The molecular characterization of the ELF strains remoted from A. schleicheri was carried out utilizing BLASTn evaluation of ITS sequences. The obtained sequences confirmed excessive similarity (> 99%) with reference sequences in GenBank. Pressure HA confirmed a 100% identification match with Apiospora kogelbergensis (CBS 113333). Strains HC, A-HF1, and B-HF1 exhibited 100% identification with Cladosporium limoniforme (CBS 140484), whereas pressure F-KS confirmed 99.80% identification with the identical species. Pressure HE matched Alternaria botrytis (CBS 197.67) with 99.39% identification, and pressure C-HC confirmed 99.57% identification with Gibellulopsis nigrescens (CBS 120949).
For genera with advanced taxonomy, BLASTn outcomes indicated shut relationships however restricted species-level decision. Pressure HB confirmed 99.79% identification with a number of species within the Aspergillus part Terrei (e.g., A. terreus, A. citrinoterreus). Equally, pressure H-I matched a number of Monascus species (M. purpureus, M. pilosus) with 100% identification. Lastly, the basidiomycetous yeast pressure HD exhibited 99.1% identification with Sporobolomyces reniformis (CGMCC 2.5627). All BLASTn outcomes are summarized in Desk 1.
Phylogenetic relationships of endolichenic fungi
To resolve the evolutionary placement of the remoted strains, phylogenetic inference was carried out utilizing separate Most Chance (ML) analyses for every fungal genus. This method allowed for genus-specific alignments and sturdy topological assist.
Phylogeny of Dothideomycetes (Cladosporium and Alternaria)
The phylogenetic evaluation of the genus Cladosporium included strains HC, A-HF1, B-HF1, and F-KS. The ML tree (Supplementary Fig. S1 on-line) recovered a strongly supported clade (ultrafast bootstrap worth [UFBoot] = 100%) grouping all 4 isolates with the sort pressure of Cladosporium limoniforme (CBS 140484). This topology confirms the conspecificity of those isolates regardless of the morphological variations noticed in tradition. Individually, the evaluation for pressure HE inside the genus Alternaria (Supplementary Fig. S2 on-line) positioned this isolate in a clade with Alternaria botrytis (CBS 197.67) and associated taxa, supported by an UFBoot of 97%.
Phylogeny of Sordariomycetes (Apiospora and Gibellulopsis)
The phylogenetic tree positioned HA in a terminal department sister to Apiospora kogelbergensis (CBS 113333), with excessive statistical assist (UFBoot = 100%), according to the BLASTn identification (Supplementary Fig. S3 on-line). For pressure C-HC, the evaluation inside the genus Gibellulopsis clustered the isolate with Gibellulopsis nigrescens (CBS 120949), forming a monophyletic group supported by a price of 95% (Supplementary Fig. S4 on-line), distinct from different species reminiscent of G. serrae.
Phylogeny of Eurotiomycetes (Aspergillus and Monascus)
Within the Aspergillus phylogeny (Supplementary Fig. S5 on-line), pressure HB clustered inside the part Terrei. Nonetheless, the ITS marker offered poor decision to position the pressure inside a strongly supported clade (i.e., UFBOOT < 95%; particularly 61%, not proven within the tree), thereby forming a monophyletic group with a number of different Aspergillus species. The evaluation of pressure H-I inside the genus Monascus revealed a detailed relationship with Monascus purpureus and M. pilosus, forming a supported clade of 96% (Supplementary Fig. S6 on-line).
Phylogeny of Basidiomycota (Sporobolomyces)
Within the Sporidiobolales phylogeny (Supplementary Fig. S7 on-line), the ML tree positioned isolate HD as an early-diverging lineage adjoining to Sporobolomyces reniformis. Nonetheless, the phylogenetic marker offered poor decision to firmly set up this sister-group relationship (i.e., UFBOOT < 95%; particularly 81%, not proven within the tree), leading to an unresolved placement concerning S. reniformis and the opposite intently associated species.
Micro- and macro-morphological characterization
Supporting the findings from the molecular identification of the strains by ITS, macro- and microscopic analyses had been accomplished, searching for morphological traits of the fungal strains.
Apiospora kogelbergensis (pressure HA)
Classification: Fungi > Dikarya > Ascomycota > Pezizomycotina > Sordariomycetes> Xylariomycetidae > Xylariales > Apiosporaceae > Apiospora > Apiospora kogelbergensis, described in 2021, by Pintos & Alvarado10. Mycobank # 837682. Genbank. Accession Quantity: PV803146.1 (HA).
Colonies on PDA exhibit fast progress, reaching 55–65 mm in diameter after 14 days of incubation. The obverse is characterised by a dense, floccose mycelium with a uniform white to pale grey coloration. A outstanding radial progress sample is noticed, that includes a extra compact central zone that signifies the onset of early reproductive growth (Fig. 1A). The reverse of the colony stays unpigmented or shows a pale cream colour (Fig. 1B). Microscopic evaluation (Fig. 1C and D) reveals hyaline, septate, and branched hyphae with a width of two.5–4.5 μm. Giemsa staining highlights a attribute pink/purple hue inside the filaments. Whereas mature conidia weren’t definitively noticed underneath the examined tradition situations, immature globose constructions measuring 6.0–10.5 μm had been recognized.
Morphological characterization of Apiospora kogelbergensis (Pressure HA). (A) Obverse: Macroscopic view on PDA medium after 7 days of incubation, highlighting a dense and floccose mycelium of uniform white to pale grey colour with a outstanding radial progress sample. (B) Reverse: Colony exhibiting a attribute lack of diffuse pigmentation or pale cream tones. (C) Microscopy (40X): Hyaline, septate, and branched hyphae with a width of two.5–4.5 μm, highlighted by Giemsa staining with a pink/purple hue. (D) Microscopic element: Presence of immature globose constructions (6.0–10.5 μm) recognized underneath managed tradition situations.
Aspergillus sp. (pressure HB)
Classification: Fungi > Dikarya > Ascomycota > Pezizomycotina > Eurotiomycetes > Eurotiomycetidae > Eurotiales > Aspergillaceae > Aspergillus. Described by Haller et al.11. Mycobank # 7248. Genbank Accession Quantity: PV803147.1 (HB).
Colonies of Pressure HB belong to part Terrei and are characterised by remoted round progress reaching a diameter of 35–45 mm after 7 days of incubation on PDA. The obverse reveals a particular radial progress sample with a white periphery surrounding a cinnamon to yellowish-beige (cinnamomeous) middle. The central space shows a powdery texture ensuing from considerable conidiation (Fig. 2A). The reverse of the colony exhibits a outstanding yellowish-brown pigmentation that diffuses into the agar medium (Fig. 2B).
Morphological characterization of Aspergillus sp. (Pressure HB), part Terrei. (A) Obverse: Remoted round colony on PDA (35–45 mm diameter) with a white periphery and a cinnamomeous (cinnamon) powdery middle resulting from considerable conidiation. (B) Reverse: Distinguished yellowish-brown pigmentation diffusing actively into the agar medium. (C) Reproductive construction: Conidiophore terminating in a globose to subglobose vesicle (15–22 μm), visualized with Lactophenol Blue staining. (D) Conidia: Discipline of small globose conidia (2.0–2.8 μm) characteristically stained mild purple with the Giemsa approach.
On the microscopic degree (Fig. 2C and D), the important thing diagnostic characteristic is the hyaline construction rising from the mycelium. Conidiophores terminate in globose to subglobose vesicles with a diameter of 15–22 μm, that are clearly visualized utilizing Lactophenol Blue staining. The conidia are globose, small (2.0–2.8 μm), and characteristically stain mild purple with the Giemsa approach. The scale of those constructions is according to reported ranges when in comparison with the 20 μm scale bar.
Cladosporium limoniforme (strains HC, A-HF1, B-HF1, F-KS)
Classification: Fungi > Dikarya > Ascomycota > Pezizomycotina > Dothideomycetes > Cladosporiales > Cladosporiaceae > Cladosporium > Cladosporium limoniforme, described by Bensch et al.12. Mycobank # 814628. Genbank Accession Numbers: PV803148.1 (HC), PV803143.1 (B-HF1), PV803142.1 (A-HF1), PV803145.1 (F-KS).
On nutrient media, colonies of C. limoniforme exhibit a velvety texture and a uniform olive-green colour, with a well-delimited, round, and symmetrical morphology (Fig. 3A). The reverse is characterised by an olive-black pigmentation resulting from melanin accumulation, a trait that could be important for resistance in opposition to excessive ultraviolet radiation within the Altiplano (Fig. 3B). This pressure demonstrates vital phenotypic plasticity on poor media reminiscent of water agar (WA4), the place progress turns into spongy, floccose, and irregular as an adaptive response to dietary stress (not proven). Moreover, this isolate was chosen for entire genome sequencing, representing the primary full genome sequence of the species worldwide13.
Morphological characterization of Cladosporium limoniforme (Pressure HC). (A) Obverse: Velvety, uniform olive-green colony with a well-delimited and symmetrical round morphology on nutrient media. (B) Reverse: Olive-black coloration as a result of accumulation of protecting melanin. (C) Conidial morphology: Ellipsoidal blastoconidia with the distinctive lemon form (limoniform), measuring roughly 4.5–12.0 × 3.0–5.0 μm, stained with Lactophenol Blue. (D) Development sample: Typical formation of branched acropetal chains with pure olive-brown pigmentation, utilizing Giemsa staining.
Microscopically, the species is characterised by ellipsoidal blastoconidia with barely truncated ends, giving them the distinctive lemon form (limoniforme) for which the species is called (Fig. 3C and D). These conidia measure roughly 4.5–12.0 × 3.0–5.0 μm, possess a finely verruculose floor, and exhibit pure olive-brown pigmentation typical of dematiaceous genera. The expansion behavior is outlined by the formation of branched acropetal chains, a main diagnostic characteristic that clearly differentiates Cladosporium from different genera reminiscent of Aspergillus.
Sporobolomyces reniformis (pressure HD)
Classification: Fungi > Dikarya > Basidiomycota > Pucciniomycotina > Microbotryomycetes > Sporidiobolales > Sporidiobolaceae > Sporobolomyces > Sporobolomyces reniformis, described by Li et al.14. Mycobank # 828,836. Genbank Accession Quantity: PV803149.1 (HD).
Colonies of Pressure HD on PDA medium exhibit a mucoid, clean, and glossy texture, famous for his or her intense coral-red or salmon coloration (Fig. 4A and B). This pigmentation serves as visible proof of huge carotenoid synthesis, offering a important photoprotective mechanism in opposition to excessive ultraviolet radiation within the Altiplano. Macroscopic progress reveals good exhaustion streaking, highlighting the pressure’s resilience in opposition to oxidative stress.
Morphological characterization of Sporobolomyces reniformis (Pressure HD). (A) Obverse: Mucoid, clean, and glossy colonies on PDA with an intense coral-red or salmon colour ensuing from huge carotenoid synthesis. (B) Reverse: Macroscopic progress illustrating the pressure’s resilience. (C) Mobile morphology: Unicellular yeast cells with ellipsoidal to ovoid form (3.5–7.0 × 2.5–4.5 μm). No staining. (D) Ballistoconidia: Kidney-shaped (reniform) constructions (6.0–9.5 × 4.0–5.5 μm) that retain their intrinsic pinkish tone, highlighted by Giemsa staining.
Microscopic examination reveals unicellular yeast cells with an ellipsoidal to ovoid morphology (3.5–7.0 × 2.5–4.5 μm) that show polar budding and a excessive proliferation charge related for its biotechnological potential. Giemsa staining successfully highlights the cell boundaries with a attribute pink/purple hue (Fig. 4D). A key diagnostic characteristic is the manufacturing of kidney-shaped (reniform) ballistoconidia measuring 6.0–9.5 × 4.0–5.5 μm. These constructions retain their pure pinkish/salmon tone with out staining (Fig. 4C), additional validating the intrinsic presence of protecting pigments.
Alternaria botrytis (pressure HE)
Classification: Fungi > Dikarya > Ascomycota > Pezizomycotina > Dothideomycetes > Pleosporomycetidae > Pleosporales > Pleosporaceae > Alternaria > Alternaria botrytis, described by Woudenberg et al.15. Mycobank # 803718. Genbank Accession Quantity: PV803150.
Colonies of Pressure HE on PDA medium reveal fast progress, that includes a darkish olive-green to blackish velvety texture on the obverse. Superior lighting within the photographic documentation permits for a high-definition view of the common colony margins (Fig. 5A). The reverse is totally black, offering visible proof of excessive melanin manufacturing (Fig. 5B). This darkish pigmentation serves as a organic sunscreen for the holobiont, which may very well be important for its resilience in opposition to oxidative stress and excessive ultraviolet radiation within the Altiplano.
Morphological characterization of Alternaria botrytis (Pressure HE). (A) Obverse: Quick-growing colony with a darkish olive-green to blackish velvety texture. (B) Reverse: Fully black pigmentation indicating excessive melanin manufacturing. (C) Diagnostic characteristic: Very massive muriform conidia (22.0–48.0 × 10.0–18.0 μm) that includes each transverse and longitudinal septa. (D) Spore association: Ovoid to obclavate spores organized briefly acropetal chains in relation to the hyphae.
Microscopically, the first diagnostic characteristic is the presence of very massive muriform conidia (22.0–48.0 × 10.0–18.0 μm) with each transverse and longitudinal septa. The spores exhibit an ovoid to obclavate form and are organized briefly acropetal chains in relation to the hyphae. The pure darkish pigmentation, as proven in Fig. 5C and D, of the conidia, the place no staining was used, serves as direct proof of melanin, reinforcing the species’ photoprotective capability in excessive high-altitude environments.
Gibellulopsis nigrescens (pressure C-HC)
Classification: Fungi > Dikarya > Ascomycota > Pezizomycotina > Sordariomycetes > Hypocreomycetidae > Trichosphaeriales > Trichosphaeriaceae > Gibellulopsis > Gibellulopsis nigrescens, described by Zare et al.16. Mycobank # 510,695. Genbank Accession Quantity: PV803144.1 (C-HC).
Colonies of Pressure C-HC on PDA medium exhibit a woolly to floccose texture with a well-defined radial progress sample in direction of the margins. The obverse shows dense zonation with pure white margins indicating energetic younger mycelium, whereas the middle transitions to grayish-black tones. This central darkening serves because the visible signal of the progressive formation of microsclerotia, that are pigmented resistance constructions (Fig. 6A and B) The colony reverse reveals a yellowish-cream pigmentation that includes a outstanding sample of radial folds or furrows originating from the inoculation middle, a trait frequent in fungi with dense and compact progress on the agar base.
Morphological characterization of Gibellulopsis nigrescens (Pressure C-HC). (A) Obverse: Woolly to floccose texture with dense zonation and a middle transitioning to grayish-black tones resulting from microsclerotia formation. (B) Reverse: Distinguished sample of radial folds or furrows with yellowish-cream tones. (C) Microsclerotia: Dense, darkish plenty of pigmented globose to irregular hyphae (30.0–65.0 μm) visualized with Lactophenol Blue staining. (D) Microconidia: Small oval spores (3.0–5.5 × 2.0–3.5 μm) dispersed all through the microscopic discipline, utilizing Giemsa staining.
Microscopically, probably the most distinctive characteristic of this pressure is the presence of globose to irregular microsclerotia measuring 30.0 to 65.0 μm. Utilizing Giemsa staining, these resistance constructions are visualized as dense, darkish plenty of pigmented hyphae, illustrating the excessive resilience of the fungus (Fig. 6D). Moreover, small oval microconidia (3.0–5.5 × 2.0–3.5 μm) are noticed dispersed all through the sphere, finishing the morphological description of the genus (Fig. 6C).
Monascus sp. (pressure H-I)
Classification: Fungi > Dikarya > Ascomycota > Pezizomycotina > Eurotiomycetes> Eurotiomycetidae > Eurotiales > Aspergillaceae > Monascus, described by Van Tieghem17. Mycobank # 3247. Genbank Accession Quantity: PV803151.1 (H-I).
Colonies of Pressure H-I on PDA medium exhibit distinctive rhythmic and concentric progress, characterised by intense crimson or reddish pigmentation on the obverse. This high-resolution documentation captures the regularity of the mycelial growth (Fig. 7A). The reverse of the colony exhibits a deep purple-red colour, demonstrating the extracellular diffusion of pigments into the agar (Fig. 7B). This secretory capability offers the idea for evaluating these pigments to be used in histological visualization strategies.
Morphological characterization of Monascus sp. (Pressure H-I). (A) Obverse: Distinctive rhythmic and concentric progress with intense crimson or reddish pigmentation on PDA. (B) Reverse: Deep purple-red colour demonstrating extracellular diffusion of pigments into the agar. (C) Sexual section: Attribute globose cleistothecia with clear partitions and related hyphae, measuring 30.0–55.0 μm in diameter. (D) Asexual section: Terminal or lateral aleurioconidia (globose spores) organized on septate hyphae.
Microscopically, the pressure demonstrates vital taxonomic rigor by exhibiting each sexual and asexual phases. The sexual section is recognized by the presence of globose cleistothecia with clear partitions and related hyphae, that includes a attribute diameter of 30.0–55.0 μm (Fig. 7C). The asexual section is characterised by terminal or lateral aleurioconidia (globose spores) organized on septate hyphae. Moreover, using Giemsa staining highlights the density of spores and creating cleistothecia by means of darkish distinction in opposition to a light-weight background (Fig. 7D).
