The Negev Desert of Israel is home to thousands of petroglyph sites, dating back at least 5,000 years (Eisenberg-Degen and Nash, 2014), representing a precious remnant of past civilizations that inhabited the desert long ago (Eisenberg-Degen et al., 2022). The petroglyphs are mostly located in the Negev Highlands ( Figures 1A, B ) and are carved into a natural thin, dark coating known as desert varnish (Perry et al., 2006; Andreae et al., 2020), covering some of the local limestone formations ( Figure 1C ). Despite the harsh and inhospitable conditions, deserts around the world are known to harbor a multitude of extremotolerant and extremophilic microorganisms, including fungi that were once thought to be less resilient than prokaryotes (Gostinčar et al., 2023). These microorganisms have adapted to withstand extreme stress factors such as very high temperatures, temperature fluctuations, high ultraviolet and infrared irradiation, low organic matter content, and extremely limited availability of water (Sterflinger et al., 2012). Consequently, even the petroglyphs of the Negev Desert undergo biogenic weathering, like any other natural rock surfaces or stone cultural heritage exposed to the environment (e.g., Steiger et al., 2014; Favero-Longo and Viles, 2020). Recent studies have investigated the biodiversity of lithobiontic microbial communities associated with petroglyph sites in the Negev Desert and their potential role in the deterioration of this valuable cultural heritage (Nir et al., 2019, Nir et al., 2021; Rabbachin et al., 2022; Nir et al., 2023). These investigations showed deterioration patterns of the rock substrate, revealing a clear weathered layer underneath the dark coating ( Figure 1D ) due to the complete lack of the stone calcite matrix in these areas (Nir et al., 2021; Rabbachin et al., 2022), indicating a feasible dissolution of the calcareous material by microbial activity (Krumbein and Jens, 1981; de Los Ríos et al., 2009). Metagenomic analyses of the stone microbial community, although bacteria-dominated, also included fungi (Nir et al., 2021; Rabbachin et al., 2022). However, most of the genera identified did not appear to be rock inhabiting species (except for a few taxa such as Exophiala, Coniosporium, and Letharia) but rather cosmopolitan airborne fungi, with minimal evidence of fungi growing on stone found through microscopic observations (Rabbachin et al., 2022). Various studies have reported airborne ubiquitous species as part of the desert endolithic communities (Gonçalves et al., 2016; Tang et al., 2016; Gómez-Silva et al., 2019; Grishkan and Temina, 2023), but this could be the result of “post-sampling contamination” (Coleine et al., 2021) or the presence of airborne spores deposited on the rock surface (Sterflinger et al., 2012). In fact, among the variety of extremotolerant and extremophilic microorganisms that inhabit the desert, fungi are specialized to resist complete desiccation through the formation of thick-walled spores (Sterflinger et al., 2012), allowing them to survive in a dormant state even in the driest environments (e.g., the Atacama Desert, Chile) (Conley et al., 2006). In the last decade, with the development of novel molecular techniques, studies on the soil mycobiota of deserts worldwide have revealed unexpectedly rich biodiversity (Zhang et al., 2016; Murgia et al., 2018; Santiago et al., 2018; Ameen et al., 2021). Griskhan and colleagues have conducted extensive research on the soil microfungi of Israel’s Negev Desert (Grishkan, 2019; Grishkan et al., 2023), highlighting that fungi with thick-walled, strongly melanized spores (e.g., Cladosporium and Chaetomium) and large multicellular conidia (e.g., Alternaria) were dominant in the majority of the Negev topsoil, while aspergilli were a fundamental part of the thermotolerant fungal community.

Compared to the soil mycobiota, desert lithobionts have received less attention, and a comprehensive understanding of both their diversity and ecological function is still in progress (Coleine et al., 2022). It is within this category, however, that the real specialists of the desert environment are found, not only able to survive but also to thrive in the lithic support (Sterflinger et al., 2012). Together with lichens, these rock specialists are the so-called microcolonial fungi (MCF) or black fungi, also referred to as rock-inhabiting fungi (RIF), when they colonize stone substrates (Liu B. et al., 2022 and references therein). This peculiar group of microorganisms is extremely resilient to hostile environments, and species belonging to this group have been isolated from hot deserts (Staley et al., 1989) to extremely cold Antarctic drylands (Selbmann et al., 2015; Coleine et al., 2021), but also in more moderate climates like the Mediterranean basin, where they have often been associated with the deterioration of stone cultural heritage (Sterflinger and Krumbein, 1997; Marvasi et al., 2012; Onofri et al., 2014; Isola et al., 2016; Paiva et al., 2023). In fact, MCF, due to their great ability to penetrate and degrade hard rocks as well as softer stones like limestone, sandstone, and marble (often used as building construction material or artistic support), is considered highly dangerous for stone artifacts. They can cause black discoloration, surface exfoliation (sugaring) due to their inter-crystalline growth, and surface erosion (biopitting) that can eventually lead to large cavities (De Leo et al., 2022). As well as MCF, lichens are also well-known to be rock deteriogens and a potential threat to stone cultural heritage (e.g., de Los Ríos et al., 2009; Cozzolino et al., 2022).

In this study, we aimed to better understand whether fungi associated with petroglyphs in the Negev Desert are transient or integral members of the microbial ecosystem. We analyzed the biodiversity of fungal communities on the rock and compared it to those in the surrounding environment (soil and air). To achieve this, we utilized high-throughput sequencing technology (nanopore sequencing) targeting the Internal transcribed spacer (ITS) fungal region alongside culture-based methods. In addition, to gain deeper insights into the potential role of fungi in the deterioration of petroglyphs (carbonate rock), the ability of the isolated strains to dissolve calcium carbonate was tested.

The combined use of culture-dependent and culture-independent approaches enabled us to gain a deeper understanding of the biodiversity of fungal communities associated with the Negev Desert petroglyphs (rock) and their surrounding environment (soil and air).

Both the metagenomic (targeted ITS long-amplicon sequencing) and cultivation studies seem to indicate that the presence of fungi on the stone samples is very limited. In fact, in the metagenomic study, after processing the raw reads, in three out of four rock samples (AV_A, AV_B, and EZ_A), almost no sequences with the correct ITS length (300–900 bp) were present ( Supplementary Table 2 ). Since no macroscopic or microscopic evidence of fungi was found in these samples, and the same DNA extraction procedure and PCR primers of the other samples were used, we assume that fungi were in such low proportion to be under the detection limit. The short fragments that were amplified in these samples ( Supplementary Table 2 ) are probably just primers and primer-dimers due to the very low template DNA concentration. The only sample in which ITS sequences have been successfully detected was the rock sample without the black varnish, EZ_D. Consistently, the highest amount of cultivable fungi was also recovered from the same sample, while no fungi could be cultivated from the other stone samples (except three colonies from AV_B). The low abundance of fungi in the microbiomes of rocks coated by desert varnish collected from petroglyph sites agrees with previous studies (Lang-Yona et al., 2018; Nir et al., 2021; Rabbachin et al., 2022). The comparison of the data obtained from the soil and rock samples by the culture-based approach and the culture-independent method showed that, as expected, the culture-dependent approach underestimates the total fungal diversity (58 genera were detected using nanopore sequencing ( Supplementary Figure 3 ), while 12 were retrieved by cultivation ( Supplementary Table 1 ); of these, 9 genera were detected by both techniques). In fact, although culture-based methods are highly valuable in biodiversity studies, they come with limitations and biases (Jeewon and Hyde, 2007), such as the different growth capabilities of fungal taxa under specific experimental conditions. This can lead, for example, to an overestimation of heavily sporulating species and, conversely, to the loss of fungi that are not cultivable or require specific growth conditions (Hamm et al., 2020). On the other end, molecular approaches present the drawback of detecting not only active or dormant members of a microbial community but also dead organisms (Grishkan et al., 2023).

While the soil showed quite high biodiversity (discussed later in this section), the rock mycobiome, according to the metagenomic study, was mostly represented by three genera: Flavoplaca (41.5%), Vermiconidia (9.9%), and Knufia (1.7%) ( Figure 3 ). In addition, almost 30% of the fungal community of the stone sample was composed of Capnodiales sp. ( Supplementary Figure 4 ), which could not be identified at lower taxonomic levels and may belong to an unclassified species. From the cultivation approach, Alternaria sp. (which was also detected by the metagenomic analyses in low abundance, 0.3%, Supplementary Figure 3 ), Cladosporium sp., Coniosporium sp., and one unidentified MCF strain were isolated. The dominant genus Flavoplaca (order Teloschistales), with the main species detected in this study Flavoplaca arcis [basionym: Caloplaca citrina var. arcis (Arup et al., 2013)] ( Supplementary Figure 4 ), is a crustose, saxicolous (rock-dwelling) lichen occurring on limestone (Fletcher and Laundon, 2009), and seems to be commonly present in the Negev Desert (Temina, 2021). Other species identified in this study within the genera Vermiconidia, Knufia, and Coniosporium, as well as Phaeotheca and Devriesia, were also identified on the stone sample, but in lower proportions (0.4% and 0.2%, respectively, Supplementary Figure 3 ). These fungi are all microcolonial fungi (Isola et al., 2016; Liu B. et al., 2022). According to Isola et al (Isola et al., 2016), in natural, harsh environments, rock fungi in the class Dothideomycetes, predominate over MCF belonging to the order Chaetothyriales, and this is in accordance with our results. Indeed, except the genus Knufia (order Chaetothyriales, class Eurotyomycetes), the other MCF identified on the stone in this study belong to the class Dothideomycetes, in which many species exhibit adaptation mechanisms to extreme conditions, such as thick melanized cellular walls, meristematic development, morphological and metabolic versatility, mycosporine-like substances, and oligotrophy (Gorbushina et al., 2003; Gostinĉar et al., 2012; Zakharova et al., 2013; De Leo et al., 2022). The ability of MCF to weather stone, which occurs through both mechanical (inter-crystalline growth) and chemical (release of organic acids) processes, poses a threat to stone cultural heritage, such as the petroglyphs of the Negev Desert (De Leo et al., 2022). For example, the species Vermiconidia calcicola, also identified in this study (5.8% of the fungal community of the stone sample EZ_D) ( Supplementary Figure 4 ), and Coniosporium sp. (isolated from sample EZ_D) ( Table 1 ) have been studied in relation to the biodeterioration of marble, and their ability to release acids and dissolve calcium carbonate (on CaCO₃ agar medium) has been demonstrated (Berti et al., 2023). Favero-Longo et al. (2011) hypothesized instead that MCF dissolves calcium carbonate through the production of siderophores. In this study, we tested the ability of the isolated strains from soil and stone to dissolve calcium carbonate through acid production in laboratory settings. Unfortunately, all tested strains, including Coniosporium sp. (strain NS14, Table 1 ), resulted in negative results, and only strain NS16, Preussia australis, isolated from soil, resulted in positive results ( Supplementary Figure 2 ). This species, together with Sporormiella spp. (Preussia/Sporormiella species complex), occurred frequently in the soil ( Table 1 ; Figure 3 ), and, indeed, they have been often reported as fungal endophytes in deserts and arid environments (Massimo et al., 2015; González-Menéndez et al., 2018; Sandberg et al., 2022). Preussia/Sporomiella species have the ability to produce a variety of bioactive secondary metabolites with antibacterial, antifungal, and antitumoral activities (Mapperson et al., 2014) (González-Menéndez et al., 2018). Within these secondary metabolites, there are also some organic acids, which, in this case, might have induced the dissolution of the calcium carbonate in the plate assays.

Nevertheless, in the soil mycobiome, the genus Alternaria was the only one consistently present in all samples and consistently detected by both metagenomic and culture-based approaches. Not only was it found in high abundance ( Figure 3 ; Table 1 ), but it also exhibited the highest biodiversity. Seven species were identified by nanopore sequencing ( Supplementary Figure 4 ), and 13 strains (NS1–NS13) belonging to seven different Alternaria sections were retrieved by cultivation ( Table 1 ). The abundant presence of Alternaria in the Negev topsoil aligns perfectly with previous results obtained by Griskhan and colleagues (Yu et al., 2012; Grishkan and Kidron, 2013; Grishkan et al., 2023) and with other biodiversity studies on desert soil (Conley et al., 2006; Bates et al., 2012; Zhang et al., 2016). Alternaria species exhibited strongly melanized, large, thick-walled, and multicellular spores (examples are shown in Figure 4 ), all characteristics that enable these fungi to thrive in high temperatures, strong UV radiation, and xeric conditions (Grishkan, 2019). In our study, the species A. chlamydosporigena, A. alternata, and A. chlamydospora were the most abundant based on the metagenomic results ( Supplementary Figure 4 ), consistent with the findings by Grishkan et al. (2023). Additionally, one-third of the cultivable Alternaria spp. belong to the Alternaria sect. Ulocladioides and Alternaria sect. Ulocladium, both known for their dark colonies ( Supplementary Figures 1E, F ) with very large, extremely resistant spores ( Figures 4B, C ).

The culture-based approach showed that Cladosporium spp. occurred frequently in the soil ( Table 1 ), while metagenomic analyses detected this genus in low abundance (< 0.5%, Supplementary Figure 3 ). Cladosporium is a common and ubiquitous group of hyphomycetes in the environment, capable of adapting to extreme temperatures and xeric conditions by developing thick-walled, melanized conidia capable of rehydration and germination within hours (Abdollahzadeh et al., 2020; Peng et al., 2021). The presence of Cladosporium, particularly species from the C. cladosporioides complex (strains NS20, NA1, NA2, NA3, and NA16; Table 1 ), as a common fungal taxon in desert soil is not surprising and has been observed by other authors (Santiago et al., 2018; Ameen et al., 2022; Grishkan et al., 2023). Additionally, Cladosporium is one of the most common airborne fungi (De Antoni Zoppas et al., 2006; Herrero et al., 2006; Bensch et al., 2012; Anees-Hill et al., 2022) and has been reported as a major component of the air mycobiota in desert environments (Ismail et al., 2002). In our study, Cladosporium spp. were the most abundant strains retrieved from the air sampling ( Table 1 ). Interestingly, species within the C. sphaerospermum complex (strains NS19, NA12, and NA13; Table 1 ), although common cosmopolitan airborne species, are often associated with extreme environments, such as hypersaline locations (Zalar et al., 2007; Sterflinger et al., 2012), and strains identified as C. sphaerospermum have been shown to grow at very low water activity (as low as 0.815) (Hocking et al., 1994; Gostinčar et al., 2010). From the air sampling, Nigrospora and Stemphylium strains were also isolated; both are melanized fungi producing large spores (Grishkan et al., 2023). Stemphylium was also present in all soil samples, according to the metagenomic analysis, although in low abundance ( Supplementary Figure 3 ).

In the four soil samples, four dominant genera were identified solely through metagenomic analysis. These genera, Iodophanus (46.6% of sample AVS_A), Circinaria (13.3% of sample AVS_B), Tricharina (60.1% of sample EZS_A), and Verrucaria (35.8% of sample EZS_D), were detected solely by the metagenomic analysis ( Figure 3 ). The genus Tricharina (order Pezizales) was previously identified as part of the microbiome associated with the rhizosphere of plants in the Atacama Desert (Fuentes et al., 2020), while the genus Iodophanus (order Pezizales), whose members are rich in carotenoid pigments, was found in desert soil by a few studies (Abed et al., 2010; Ameen et al., 2021; Wang et al., 2022). The genera Circinaria and Verrucaria, together with the genus Flavoplaca, detected on the stone sample, are instead lichen-forming fungi (Miadlikowska et al., 2006; Thüs et al., 2011) and therefore cannot be isolated under laboratory conditions. In lower abundance in the soil samples, other lichen-forming species were also identified, such as Catapyrenium sp., Willeya sp., Candelariella spp., and Gloeoheppia turgida ( Supplementary Figure 4 ). Most lichens in the Negev Desert are saxicolous species that colonize limestone substrates (Temina, 2021). In this study, except for Catapyrenium and Gloeoheppia, the other lichens identified belong to this category.

As for MCF, lichens are able to withstand extreme conditions (Armstrong, 2017; Gostinčar et al., 2023), they are rock specialists, and are well-known stone deteriogens (e.g. 494 Salvadori and Casanova, 2016; Cozzolino et al., 2022). A few studies report serious damage to numerous rock art sites due to lichen colonization (Knight et al., 2004; Dandridge, 2006). On the other hand, lichens, along with other biofilms, can have a dual role and, in some cases, can be considered protectants of stone monuments (Pinna, 2014) (Mcllroy de la Rosa et al., 2013) (Favero-Longo and Viles, 2020). In fact, research has shown that lichens have bioprotective effects through physical shielding, which can mitigate the impact of abiotic weathering (e.g., rain, wind, thermal stress) on the rock, or through the formation of an insoluble crust, typically calcium oxalate, at the lichen-rock interface, thereby safeguarding the underlying stone (Mcllroy de la Rosa et al., 2013). (Pinna, 2014). Nevertheless, in order to assess whether a bioprotective or biodeteriorative action is prevailing, both the abiotic and biogenic weathering processes should be properly weighed and evaluated case by case (Liu X. et al., 2022). In the context of this study, we believe that biodeteriorative effects are prevailing. Indeed, while, for example, in a humid and rainy environment, limestone dissolution rates are high, thus a lichen cover might protect the stone substrate, in a dry environment such as the Negev Desert, where dissolution rates are extremely low, lichen-induced surface loss may exceed surface loss from dissolution, leading to biodeterioration (Mcllroy de la Rosa et al., 2013). Moreover, at the Negev Desert rock art sites, some stones are visibly colonized by epilithic lichens, obscuring the black rock surface on which the petroglyphs are carved and causing an unacceptable aesthetic impairment.

In a recent study on the biodeterioration of Negev petroglyph sites, Nir et al. (2021) suggested that lichens play a role in both the deterioration of the bedrock (calcium carbonate) and the disintegration of the external desert varnish. They observed that in samples colonized by lichens, the black layer was no longer present. Additionally, lichens can accelerate the weathering of the desert varnish, which is composed of clay minerals and amorphous silica in a matrix of manganese and iron oxides, through mineral dissolution of manganese and iron oxides (Nir et al., 2021).

Finally, the molecular approach also allowed the detection of other phyla in the soil samples, namely, Basidiomycota, Chytridiomycota, and Mucoromycota ( Figure 2A ). While Basydiomycota taxa, such as Arrhenia, Lichenomphalia, a lichenized agaric genus (Sandoval-Leiva et al., 2017), and Laccaria ( Figure 3 ) are rarely found in desert environments, members of Chytridiomycota, such as Rhizophlyctis spp., and members of Mucoromycota, such as Mortierella spp., were previously identified in desert soil (Murgia et al., 2018; Grishkan et al., 2023).

The Negev Desert soil showed high fungal biodiversity, formed by many cosmopolitan species that are resistant to harsh environmental conditions due to, for example, the production of melanized, large, thick-walled spores ( Figure 4 ). Our results indicate that the rock mycobiota, if detectable, is instead highly specialized and consists of extremotolerant fungal species known to deteriorate limestone (MCF and lichens). The comparison of the rock mycobiota to the fungal communities in the environment (soil and air) clearly shows that Cladosporium and Alternaria, detected in low abundance on the stone sample, as well as other cosmopolitan species previously found on those petroglyphs (Rabbachin et al., 2022), come from the surrounding environment and should be regarded as transient. The three stone samples presenting the black varnish (AV_A, AV_B, and EZ_B) show no apparent fungal colonization, or if present, fungi are below the detection limit of the techniques used in this study. On the other hand, on the stone sample without the black crust (EZ_D), fungi were detected both by ITS sequencing and cultivation. Based on this, together with results from previous studies including microscopical and element analysis (Nir et al., 2021; Rabbachin et al., 2022), we can conclude that in this sample, the exfoliation of the black layer has been enhanced by the detected highly biodeteriorative fungi.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

LR: Conceptualization, Data curation, Investigation, Visualization, Writing – original draft, Writing – review & editing. IN: Data curation, Investigation, Resources, Writing – review & editing. MW: Data curation, Formal Analysis, Visualization, Writing – review & editing. YV: Investigation, Writing – review & editing. GP: Conceptualization, Supervision, Writing – review & editing. AG: Supervision, Writing – review & editing. AK: Supervision, Writing – review & editing. KS: Conceptualization, Supervision, Writing – review & editing.