A New Extremotolerant Ecotype of the Fungus Pseudotaeniolina globosa Isolated from Djoser Pyramid, Memphis Necropolis, Egypt

Most of the rock-inhabiting fungi are meristematic and melanized microorganisms often associated with monument biodeterioration. In previous microbial profiling of the Egyptian Djoser pyramid, a Pseudotaeniolina globosa isolate was found. The current study aimed to characterize the P. globosa isolated from the Djoser pyramid compared with an Italian isolate at morphological, physiological, and molecular levels. Experiments were carried out to test temperature, salinity, and pH preferences, as well as stress tolerance to UV radiation and high temperature, in addition to a multi-locus genotyping using ITS, nrSSU or 18S, nrLSU or 28S, BT2, and RPB2 markers. Morphological and molecular data confirmed the con-specificity of the two isolates. However, the Egyptian isolate showed a wider range of growth at different environmental conditions being much more tolerant to a wider range of temperature (4–37 °C) and pH values (3.0–9.0 pH) than the Italian (10–30 °C, 4.0–6.0 pH), and more tolerant to extreme salinity levels (5 M NaCl), compared to the lowest in the Italian isolate (0.2 M NaCl). Besides, the Egyptian isolate was more tolerant to high temperature than the Italian isolate since it was able to survive after exposure to up to 85 °C for 5 min, and was not affected for up to 9 h of UV exposure, while the Italian one could not regrow after the same treatments. The Pseudotaeniolina globosa species was attributed to the family Teratosphaeriaceae of the order Capnodiales, class Dothideomycetes. Our results demonstrated that the Egyptian isolate could be considered an ecotype well adapted to harsh and extreme environments. Its potential bio-deteriorating effect on such an important cultural heritage requires special attention to design and conservation plans and solutions to limit its presence and extension in the studied pyramid and surrounding archaeological sites.


Introduction
Black meristematic fungi or micro-colonial fungi [1,2], which were more recently referred to as "rock-inhabiting fungi" (RIF) [3], are slow-growing microorganisms often associated with natural rocky substrates [4,5]. To economize the energy requirements in extreme environments, their life cycles are simplified [6], lacking morphologically differentiated sexual phases, and producing only a few metabolites and morphological structures crucial for survival [7]. This group of fungi possesses peculiar characters related to stress tolerance (e.g., melanized cell walls), enabling them to successfully reside under harsh climatic conditions of prolonged desiccation, extreme temperatures (even in Arctic Valley, situated at 29 • 52 10.17 N and 31 • 13 8.70 E in the Giza governorate, Egypt. The Pseudotaeniolina globosa isolate used in the current study, coded as DPS10, was obtained from soil particles collected at the Djoser pyramid's ground entrance using Dichloran Rose Bengal Chloramphenicol (DRBC) agar medium (#CM0727, Oxoid, Ontario, Canada). The nomenclature of fungi follows Index Fungorum (http://www.indexfungorum.org).

Morphological Characterization of Pseudotaeniolina Globosa
Hyphal maturation and conidiogenesis were observed. Culture media preference experiments were also performed.

Hyphal Maturation and Conidiogenesis
These were studied using light and phase contrast microscopy on slide cultures performed by inoculating the DPS10 in squared agar blocks of Malt Extract Agar (MEA; #1038, Condalab, Madrid, Spain). The slide cultures were incubated for one month at 25 • C in wet sterile Petri dishes with filter paper to avoid media dehydrating and observed using lactophenol by a Leica DMLB Tilting Trinocular Phase Contrast (100×) and Dark Field Light Microscope (100×). Digital images were captured using a Leica DFC500 digital color camera optimized with the software Micromax Arkon (v. 8.12.05).

Physiological Characterization of Pseudotaeniolina Globosa
Temperature preferences, growth at different salt concentrations, and growth at different pH were performed according to Selbmann et al. [8]. Colonies with diameter >2 mm were considered positive, according to Kane & Summerbell [31]. Comparisons with an Italian isolate (MC769) of the species were made based on the data reported by De Leo et al. [16].

Temperature Preferences
PDA Petri dishes divided into four sections in duplicates containing the DPS10 isolate were incubated at 4, 10, 25, 30, 34, and 37 • C. The colony diameter was recorded after one month of incubation to detect the isolate's optimum growth among the different selected temperatures.

Growth at Different Salt Concentrations
An experiment was performed to test the ability of the fungus to grow in the presence of a different concentration of salts and to recognize its salinity preference by inoculating the DPS10 isolate on four spots in duplicates of MEA plates supplemented with a scale of NaCl concentrations of 1.2, 1.5, 3.0, 5.0, 7.0, 10.0, 12.0, 15.0, 18.0, 25.0, and 30.0%, incubated at 25 • C for one month.

Growth at Different pH
The DPS10 isolate capacity to grow at different pH values was tested in triplicates of MEB medium at pH levels of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0. At standard conditions for RIFs pH growth [8], the experiment was initially performed at pH 5.0 by 1N HCl; then media was adjusted for pH 1.0 using HCl/KCl buffer and for pH 2.0-7.0 by using McIlvaine solution and for pH 8.0-9.0 by applying Clark & Lubs solution. DPS10 isolate was incubated at 25 • C for one month in a shaken culture at 70 rpm [32].

Tolerance Assessment of Pseudotaeniolina Globosa
As P. globosa is exposed to the sun and dryness prevailing in the Djoser pyramid area, it is supposed to be adapted to this arid environment. In addition to the DPS10, isolate MC769 of P. globosa (CBS 109889T) [16] was used for tolerance assessment and molecular analyses. The MC769 was obtained from the fungal collection of the Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Italy, and freshly re-cultivated using the culture medium Malt Extract Broth (MEB; #CM0057, Oxoid, USA). Experiments were designed to recognize the extreme UV radiation levels and temperature that the DPS10 and MC769 isolates can tolerate.

UV Tolerance under Wet and Dry Conditions
The ability of the fungus to survive and reproduce after exposure to germicide UV-C radiation (253.7 nm) in wet and dry conditions was tested. The experiment material was prepared in replicates by placing about 1 cm 2 mycelia of one month old cultures of DPS10 and MC769 isolates in an open sterile Petri dish each along with 1 mL physiological solution (0.9% NaCl), where the solution was not more than 1 mm deep (wet condition), or without the physiological solution (dry condition). The Petri dishes were then placed at 35 cm from the UV lamp (Philips TUV T8 30W). The sampling was done in duplicates after 10 and 30 min, 1, 3, 6, 9, 12, and 24 h. The treated mycelia under wet and dry conditions were re-planted in PDA medium and incubated at 25 • C for one month to record their regrow ability.

Heat Stress Tolerance
The experimental aim was to identify the degree of the fungus ability to survive and resume growth after the exposure of the cells to high-temperature stress. The experiment was performed in duplicates by harvesting about 1 cm 2 mycelia of one month old cultures of DPS10 and MC769 isolates and transfer them to 1 mL of physiological solution in 2 mL tubes. The tubes were put in a water bath adjusted to 65 • C up to 85 • C in 5 • C intervals. The sampling was done in duplicates after 2, 5, 10, and 15 min of exposure at each temperature. The same was repeated in dry conditions using hot blocks without a physiological solution. The treated mycelia from both wet and dry conditions were re-planted in PDA medium and incubated at 25 • C for one month.

Molecular Characterization of Pseudotaeniolina Globosa
Molecular characterization of both isolates and family assignment of P. globosa were performed using a PCR based approach combined with Sanger sequencing, commonly known as "multi-locus genotyping" using five molecular markers.

DNA Extraction
DNA for both fungal isolates was extracted using cetyltrimethylammonium bromide (CTAB) acid-washed beads using the manual protocol of Möller et al. [33] and modified by Urzì et al. [34]. DNA was quantified with Qubit TM and the Qubit TM dsDNA BR assay kit (Invitrogen, Thermo Fisher Scientific, Massachusetts, MA, USA).

Multi-Locus Genotyping
The loci that were amplified for multi-locus comparison between the two isolates were the ITS, the small subunit of the nuclear ribosomal RNA (nrSSU or 18S), the large subunit of the nuclear ribosomal RNA (nrLSU or 28S), B-tubulin (BT2), and RNA polymerase II (RPB2) [21]. Primers are listed in Table 1. PCR reactions were performed using the MyTaq™ Red Mix (Cat# BIO-25043, BioLine, London, UK). Each 25 µL reaction tube included 5 pmol of each primer, and 40 ng of template DNA. The amplification was carried out using a Techne™ 512 thermocycler (Techne, Staffordshire, UK). The PCR programs were adjusted according to the primer pair melting temperature (Tm) as follows: the first denaturation step at 95 • C for 5 min was followed by 33 cycles of denaturation at 95 • C for 30 s, annealing at the assigned temperature (see Table 1) for 30 s, extension at 72 • C for 30 s and a final extension 72 • C for 5 min.
Taxonomic ranking and phylogenetic relationships were retrieved from the NCBI taxonomy database (https://www.ncbi.nlm.nih.gov/taxonomy). Alignments of the target sequences and the BLAST query results were performed using ClustalW [36] using the default settings, while repeated sequences were discarded. Phylogenetic trees were constructed by the maximum likelihood method based on the Generalized Time-reversible (GTR) model using Fasttree v2.1.5 [37].

Hyphal Maturation and Conidiogenesis
The DPS10 isolate was anamorphic. Colonies grew slowly as they reached 21.0 ± 0.8 mm in diameter after one month. Yeast-like cell forms were absent, and the teleomorph was not observed. Black colonies were butter-like (butyreae); they became shiny and of more rigid structure and wrinkled, with a cauliflower shape, with age. The mycelium was composed of pale brown, thick-walled immersed, branched hyphal cells, 7.0-12.0 µm long, and 4.0-6.0 µm wide. The meristematic mycelium eventually converted into multicellular clumps, 5.0-12.0 µm in diameter. Conidia were produced by arthric disarticulation of hyphae; these were uni-or bi-cellular, pale brown, constricted at the septa, and 4.8-5.5 µm in diameter ( Figure 1). more rigid structure and wrinkled, with a cauliflower shape, with age. The mycelium was composed of pale brown, thick-walled immersed, branched hyphal cells, 7.0-12.0 μm long, and 4.0-6.0 μm wide. The meristematic mycelium eventually converted into multicellular clumps, 5.0-12.0 μm in diameter. Conidia were produced by arthric disarticulation of hyphae; these were uni-or bi-cellular, pale brown, constricted at the septa, and 4.8-5.5 μm in diameter ( Figure 1).

Culture Media Preferences
The isolate DPS10 was able to grow and reproduce on different culture media after one month of incubation at 25 °C. On PDA, it showed a visible optimum growth with a diameter record of 21.0 ± 0.8 mm, with black, shiny, hard, wrinkled structure and a cauli-

Culture Media Preferences
The isolate DPS10 was able to grow and reproduce on different culture media after one month of incubation at 25 • C. On PDA, it showed a visible optimum growth with a diameter record of 21.0 ± 0.8 mm, with black, shiny, hard, wrinkled structure and a cauliflower shape, slightly raised at the center (umbonate), radially folded, with a unique appearance of an olive-green glow surrounding the colony edge. On MEA and CzA media, it showed a small reduction of 19.5 ± 0.7 mm in diameter, while the same morphological appearance was observed, except an olive-green color on MEA, and change from umbonate to crateriform with leathery texture on CzA. The smallest ultimate colony diameters were observed on the OMA medium, being 18.5 ± 0.8 mm in diameter, with a different appearance of a flat, hairy, gray growth on the colony top. No changes in the conidia morph among the tested media were observed ( Figure 2). nate to crateriform with leathery texture on CzA. The smallest ultimate colony diameters were observed on the OMA medium, being 18.5 ± 0.8 mm in diameter, with a different appearance of a flat, hairy, gray growth on the colony top. No changes in the conidia morph among the tested media were observed ( Figure 2).
As reported by De Leo et al. [16], MC769 isolate colony on PDA showed the highest growth record of 28.0 mm in diameter with flat, regular margin, slightly raised at the center, cerebriform, and radially folded. On MEA, the colony was black and shiny, buttery, flat, slightly raised at the center and of 25.0 mm in diameter. It had a sharp, regular margin of 21.0 mm diameter on OMA, while the colony on CzA was flat, with a fimbriate margin, attaining up to 10.0 mm diameter ( Figure 2).

Temperature Preferences
The DPS10 isolate grew and reproduced at a wide range of temperatures, ranging from 4 to 37 °C, with the optimum growth observed at 25 °C where the colony reached 21.0 ± 0.7 mm in diameter. Above this temperature, a considerable reduction of colony As reported by De Leo et al. [16], MC769 isolate colony on PDA showed the highest growth record of 28.0 mm in diameter with flat, regular margin, slightly raised at the center, cerebriform, and radially folded. On MEA, the colony was black and shiny, buttery, flat, slightly raised at the center and of 25.0 mm in diameter. It had a sharp, regular margin of 21.0 mm diameter on OMA, while the colony on CzA was flat, with a fimbriate margin, attaining up to 10.0 mm diameter ( Figure 2).

Temperature Preferences
The DPS10 isolate grew and reproduced at a wide range of temperatures, ranging from 4 to 37 • C, with the optimum growth observed at 25 • C where the colony reached 21.0 ± 0.7 mm in diameter. Above this temperature, a considerable reduction of colony diameter occurred at 30 • C with 13.7 ± 0.3 mm, and 37 • C with 4.8 ± 0.4 mm in diameter. At the lowest temperature at which the colony was able to grow, it reached 7.0 ± 0.7 mm in diameter. Considering these data, it can be referred to as a mesophilic-psychrotolerant fungus. In comparison, as De Leo et al. [16] reported, the isolate MC769 showed the same diameter of optimum growth at 25 • C and near same diameter, between 10 to 30 • C, without any record of growth at lower and higher temperature degrees ( Figure 3). diameter occurred at 30 °C with 13.7 ± 0.3 mm, and 37 °C with 4.8 ± 0.4 mm in diameter. At the lowest temperature at which the colony was able to grow, it reached 7.0 ± 0.7 mm in diameter. Considering these data, it can be referred to as a mesophilic-psychrotolerant fungus. In comparison, as De Leo et al. [16] reported, the isolate MC769 showed the same diameter of optimum growth at 25 °C and near same diameter, between 10 to 30 °C, without any record of growth at lower and higher temperature degrees ( Figure 3).

Growth at Different NaCl Concentrations
The DPS10 isolate could grow at all the tested NaCl concentrations, even at 30.0%, the maximum checked. It showed colonies of 22.0 ± 0.5 to 18.5 ± 1.2 mm diameter up to 10.0% of NaCl concentration and showed a limited reduction of colony diameter of 17.0 ± 1.9 and 14.6 ± 1.6 mm at 12.0% and 15.0% NaCl concentration, respectively; and a considerable reduction to 9.5 ± 1.7 and 7.5 ± 0.8 mm at 18.0% and 30.0% NaCl concentration, respectively. Differently, MC769 isolate only grew at a maximum of 1.2% NaCl concentration as reported by De Leo et al. [16] (Figure 4).

Growth at Different NaCl Concentrations
The DPS10 isolate could grow at all the tested NaCl concentrations, even at 30.0%, the maximum checked. It showed colonies of 22.0 ± 0.5 to 18.5 ± 1.2 mm diameter up to 10.0% of NaCl concentration and showed a limited reduction of colony diameter of 17.0 ± 1.9 and 14.6 ± 1.6 mm at 12.0% and 15.0% NaCl concentration, respectively; and a considerable reduction to 9.5 ± 1.7 and 7.5 ± 0.8 mm at 18.0% and 30.0% NaCl concentration, respectively. Differently, MC769 isolate only grew at a maximum of 1.2% NaCl concentration as reported by De Leo et al. [16] (Figure 4).

Growth at Different pH
The DPS10 isolate showed an ability to grow at a wide range of pH values (from pH 3.0 to pH 9.0). The maximal growth was recorded between pH 5.0 and pH 7.0, and the minimal growth was recorded at pH 3.0 and pH 9.0. Based on the data from De Leo et al. [16], MC769 isolate showed growth at a narrower range of pH values (from pH 4.0 to pH 7.0), with maximal growth at pH 5.0 and minimal growth at pH 7.0. Comparing the two isolates, both started to grow at pH 4.0 with optimal growth up to pH 5.0 for MC769 and up to pH 7.0 for DPS10 and continued with growth reduction to pH 9.0. On the contrary, MC769 showed no growth at pH 8.0 ( Figure 5).

UV Tolerance under Wet and Dry Conditions
The isolates DPS10 and MC769 showed an equal maximum growth rate after UV exposure of 10 min until 6 h; the isolates maintained the same colony diameter (21.0 and 25.0 mm, respectively). MC769 showed a reduction of 50% of average colony diameter (after 9, 12, and 24 h of UV exposure), while DPS10 showed 50% reduction after 12 and 24 h of UV exposure (Figure 6). the maximum checked. It showed colonies of 22.0 ± 0.5 to 18.5 ± 1.2 mm diameter up to 10.0% of NaCl concentration and showed a limited reduction of colony diameter of 17.0 ± 1.9 and 14.6 ± 1.6 mm at 12.0% and 15.0% NaCl concentration, respectively; and a considerable reduction to 9.5 ± 1.7 and 7.5 ± 0.8 mm at 18.0% and 30.0% NaCl concentration, respectively. Differently, MC769 isolate only grew at a maximum of 1.2% NaCl concentration as reported by De Leo et al. [16] (Figure 4).

Growth at Different pH
The DPS10 isolate showed an ability to grow at a wide range of pH values (from pH 3.0 to pH 9.0). The maximal growth was recorded between pH 5.0 and pH 7.0, and the minimal growth was recorded at pH 3.0 and pH 9.0. Based on the data from De Leo et al. [16], MC769 isolate showed growth at a narrower range of pH values (from pH 4.0 to pH 7.0), with maximal growth at pH 5.0 and minimal growth at pH 7.0. Comparing the two isolates, both started to grow at pH 4.0 with optimal growth up to pH 5.0 for MC769 and up to pH 7.0 for DPS10 and continued with growth reduction to pH 9.0. On the contrary, MC769 showed no growth at pH 8.0 ( Figure 5).

UV Tolerance under Wet and Dry Conditions
The isolates DPS10 and MC769 showed an equal maximum growth rate after UV exposure of 10 min until 6 h; the isolates maintained the same colony diameter (21.0 and 25.0 mm, respectively). MC769 showed a reduction of 50% of average colony diameter (after 9, 12, and 24 h of UV exposure), while DPS10 showed 50% reduction after 12 and 24 h of UV exposure ( Figure 6).

Heat Stress Tolerance
Heat stress on isolates DPS10 and MC769 at 65, 70, and 75 °C after 2 and 5 min showed the maximum growth rate a colony can reach in one month (colony diameter of 21.0 and 25.0 mm, respectively). However, when the exposure extended to 10 and 15 min, a lower growth rate was observed at all the tested temperatures for both isolates. At 70 and 75 °C, after 15 min of exposure, the MC769 showed a slower rate than the low rate described for DPS10. When the 80 °C was tested, both isolates were grown at a low rate after 2 and 5 min of exposure; however, only DPS10 maintained the same rate after 10 min and showed a slow rate after 15 min of exposure contrast to MC769, which showed no growth. When the 85 °C was tested, only DPS10 grew at a slow rate after 2 and 5 min, while showed no growth after 10 and 15 min of exposure (Figure 7).

Heat Stress Tolerance
Heat stress on isolates DPS10 and MC769 at 65, 70, and 75 • C after 2 and 5 min showed the maximum growth rate a colony can reach in one month (colony diameter of 21.0 and 25.0 mm, respectively). However, when the exposure extended to 10 and 15 min, a lower growth rate was observed at all the tested temperatures for both isolates. At 70 and 75 • C, after 15 min of exposure, the MC769 showed a slower rate than the low rate described for DPS10. When the 80 • C was tested, both isolates were grown at a low rate after 2 and 5 min of exposure; however, only DPS10 maintained the same rate after 10 min and showed a slow rate after 15 min of exposure contrast to MC769, which showed no growth. When the 85 • C was tested, only DPS10 grew at a slow rate after 2 and 5 min, while showed no growth after 10 and 15 min of exposure (Figure 7).

Molecular Characterization and Phylogeny of Pseudotaeniolina Globosa
The amplification of the five loci for the two isolates was successful, showing the expected molecular size as previously reported. Sequences were deposited in GenBank repository under the accession numbers detailed in Table 2. Based on the BLAST results, similar top results were retrieved and filtered by query coverage (i.e., <95% query coverage were discarded) and matched with "Popsets" files downloaded from the NCBI database for each locus prior to alignment and phylogenetic analysis. All the retained accessions belonged to the order Capnodiales. The maximum likelihood (ML) phylogenetic trees were rooted with the Extremaceae family species, while all the remaining sequences belonged to the families Teratosphaeriaceae and Mycosphaerellaceae for all the genotyped loci. In the case of ITS, a total of 30 accessions belonging to the order Capnodiales in addition to the current two isolates of P. globosa (DPS10 and MC769) showed an alignment length of 566 bp, including partial 5 and 3 ends of both ITS1 and ITS2, respectively, and the complete 5.8S locus.

Molecular Characterization and Phylogeny of Pseudotaeniolina Globosa
The amplification of the five loci for the two isolates was successful, showing the expected molecular size as previously reported. Sequences were deposited in GenBank repository under the accession numbers detailed in Table 2. Based on the BLAST results, similar top results were retrieved and filtered by query coverage (i.e., <95% query coverage were discarded) and matched with "Popsets" files downloaded from the NCBI database for each locus prior to alignment and phylogenetic analysis. All the retained accessions belonged to the order Capnodiales. The maximum likelihood (ML) phylogenetic trees were rooted with the Extremaceae family species, while all the remaining sequences belonged to the families Teratosphaeriaceae and Mycosphaerellaceae for all the genotyped loci. In the case of ITS, a total of 30 accessions belonging to the order Capnodiales in addition to the current two isolates of P. globosa (DPS10 and MC769) showed an alignment length of 566 bp, including partial 5′ and 3′ ends of both ITS1 and ITS2, respectively, and the complete 5.8S locus.
The number of identical sites was 253 bp (50%), while the un-gapped average length was 446 bp with GC% = 55.9%. A major observation is that the family Teratosphaeriaceae members were highly clustered into two sub-clades (I and II; bootstrap value ≥ 0.80). The isolate P. globosa DPS10 was highly clustered to eight P. globosa accessions (bootstrap value = 0.96). The most similar sequence to the Egyptian isolate (with bootstrap value = 0.81) was the specimen CBS303.84, which was early registered in GenBank as Trimmatostroma sp. [44] and corrected to P. globosa in a later publication [8]. The geolocation of the samples ( Table 2) does not reflect the phylogenetic relationships based on this marker (Figure 8, ITS).  The number of identical sites was 253 bp (50%), while the un-gapped average length was 446 bp with GC% = 55.9%. A major observation is that the family Teratosphaeriaceae members were highly clustered into two sub-clades (I and II; bootstrap value ≥ 0.80). The isolate P. globosa DPS10 was highly clustered to eight P. globosa accessions (bootstrap value = 0.96). The most similar sequence to the Egyptian isolate (with bootstrap value = 0.81) was the specimen CBS303.84, which was early registered in GenBank as Trimmatostroma sp. [44] and corrected to P. globosa in a later publication [8]. The geolocation of the samples (Table 2) does not reflect the phylogenetic relationships based on this marker (Figure 8, ITS).  In the case of the nrSSU (18S), a total of 18 accessions belonging to the order Capnodiales in addition to the current two isolates of P. globosa (DPS10 and MC769) showed an alignment length of 1670 bp. The number of identical sites was 1541 bp (92.3%), while the un-gapped average length was 1667 bp with GC% = 48.5%. The P. globosa isolates were included within the clades identified as order Capnodiales. In addition to DPS10 and MC769 isolates, a single P. globosa accession CCFEE 5734 was highly clustered in the same clade (bootstrap value = 0.92). The P. globosa accessions were sister to Hortaea werneckii (Horta) Nishim. & Miyaji (CBS 107.67), a very peculiar species known as an extremophilic halotolerant fungus, and Stenella araguata Syd. (CBS 105.75) (family Mycosphaerellaceae) a biological causal agent for Tinea nigra dermatological disease (bootstrap value = 0.79; Figure 8, nrSSU 18S) [50]. For the nrLSU (28S), a total of 28 accessions in addition to the current two isolates of P. globosa (DPS10 and MC769) showed an alignment length of 782 bp (incomplete sequence, partial at 3 terminal). The number of identical sites was 573 bp (73.8%), while the un-gapped average length was 760 bp with GC% = 53.9%. The ML tree showed a monophyletic clustering of the families within order Capnodiales with the Extremaceae clade as root, followed by the Mycosphaerellaceae clade and the Teratosphaeriaceae clade. In the latter, the P. globosa DPS10 and MC769 isolates were highly clustered with two P. globosa accessions recorded as Capnodiales incertae sedis (bootstrap value = 0.99; Figure 8, nrLSU 28S).
Based on BT2, all retrieved sequences by BLAST without the intronic regions were discarded. A total of nine accessions and the current two isolates of P. globosa (DPS10 and MC769) showed an alignment length of 425 bp. The number of identical sites was 197 bp (48.2%), while the un-gapped average length was 385 bp with GC% = 55.6%. The isolates of P. globosa were included within the clades identified as order Capnodiales, family Teratosphaeriaceae (Figure 8, BT2). In RPB2, a total of 19 accessions and the current two isolates of P. globosa (DPS10 and MC769) showed an alignment length of 239 bp. The number of identical sites was 107 bp (44.8%), while the un-gapped average length was 239 bp with GC% = 53.1%. The isolates of P. globosa were included within the clades identified as order Capnodiales, family Teratosphaeriaceae with bootstrap value = 0.70 (Figure 8, RPB2).

Discussion
Black meristematic fungi or RIFs are remarkably extremotolerant microorganisms, frequently isolated from stone surfaces and habitats where minimal nutrients are available [51]. RIF survive under extreme conditions and grow to form micro-colonial fungal life forms (MCF) [52]. The selective pressure of such harsh conditions directed the evolution of their genomes to develop tolerant alleles through adaptive genetic variation and accumulation of beneficial mutations; the genetic pools of those organisms deviated significantly from sister fungal lineages to overcome the rock surface conditions (e.g., high UV radiation and temperatures) [53]. For instance, the highly concentrated black pigments in the MCF cell walls serve as a UV-protective substance (e.g., [54]). This is the first report of Pseudotaeniolina globosa on Egyptian archaeological remains. Pseudotaeniolina globosa is one of the uncommonly recorded RIF; only a few records were previously reported. All the reported isolates are known as environmental saprobes, predominantly found in water-limited ecological niches. Except for a unique strain isolated from the aortic wall of a patient with an aortic aneurysm, its clinical significance has not been confirmed [45].
In the current work, the morphological measurements of the hyphal length and width on MEA media of DPS10 isolate (7.0-12.0 × 4.0-6.0 µm) were smaller than the MC769 (8.0-15.0 × 6.0-7.0 µm) [16]. However, the DPS10 produces single cells or asymmetrically septate cells after conidiogenesis, a unique character to P. globosa [16]. The current isolate is slimy and forms yeast-like colonies after 4-5 days of growth, which differentiate the P. globosa from a micro-morphologically similar species known as Knufia petricola (Wollenz. & de Hoog) Gorbushina & Gueidan (=Sarcinomyces petricola Wollenz. & de Hoog) [16,55,56]. Regardless of the morphological variation between both isolates, the molecular characterization of the DPS10 isolate confirms its identity and high similarity to the P. globosa MC769 isolate. However, under abiotic stress (pH, salinity, and temperature) the DPS10 showed tolerance to extreme conditions. The pH of a medium is a decisive factor for fungal presence and diversity; for example, the soil pH was the most significant factor correlated to the fungal community composition in Svalbard, High Arctic [57]. The DPS10 optimum growth was at 5.0-7.0 pH and it grew on a broader range from 3.0 to 9.0 pH compared to MC769 isolate, which grew well between 4.0-6.0 and scarcely at 7.0 pH. Thus, the ability of DPS10 to colonize different media with different pH ranges is worth further studying.
The DPS10 optimum growth was at 0.25 M (1.5%) NaCl and growth continued, although at lower rates, up to 5.2 M (30.0%) NaCl, while MC769 was strictly limited to 0.20 M (1.2%) NaCl supplemented MEA. Fungi are considered halophilic if they are isolated from sites with 1.7 M (~10.0%) NaCl, and halotolerant if sporadic isolates can grow in vitro at 3 M NaCl supplemented medium [58]. According to previous reports of model halotolerant fungi, Debaryomyces hansenii (Zopf) Lodder & Kreger-van Rij can grow in up to 3 M NaCl [59], Hortaea werneckii in up to 5 M NaCl [60], while Wallemia ichthyophaga Johan-Olsen thrives in 5.2 M NaCl, the latter of which is considered the most halophilic fungus; however, it is unable to grow without salt in its medium [61]. The MC769 isolate was previously described as halo-sensitive [16]. However, according to our results, the DPS10 isolate can be considered halotolerant [58]. At the intracellular Na + and K + ion accumulation level, it was shown that D. hansenii and W. ichthyophaga are Na + -intruder fungi and accumulate higher amounts of Na + ions than H. werneckii, which excludes these ions from its cells [58]. Halophily is expressed in several groups of the same order, but not the closest at the taxonomic level [58]. Hortaea werneckii is the nearest phylogenetic neighbor to P. globosa and, most probably, follows the same mechanism. Alternatively, they are extreme xerotolerants whose growth on substrates is determined by water potential and not by the solute's chemical nature [62].
DPS10 and MC769 showed optimum growth rate at 25 • C, but DPS10 had a broader range of growth temperatures (4-37 • C) than MC769 (10-30 • C). Additionally, DPS10 isolate was able to regrow after being exposed to 85 • C for 5 min while MC769 regrow only after being exposed to 80 • C for 5 min. Both P. globosa isolates are melanized, thus constitute a chemical ability to absorb and tolerate UV light [63]. Both isolates were tolerant to UV exposure up to 24 h; however, the growth rate was not affected up to 9 h in the DPS10 case compared to MC769, which was affected after 9 h of exposure to UV. Melanin serves as an extracellular electron-dense granular layer for black fungi and yeasts and forms an intracolonial matrix structure [52]. The results suggest that DPS10 is an extremotolerant P. globosa ecotype adapted to the harsh and extreme environments as arid and desert climate. It can survive pH changes, high salt concentrations, and tolerate high temperatures and UV radiation, demonstrating its potential as a persistent bio-deteriorating biological agent. Thus, further studies are required to understand its nature fully and develop counter strategies and conservation plans for one of Egypt's oldest pyramids (i.e., the Djoser pyramid).
Within Ascomycota, the main orders with halophilic and halotolerant representatives are Capnodiales, Dothideales, and Eurotiales [58]. Among them, the Capnodiales have a xero-tolerant tendency, as they contain a large number of extremotolerant species that can grow as epilithic or crypto-endolithic species at high or low temperatures [6]. Most RIF possesses convergent morphological and physiological characteristics; however, many RIF subgroups are phylogenetically uncertain within the class Dothideomycetes [3]. According to the NCBI taxonomy database, the genus Pseudotaeniolina is uncertainly positioned within the order Capnodiales (incertae sedis) with no family assigned. When first reported, P. globosa was found clustered with species associated with low water availability (halophilic, epilithic, or epiphytic) and defined as RIF within the Dothideales but was poorly supported by the phylogenetic analysis using ITS sequences (>50%) [16]. In the current study, based on five molecular markers (ITS, SSU, LSU, BT2, and RPB2), P. globosa was clustered within the Teratosphaeriaceae family with bootstrap values of 0.70-0.98.
Ruibal et al. [3] conducted a phylogenetic analysis using a multi-locus approach (nrSSU, nrLSU, and mtSSU) to resolve several taxonomic complexes of Dothideomycetes families. Among other families, the Teratosphaeriaceae was the most diverse and phylogenetically conflicting. The family Teratosphaeriaceae was represented as two separated clades, numbered as (1) and (2); however, the study did not include any P. globosa records. When we matched the species, the P. globosa isolates should be part of Teratosphaeriaceae group 1. When locus analyses conducted separately, the clustering was apparent at the ribosomal cistron sequences (ITS, SSU, and LSU) and resulted in a better resolution for the family compared to the closest one (Mycosphaerellaceae), especially the LSU. In contrast to other markers, the SSU phylogenetic signal was the only marker to show a paraphyletic clustering among the two proximate families, Mycosphaerellaceae and Teratosphaeriaceae; however, the taxonomical resolution within the Teratosphaeriaceae was not as structured as the ITS based phylogeny. The current study focused on the taxonomical family assignment of the P. globosa rather than on the taxonomical analysis of the order or on finding the best marker for such an assignment. However, many accessions were discarded and are missing in our analysis (e.g., RPB2) due to wrong taxonomic assignment or incomplete metadata about the accessions, which require urgent revision to avoid misleading phylogenetic signals. For instance, the P. globosa CCFEE 5734 RPB2 sequence (GenBank accessions KF310073) seems confused with Extremus antarcticus Quaedvl. & Crous isolate CCFEE 451 RPB2 sequence (GenBank accessions KF310085 [21]).
Future research will target the genomic signatures of the extremo-tolerance characters of the DPS10 isolate to accurately detect the transcriptional responses and metabolic suspension and reactivation under the fluctuation of water availability and its extreme ability to maintain viability in a high salinity medium. This will be achievable after acquiring additional P. globosa isolates and performing a more sophisticated comparison at genomic, transcriptomic, and metabolic levels using NGS-based approaches.