bradyrhizobium only in L8. The genus Sphingopyxis was shown to be present in L7 and L8, while Paracoccus was only identified in L7 and Pannonibacter only in L8 (Figure 6).

The Actinobacteria were the second most represented phylum within the bacteria, with all members identified belonging to the Actinobacteria class (Figure 4B). Within the order Corynebacteriales, the genus Mycobacterium was present in drawings L3–L7 while the genus Nocardia was only identified in L7 and L8. Members of the order Micrococcales were identified in almost all the drawings, being the genus Intrasporangium only present in L2 and L7 and the genus Cryobacterium widely distributed among L3–L6. In addition, the genus Streptomyces (order Streptomycetales) was well represented in drawings L7 and L8 but also in lower proportion in L5 while Cutibacterium (order Propionibacteriales) was identified in L3 and L5 (Figure 6).

The phylum Firmicutes was mainly represented by members of the Bacilli class (Figure 4B), with genera of the order Bacillales, such as Bacillus present exclusively in L3 and Staphylococcus, present in a relative high abundance in L3 (over 8% of total reads) but also well represented in L5 and L6 (4 and 3.15% of total reads, respectively) and in lower proportion in L8 (Figure 6). Members of the order Lactobacillales showed to be widely represented in almost all drawings, with the genera Streptococcus (in drawings L3–L6), Lactobacillus (in drawings L5 and L6), Lactococcus (in L4, L6, and L7) and Leuconostoc (solely in L6). Members of the Clostridia class were detected (Figure 4B), but each of the identified genera contributed less than 0.5% of the total reads.

The same occurred with the Flavobacteria and Cytophagia classes within the phylum Bacteroidetes, no single genus accounted for more than 0.5% of the total reads (Figure 4B). The phylum Planctomycetes showed to be represented only in drawings L7 and L8 with members of the Planctomycetia class. Inside this class, only the genus Isosphaera accounted for more than 0.5% of the total reads in L7. Finally, the phylum Cyanobacteria was only significantly represented in L3 with the genera Anabaena and Oscillatoria, both accounting for more than 0.5% of total reads in this drawing (Figure 6).

First of all, it is important to note that an average of 34% of the eukaryotic sequences that could be identified in the drawings showed to be affiliated with the Chordata phylum (Figure 5), being all related to human sequences, with the exception of drawing L4, where these sequences represented more than 96% of the eukaryotic sequences. The remaining eukaryotic sequences were affiliated with fungi distributed in different phyla, i.e., Ascomycota (3–56% of eukaryotes) and in lower proportions Basidiomycota, Mucoromycota and Microsporidia (0.5–5% of eukaryotes), in addition to some other phyla, each representing less than 0.5% of the microbiomes.

Only a few taxa of the phylum Ascomycota represented more than 0.5% of the total reads in the microbiomes of the drawings (Figure 6). Thus, members of the Eurotiomycetes class proved to be widely distributed among the drawings, with species of the genus Aspergillus identified in L3, L5–L8, while species of the genera Penicillium (in L3) and Penicilliopsis (L7) were representative in only one of the drawings. Members of the Sordariomycetes class of the genus Colletotrichum showed to be representative in L2, L7, and L8. The genus Trichoderma was representative only in the last two drawings (L7 and L8) while the genus Grosmannia was solely identified in L5 in a relatively high proportion (12.6% of total reads) (Figure 6). Within the Dothideomycetes class, species of the genus Alternaria were representative in drawings L3 and L5 and of the genus Bipolaris in L7 and L8. Finally, members of the Saccharomycetes class showed to be widespread in drawings L2, L3, L5, and L6, with species of the genera Ascoidea and Candida (Figure 6).

The results derived from bacteria showed a dominance of Gammaproteobacteria of the order Pseudomonadales. The genus Moraxella was shown to be most abundant at L2, but was also well represented in the L3–L5 drawings, with almost all sequences identified as M. osloensis, a saprophytic species in human skin and mucous membranes (Alkhatib et al., 2017). Its high abundance in these drawings can be explained by contamination due to human handling. Nevertheless, this species was also identified in a survey investigating bulls of indulgence from the fifteenth to sixteenth centuries (Jurado et al., 2010). The genus Acinetobacter was present in drawings L2, L3, and L7, being most of the sequences related to A. baumannii. This species is frequently isolated from hospital environments, although it has been found in environmental samples (Antunes et al., 2014). Nevertheless, sequences related to Acinetobacter spp. were previously identified in a cloning and sequencing survey performed on a deteriorated Italian manuscript dated from the thirteenth century (Michaelsen et al., 2010). The genus Pseudomonas was relative abundant in drawings L7 and L8, with the dominant species P. parafulva, and the genus Psychrobacter was representative solely in L5 with most of the sequences affiliating with Psychrobacter sp. P11G5, which is a psychrophilic species exhibiting laccase activity (Moghadam et al., 2016). Bacterial laccases (Chauhan et al., 2017) are produced either extracellularly or intracellularly and their activity is in a wide range of temperature and pH. They are different from fungal laccases that are multicopper enzymes belonging to the group of blue oxidases (Janusz et al., 2020).

Members of the Enterobacteriales were detected in drawings L2–L6 but interestingly absent in drawings L7–L8. The genus Salmonella was detected in L2 and L5, being Salmonella enterica subsp. enterica serovars identified in both drawings (Vila Nova et al., 2019). In contrast, Escherichia was identified in L4–L6, with the species E. coli, which is a common inhabitant of the gut microbiota. The genus Proteus, with the species P. mirabilis, which frequently causes urinary tract infections in humans (Pearson et al., 2008), was present exclusively in L3. Interestingly, two genera identified only in this last drawing, namely Buchnera and Xenorhabdus, showed the presence of species that are obligate endosymbionts of aphids, such as B. aphidicola (Shigenobu and Wilson, 2011) and nematodes, such as X. bovienii (Murfin et al., 2015). Interestingly, X. bovienii is a bacterium which has been isolated from several nematode Steinernema species. Steinernema affects many insects and also flies (Archana et al., 2017). Therefore, it is plausible to relate the presence of these species with the presence of insect’s droppings in this drawing.

The genus Methylovulum was detected only in drawing L3 and all identified sequences affiliated with M. psychrotolerans, a cold-adapted methanotroph from terrestrial environments (Oshkin et al., 2016). Interestingly, the genus Alcanivorax was present in drawings L7 and L8, with the dominant species A. xenomutans, which has capabilities for alkane degradation and heavy-metal resistance (Fu et al., 2018). Finally, the genus Stenotrophomonas, with the species S. rhizophila and S. maltophilia, was only present in L8 with a high relative abundance. It is important to remark that species of this genus have been identified as colonizers and potential spoilers of oil paintings on canvas support (López-Miras et al., 2013), tempera paintings (Zhgun et al., 2020) as well as ancient paper manuscripts (Michaelsen et al., 2010) and book collections (Kraková et al., 2017; Okpalanozie et al., 2018). In addition, Stenotrophomonas sp. has been identified in relation to the biodeterioration of other cultural heritage materials, such as parchments (Piñar et al., 2015a) and historical stone objects (Alakomi et al., 2006; Ettenauer et al., 2011; Rosado et al., 2020). The reason for the perseverance of Stenotrophomonas in this type of materials may be due to their extracellular enzyme activities such as proteases and chitinases (Kobayashi et al., 2002) and their capability to produce extracellular polysaccharide, enabling their adhesion to different materials and providing biofilm formation on their surfaces (Alakomi et al., 2006).

The Betaproteobacteria were present in all drawings with the dominant genus Ralstonia, with species affiliating to R. solanacearum, R. pickettii, R. insidiosa, and R. mannitolilytica. Ralstonia species, especially R. pickettii and R. solanacearum are prevalent colonizers of printing paper machines (Väisänen et al., 1998) and have been detected as dominant species inhabiting damaged oil paints on canvas (Capodicasa et al., 2010; Piñar et al., 2020a). Furthermore, R. solanacearum is a phytopathogenic bacterium and may exert an active deterioration on paper materials due to the secretion of extracellular proteins, such as cellulolytic enzymes needed for virulence in plants (Liu et al., 2005). Other Betaproteobacteria showed to be relevant only in drawings L7 and L8, as the genera Burkholderia and Cupriavidus. The species B. pseudomallei and B. multivorans were dominant in L7 and Burkholderia sp. MSMB0852 and B. mesoacidophila in L8. Species of this genus were the most frequently isolated from paper products and printing paper machines (Väisänen et al., 1998). Cupriavidus sp. are generally heavy metal tolerant bacteria with the ability to degrade a variety of aromatic hydrocarbon compounds (Wang et al., 2015). The species Cupriavidus sp. USMAA2-4, C. metallidurans and C. gilardii were identified in these two drawings.

The Alphaproteobacteria class showed to be well represented in drawing L5, with a relative high abundance of species of the genus Caulobacter, with the dominance of C. segnis. Caulobacter species exert a role in decomposition, including the degradation of cellulose, lignin and polyaromatic hydrocarbons and have the capacity to grow on cellulose (Song et al., 2013). This fact may explain the presence of Caulobacter species in this paper-supported drawing and highlights the risk that this bacterium poses to their integrity. This class showed to be relevant also in drawings L7 and L8. The genus Sphingopyxis dominated in both drawings. Sphingopyxis isolates have been reported to degrade aromatic compounds in various habitats (Yang F. et al., 2020). In addition, the genus Paracoccus was only relevant in drawing L7 with the dominance of P. yeei, which has been isolated from diverse natural environments, including human skin (Lim et al., 2018). In contrast, the genus Pannonibacter was only relevant in L8, being all reads affiliated with P. phragmitetus, which is a halotolerant polycyclic aromatic hydrocarbon (PAH)-degrading bacterium (Wang et al., 2016), but it has been also described as a human pathogen (Wang et al., 2017).

The Actinobacteria were the second most represented phylum within the bacteria. The genus Cryobacterium, with C. articum, showed relatively high abundance in drawings L3–L6. C. articum is a psychrotolerant bacterium isolated from extreme cold environments (Bajerski et al., 2011). This species was previously identified in two oil paintings on canvas (Piñar et al., 2020a). The genus Mycobacterium, with the dominant species M. haemophilum, was present in all the above-mentioned drawings in addition of L7. M. haemophilum is an emerging pathogen associated most commonly with skin infections (Tufariello et al., 2015). The genus Intrasporangium was present in L2 and L7 with the dominant species I. calvum, firstly isolated from the indoor environment (Kalakoutskii et al., 1967). The genus Nocardia was identified in drawings L7 and L8 with the dominant species N. farcinica. This species causes human infections acquired through the respiratory tract or skin (Torres et al., 2000). The genus Streptomyces was also represented in drawings L7 and L8 and in lower proportion in L5. Streptomycetes are ubiquitous in nature and many species are potent producers of lignocellulolytic enzymes including cellulases, hemicellulases and ligninolytic enzymes (Saini et al., 2015). The most dominant species in L7 and L8 was Streptomyces sp. CNQ-509, while Streptomyces albus dominated in drawing L5. The last species is well-known for its capability to produce xylanases (Saini et al., 2015), which are enzymes responsible for the hydrolysis of β-1,4 bonds in plant xylan, the main component of hemicellulose. The detection of species of this genus in these drawings show a latent risk of deterioration due to the potential cellulolytic activity of these strains. Finally, the genus Cutibacterium (Propionibacterium) was identified in L3 and L5, with the species C. acnes in both drawings. This species is considered as human microbial marker, however, its detection should be considered with caution, as it is a potential pathogen (Fitz-Gibbon et al., 2013).

The phylum Firmicutes was mainly represented by members of the order Bacillales, with the dominance of members of the genus Staphylococcus, present in L3, L5, L6, and L8 (Figure 6). The dominant species in all mentioned drawings was S. aureus, but other species were also identified in high proportions, such as S. equorum, S. capitis, S. pasteuri, and S. epidermidis, among others. The genus Bacillus was present exclusively in L3, with the dominant species B. mycoides and B. thuringiensis. Species belonging to Bacillus and Staphylococcus have been previously identified as the main bacterial species colonizing paper archival materials (Michaelsen et al., 2010; Kraková et al., 2012, 2017; Karakasidou et al., 2018; Okpalanozie et al., 2018).

Members of the order Lactobacillales showed to be widely represented in almost all drawings, with the genera Streptococcus (in drawings L3–L6), being the emerging pathogen S. pseudopneumoniae dominant in L3–L5. This species is associated with lower-respiratory-tract infections and a high prevalence of antimicrobial resistance (Garriss et al., 2019). In contrast, S. thermophilus showed to be the dominant species in L6, which is a fermentative lactic acid bacterium (Cui et al., 2016). This species shows acidifying capacity, proteolytic activity, fast growth and production of exopolysaccharide (EPS), with all these capabilities posing a risk to the integrity of the paper support of the drawings. The genus Lactobacillus was identified in drawings L5 and L6, with L. salivarius and L. gallinarum as dominant species in L5 and L6, respectively, which are species frequently isolated from the digestive tract of mammals (Neville and O’Toole, 2010) and chicken. The genus Lactococcus was identified in L4, L6, and L7 with L. piscium as dominant species in L4 and L. lactis in L6 and L7. L. lactis has been isolated from a variety of environmental sources but is most commonly associated with fresh or fermented plant material or with milk products (Kelly et al., 2010). L. piscium is a psychrotrophic lactic acid bacterium and is known to be one of the predominant species within spoilage microbial communities in cold-stored packaged foods (Andreevskaya et al., 2015). Finally, members of the Leuconostoc genus were solely identified in L6. Three species showed to be dominant in this drawing, namely L. mesenteroides subsp. dextranicum, L. kimchii, and L. citreum. All of them are commonly used for souring vegetables, producing fermented foods (Dols et al., 1997). However, it is important to note that Leuconostoc species have been detected in association with the fruit fly (Drosophila melanogaster), whose diet may contain communities of fermentation microbes, including a high percentage of Leuconostoc in the fly microbiome (Chandler et al., 2011). Also, the housefly (Musca domestica L.) lives in close association with Leuconostoc (de Jonge et al., 2020), which was detected mainly in adult flies and associated to the buccal parts of the insects. It is conceivable that the discovery of this bacterium is associated with the presence on the drawings of droppings of insects, mainly related to synanthropic diptera (Jones et al., 1999) and dating back to the time when the drawings were not protected and preserved in an appropriate manner (see section “SEM-EDS”).

Regarding the members of other phyla relatively less abundant in the microbiomes of the drawings, such as Bacteroidetes, Planctomycetes or Cyanobacteria (Figure 4A), it should be mentioned that most of their genera represented less than 0.5% of the total reads. Thus, only the genus Isosphaera, with the species I. pallida (Planctomycetes phylum) accounted for more than 0.5% of the total reads in L7. Finally, the phylum Cyanobacteria was only significantly represented in L3 with species of the genera Anabaena (A. cylindrica) and Oscillatoria (O. nigro-viridis), both accounting for more than 0.5% of the total reads in this drawing. Airborne dispersal of microalgae occur at very low concentration in the atmosphere and their occurrence is little known due to technical limitations in investigating microalgae from air samples (Tesson et al., 2016). However, recent studies showed that microalgae can actually be air transported and form a consistent component of airborne biological particulate, depending on the environmental conditions and the local deposition rates (Genitsaris et al., 2011). Furthermore, both the O. nigro-viridis and the A. cylindrica are cyanobacteria whose tissue can be mineralized with calcium carbonate (Benzerara et al., 2014). Characteristic stromatolite and travertine formations are produced by species precipitating calcium carbonate in their sheaths and within colonial mucilage (Komárek et al., 2003). Some fillers in paper manufacture are based on carbonates from natural sources, such as fossil sedimentary rocks (i.e., biomicrite) (Gerteiser and Laufmann, 1989), which can sometimes be recognized in the form of microfossils among the paper fibers (Bicchieri et al., 2019). It is therefore possible to speculate that the presence of cyanobacteria is due to the use of carbonate-based filler material and natural rocks, perhaps from recent sedimentation, in the manufacture -if not of the Leonardo drawing- perhaps of the cartoons supporting the drawing itself. Both in the case of airborne cyanobacterial propagules and, even more, of biological traces in the sizing minerals, the data confirm the sensitivity of the molecular method used.

It is important to note that it is difficult to compare the data generated in this study with those published in the literature, due to the different technologies applied. As mentioned above, there are not many studies focused on the detection of bacteria on paper-materials and even fewer conducting culture-independent approaches. However, the data generated in this study through third-generation sequencing analyses are somewhat consistent with previous studies where first- and second-generation sequencing was performed. The core microbiome found in this study includes mainly bacteria belonging to the phyla Proteobacteria, Actinobacteria, and Firmicutes, which correlates well with the few previously existing molecular studies. These three phyla were previously detected by different cloning approaches performed on photographic material, including cellulose supports (Bučková et al., 2014; Puškárová et al., 2016) as well as on an ancient Italian manuscript (Michaelsen et al., 2010). Principi et al. (2011) also showed the presence of members of the phyla Proteobacteria and Firmicutes in the pages of Leonardo da Vinci’s Atlantic Codex. Later, Kraková et al., 2017 confirmed that the core microbiome of various archival materials, investigated with next generation sequencing technology (Illumina MiSeq), consisted mainly of members of the three phyla mentioned above.

To summarize our data, the bacterial species detected in this study can be roughly grouped according to their origin on the paper-support of the drawings. A first group is formed by bacteria that are mainly contaminants introduced by human manipulation, mainly species of the genera Staphylococcus, Streptococcus, and Cutibacterium (Propionibacterium), but also species of the genus Moraxella, Paracoccus, Mycobacterium, and Nocardia could have been introduced by a mishandling of the drawings. This result is reinforced by a large amount of human DNA found in all the drawings. A second group is formed by bacteria that may have been deposited in the drawings through vectors, such as insects. These bacteria belong to the genera Salmonella, Escherichia, Buchnera, Xenorhabdus, and Leuconostoc. This result is supported by the finding of insect droppings on the drawings. The third group consists of bacteria that are actually potential spoilers of the paper-support due to their capabilities of producing extracellular enzymes, and they carry a latent risk to the integrity of the drawings. They are members of the genera Ralstonia, Stenotrophomonas, Burkholderia, Caulobacter, Streptomyces, and Bacillus. Some of them could have been introduced into the paper during the production process and others have been deposited on its surface later through the air. However, it is important to note that the bacterial community was influenced by the geographical location of the drawings, Rome or Turin, denoting an environmental influence.

Among the detected fungi, only some members of the Ascomycota phylum represented more than 0.5% of total reads in the microbiomes of the drawings, among them, members of Sordariomycetes, Eurotiomycetes, Dothideomycetes, and Saccharomycetes. Within the Sordariomycetes, the genus Colletotrichum showed to be present in L2, L7–L8. This genus includes several plant pathogens (Cannon et al., 2012). The species C. higginsianum was dominant in L7–L8 while C. orchidophilum was dominant in L2. Interestingly, the genus Grosmannia was only present at L5 in a very high proportion, with G. clavigera being the sole species identified. This species is the main fungal pathogen associated with the mountain pine beetle and causes blue stain in wood (Wang et al., 2014). The fungus is linked to American conifers. However, it is important to note that individuals of Pseudotsuga menziesii are used as a perennial ornamental conifer species in the northern regions of Italy. It is presumed that the presence of this fungus could be due to a contamination event related to its presence in Rome at the conservation institute where the drawings were analyzed and sampled. In the historical garden of the institute there are in fact ornamental evergreen conifer species. The fungus can also live as a saprotroph in its anamorphic state on bark and cellulosic debris (Leptographium form). A further hypothesis is that the drawing came into contact with furniture or woody material, possibly raw, contaminated by the fungus. The DNA in G. clavigera mycelium remains stable for more than 10 years also in heat-treated wood samples (Wong et al., 2020). Finally, the genus Trichoderma was present in L7 and L8, with the species T. reesei as the dominant species in both drawings. Trichoderma reesei is one of the main producers of cellulolytic and xylanolytic enzymes, being the most studied fungus involved with lignocellulosic degradation (Bischof et al., 2016). The detection of this fungus supposes a great latent risk of deterioration for these paper-supported drawings if the environmental conditions become suitable for its germination.

Members of the Eurotiomycetes were present in drawings L3, L5–L8, being Aspergillus widespread in all these drawings, while Penicillium showed to dominate in L3 and Penicilliopsis in L7. The species A. glaucus, A. clavatus, and A. niger were dominant in all those drawings. Aspergillus species are well known for the production of cellulases and their main role in the biodeterioration of paper (Das et al., 1997). The most dominant species found in the drawings, namely A. glaucus, is known to have a wide environmental distribution due to its physiological hardiness under extreme conditions and to its ability to grow on substrates with a low water activity (Hubka et al., 2013). A. glaucus has been detected in cultural assets made of wooden (Sterflinger et al., 2018b) and canvas support (Piñar et al., 2020a), but its isolation and undistinguishable morphological identification were difficult in the past (Hubka et al., 2013). Penicillium rubens showed to be the dominant species in L3. This species is a common fungus of the indoor environment and well known for its resilience under low water activities (van Laarhoven et al., 2017). Finally, Penicilliopsis zonata was the dominant members of Eurotiomycetes in L7. It is important to note that in the indoor environments, the relative ratio and abundances of fungal species belonging to Aspergillus, Eurotium and Penicillium genera are used along with indicator species to evaluate the presence of hidden moisture damage in buildings (ISO/DIS 16000-17:2006, 2006; Baudisch et al., 2009). Most indoor contamination studies are based on the composition of the dust in suspension. The fungal species in the dust deposited on surfaces are less studied and there are less significant statistics. However, the fact that the drawings, even those preserved in the same environment, presented a different mycoflora is an interesting fact as it allows to formulate hypotheses on the conservation conditions to which the objects have been exposed. The predominance of xerophilic fungal species such as Penicillium rubens and Aspergillus glaucus, indicates that the drawings have not been preserved in humid environments.

Within the Dothideomycetes only members of two genera accounted for more than 0.5% of total reads, namely Alternaria and Bipolaris. Alternaria, with the only detected species A. alternata was present in L3 and L5. This fungus is a well-known paper-spoiler (Das et al., 1997; Michaelsen et al., 2006; Mesquita et al., 2009) and also considered as an allergen, imposing a severe health threat on librarians, conservators, and scholars who might be in contact with infected paper-supported materials (Karakasidou et al., 2018). It must be mentioned, however, that the Alternaria is a fungus that needs water available in the materials and that it typically develops after flooding and wetting events in building materials, including wood and paper. Therefore, the presence of hydrophilic fungal species on the drawings, in the absence of any visible manifestation of water damage, is due to their common presence in the air as airborne contaminants, also coming from the external environment, from the decomposing plant material typically present in parks and gardens. Bipolaris sorokiniana was the main species identified in L7 and L8. B. sorokiniana (anamorph of Cochliobolus sativus) is a phytopathogenic fungus that produce cell wall-degrading enzymes, such as cellulases, which aid invasion into host cells (Aich et al., 2017). As far as we know, B. sorokiniana has not been considered as a paper degrader but, in fact, the fungus is provided with cellulases that can degrade paper fibers and thus, this species poses a latent risk of deterioration for the drawings in which it has been detected.

Members of the Saccharomycetes, with Ascoidea and Candida species were detected in L2–L3 and L5–L6 but curiously, not in drawings L7–L8. A. rubescens, C. orthopsilosis, and C. dubliniensis were the dominant species detected in all four mentioned drawings. Ascoidea species appear primarily to be disseminated by insect vectors (Batra, 1963) and have not been identified in paper materials so far. In contrast, Candida spp. have been previously identified in archival documents and photographic material, but no evidence regarding implication in paper deterioration has been provided (Principi et al., 2011; Borrego et al., 2015; Karakasidou et al., 2018). The detection and prevalence of species of the genus Candida in repositories and archives may be an indication of their ubiquity in this environment, where they can be introduced from the outside environment or contaminated materials after production. Moreover, certain species of Candida are typical human microbiota, which might suggest anthropogenic pollution by handling the collections (Borrego et al., 2015, 2017).

In summary, most of the fungi detected in this study have been previously identified in other studies focused on paper-supported materials and include species of the genera Trichoderma, Aspergillus, Penicillium, Penicilliopsis, Alternaria, and Candida. On the contrary, as far as we know, species of other genera, such as Colletotrichum and Bipolaris, which are mainly plant pathogens, have not been described as common in paper materials. However, they possess extracellular enzymes that may be directly responsible for paper deterioration. Finally, the genera Grosmannia and Ascoidea include typical species associated with insects and could have been introduced to the surface of the drawings through these vectors. As observed with the bacterial communities, it was also possible to see a correlation of the fungal communities with their geographical location, highlighting an environmental and geographical influence on the microbiomes of the drawings.