Wide divergence of fungal communities inhabiting rocks and soils in a hyper-arid Antarctic desert.

Highly simplified microbial communities colonise rocks and soils of continental Antarctica ice-free deserts. These two habitats impose different selection pressures on organisms, yet the possible filtering effects on the diversity and composition of microbial communities have not hitherto been fully characterised. We hence compared fungal communities in rocks and soils in three localities of inner Victoria Land. We found low fungal diversity in both substrates, with a mean species richness of 28 across all samples, and significantly lower diversity in rocks than in soils. Rock and soil communities were strongly differentiated, with a multinomial species classification method identifying just three out of 328 taxa as generalists with no affinity for either substrate. Rocks were characterised by a higher abundance of lichen-forming fungi (typically Buellia, Carbonea, Pleopsidium, Lecanora, and Lecidea), possibly owing to the more protected environment and the porosity of rocks permitting photosynthetic activity. In contrast, soils were dominated by obligate yeasts (typically Naganishia and Meyerozyma), the abundances of which were correlated with edaphic factors, and the black yeast Cryomyces. Our study suggests that strong differences in selection pressures may account for the wide divergences of fungal communities in rocks and soils of inner Victoria Land.

Antarctic Soil Metabolomics: A Pilot Study.

In Antarctica, ice-free areas can be found along the coast, on mountain peaks, and in the McMurdo Dry Valleys, where microorganisms well-adapted to harsh conditions can survive and reproduce. Metabolic analyses can shed light on the survival mechanisms of Antarctic soil communities from both coastal sites, under different plant coverage stages, and inner sites where slow-growing or dormant microorganisms, low water availability, salt accumulation, and a limited number of primary producers make metabolomic profiling difficult. Here, we report, for the first time, an efficient protocol for the extraction and the metabolic profiling of Antarctic soils based on the combination of NMR spectroscopy and mass spectrometry (MS). This approach was set up on samples harvested along different localities of Victoria Land, in continental Antarctica, devoid of or covered by differently developed biological crusts. NMR allowed for the identification of thirty metabolites (mainly sugars, amino acids, and organic acids) and the quantification of just over twenty of them. UPLC-MS analysis identified more than twenty other metabolites, in particular flavonoids, medium- and long-chain fatty acids, benzoic acid derivatives, anthracenes, and quinones. Our results highlighted the complementarity of the two analytical techniques. Moreover, we demonstrated that their combined use represents the "gold standard" for the qualitative and quantitative analysis of little-explored samples, such as those collected from Antarctic soils.

Fungal communities in European alpine soils are not affected by short-term in situ simulated warming than bacterial communities.

The impact of global warming on biological communities colonizing European alpine ecosystems was recently studied. Hexagonal open top chambers (OTCs) were used for simulating a short-term in situ warming (estimated around 1°C) in some alpine soils to predict the impact of ongoing climate change on resident microbial communities. Total microbial DNA was extracted from soils collected either inside or outside the OTCs over 3 years of study. Bacterial and fungal rRNA copies were quantified by qPCR. Metabarcoding sequencing of taxonomy target genes was performed (Illumina MiSeq) and processed by bioinformatic tools. Alpha- and beta-diversity were used to evaluate the structure of bacterial and fungal communities. qPCR suggests that, although fluctuations have been observed between soils collected either inside and outside the OTCs, the simulated warming induced a significant (p < 0.05) shift only for bacterial abundance. Likewise, significant (p < 0.05) changes in bacterial community structure were detected in soils collected inside the OTCs, with a clear increase of oligotrophic taxa. On the contrary, fungal diversity of soils collected either inside and outside the OTCs did not exhibit significant (p < 0.05) differences, suggesting that the temperature increase in OTCs compared to ambient conditions was not sufficient to change fungal communities.

Knufia obscura sp. nov. and Knufia victoriae sp. nov., two new species from extreme environments.

Six strains of black meristematic fungi were isolated from Antarctic soils, gasoline car tanks and from the marine alga Flabellia petiolata. These fungi were characterized by morphological, physiological and phylogenetic analyses. According to the maximum-likelihood analysis reconstructed with ITS and LSU sequences, these strains belonged to the genus Knufia. Knufia obscura sp. nov. (holotype CBS 148926) and Knufia victoriae sp. nov. (holotype CBS 149015) are proposed as two novel species and descriptions of their morphological, physiological and phylogenetic features are presented. Based on the maximum-likelihood analyses, K. obscura was closely related to Knufia hypolithi (99 % bootstrap support), while K. victoriae clustered in the clade of Knufia cryptophialidica and Knufia perfecta (93 % bootstrap support). Knufia victoriae, recorded in Antarctic soil samples, had a psychrophilic behaviour, with optimal growth between 10 and 15 °C and no growth recorded at 20 °C. Knufia obscura, from a gasoline car tank and algae, displayed optimal growth between 20 and 25 °C and was more tolerant to salinity than K. victoriae.

Intra- and inter-cores fungal diversity suggests interconnection of different habitats in an Antarctic frozen lake (Boulder Clay, Northern Victoria Land).

A perennially frozen lake at Boulder Clay site (Victoria Land, Antarctica), characterized by the presence of frost mounds, have been selected as an in situ model for ecological studies. Different samples of permafrost, glacier ice and brines have been studied as a unique habitat system. An additional sample of brines (collected in another frozen lake close to the previous one) was also considered. Alpha- and beta-diversity of fungal communities showed both intra- and inter-cores significant (p < 0.05) differences, which suggest the presence of interconnection among the habitats. Therefore, the layers of frost mound and the deep glacier could be interconnected while the brines could probably be considered as an open habitat system not interconnected with each other. Moreover, the absence of similarity between the lake ice and the underlying permafrost suggested that the lake is perennially frozen based. The predominance of positive significant (p < 0.05) co-occurrences among some fungal taxa allowed to postulate the existence of an ecological equilibrium in the habitats systems. The positive significant (p < 0.05) correlation between salt concentration, total organic carbon and pH, and some fungal taxa suggests that a few abiotic parameters could drive fungal diversity inside these ecological niches.

Mrakia stelviica sp. nov. and Mrakia montana sp. nov., two novel basidiomycetous yeast species isolated from cold environments.

Five yeast strains were isolated from soil and sediments collected from Alps and Apennines glaciers during sampling campaigns carried out in summer 2007 and 2017, respectively. Based on morphological and physiological tests and on phylogenetic analyses reconstructed with ITS and D1/D2 sequences, the five strains were considered to belong to two related but hitherto unknown species within the genus Mrakia, in an intermediate position between Mrakia cryoconiti and Mrakia arctica. The names Mrakia stelviica (holotype DBVPG 10734T) and Mrakia montana (holotype DBVPG 10736T) are proposed for the two novel species and a detailed description of their morphological, physiological and phylogenetic features are presented. Both species fermented glucose, sucrose and trehalose, which is an uncommon feature in basidiomycetous yeasts, and showed septate hyphae with teliospore formation.

Effect of environmental parameters on biodiversity of the fungal component in lithic Antarctic communities.

A wide sampling of rocks, colonized by microbial epi-endolithic communities, was performed along an altitudinal gradient from sea level to 3600 m asl and sea distance from the coast to 100 km inland along the Victoria Land Coast, Antarctica. Seventy-two rock samples of different typology, representative of the entire survey, were selected and studied using denaturing gradient gel electrophoresis to compare variation in fungal diversity according to environmental conditions along this altitudinal and sea distance transect. Lichenized fungi were largely predominant in all the samples studied and the biodiversity was heavily influenced even by minimal local variations. The n-MDS analysis showed that altitude and sea distance affect fungal biodiversity, while sandstone allows the communities to maintain high biodiversity indices. The Pareto-Lorenz curves indicate that all the communities analyzed are highly adapted to extreme conditions but scarcely resilient, so any external perturbation may have irreversible effects on these fragile ecosystems.

BIOMEX Experiment: Ultrastructural Alterations, Molecular Damage and Survival of the Fungus Cryomyces antarcticus after the Experiment Verification Tests.

The search for traces of extinct or extant life in extraterrestrial environments is one of the main goals for astrobiologists; due to their ability to withstand stress producing conditions, extremophiles are perfect candidates for astrobiological studies. The BIOMEX project aims to test the ability of biomolecules and cell components to preserve their stability under space and Mars-like conditions, while at the same time investigating the survival capability of microorganisms. The experiment has been launched into space and is being exposed on the EXPOSE-R2 payload, outside of the International Space Station (ISS) over a time-span of 1.5 years. Along with a number of other extremophilic microorganisms, the Antarctic cryptoendolithic black fungus Cryomyces antarcticus CCFEE 515 has been included in the experiment. Before launch, dried colonies grown on Lunar and Martian regolith analogues were exposed to vacuum, irradiation and temperature cycles in ground based experiments (EVT1 and EVT2). Cultural and molecular tests revealed that the fungus survived on rock analogues under space and simulated Martian conditions, showing only slight ultra-structural and molecular damage.

Rock black fungi: excellence in the extremes, from the Antarctic to space.

This work focuses on rock-inhabiting fungi (RIF) of Antarctic rocky deserts, considered the closest to a possible Martian habitat, as the best example of adaptation to the extremes. The study of RIF ecophysiology, resistance and adaptation provides tools that shed light on the evolution of extremophily. These studies also help define the actual limits for life and provide insight for investigating its existence beyond our planet. The scientific results obtained from over 20 years of research on the biodiversity, phylogeny and evolution toward extremotolerance reviewed here demonstrate how these fascinating organisms can withstand conditions well beyond those in their natural environment. A final focus is given on results and perspectives arising from a recent proteomic approach, and from astrobiological experiments and their significance for future space exploration. These studies demonstrate that Antarctic RIF offer an excellent opportunity to investigate many basic, but also applicative areas of research on extremophily.

Cryptococcus vaughanmartiniae sp. nov. and Cryptococcus onofrii sp. nov.: two new species isolated from worldwide cold environments.

Twenty yeast strains, representing a selection from a wider group of more than 60 isolates were isolated from cold environments worldwide (Antarctica, Iceland, Russia, USA, Italian and French Alps, Apennines). The strains were grouped based on their common morphological and physiological characteristics. A phylogeny based on D1/D2 ribosomal DNA sequences placed them in an intermediate position between Cryptococcus saitoi and Cryptococcus friedmannii; the ITS1 and ITS2 rDNA phylogeny demonstrated that these strains belong to two related but hitherto unknown species within the order Filobasidiales, albidus clade. These two novel species are described with the names Cryptococcus vaughanmartiniae (type strain DBVPG 4736(T)) and Cryptococcus onofrii (type strain DBVPG 5303(T)).

Description of Taphrina antarctica f.a. sp. nov., a new anamorphic ascomycetous yeast species associated with Antarctic endolithic microbial communities and transfer of four Lalaria species in the genus Taphrina.

In the framework of a large-scale rock sampling in Continental Antarctica, a number of yeasts have been isolated. Two strains that are unable to grow above 20 °C and that have low ITS sequence similarities with available data in the public domain were found. The D1/D2 LSU molecular phylogeny placed them in an isolated position in the genus Taphrina, supporting their affiliation to a not yet described species. Because the new species is able to grow in its anamorphic state only, the species Taphrina antarctica f.a. (forma asexualis) sp. nov. has been proposed to accommodate both strains (type strain DBVPG 5268(T), DSM 27485(T) and CBS 13532(T)). Lalaria and Taphrina species are dimorphic ascomycetes, where the anamorphic yeast represents the saprotrophic state and the teleomorph is the parasitic counterpart on plants. This is the first record for this genus in Antarctica; since plants are absent on the continent, we hypothesize that the fungus may have focused on the saprotrophic part of its life cycle to overcome the absence of its natural host and adapt environmental constrains. Following the new International Code of Nomenclature for Algae, Fungi and Plants (Melbourne Code 2011) the reorganization of Taphrina-Lalaria species in the teleomorphic genus Taphrina is proposed. We emend the diagnosis of the genus Taphrina to accommodate asexual saprobic states of these fungi. Taphrina antarctica was registered in MycoBank under MB 808028.

Trends in Antarctic soil fungal research in the context of environmental changes.

Antarctic soils represent one of the most pristine environments on Earth, where highly adapted and often endemic microbial species withstand multiple extremes. Specifically, fungal diversity is extremely low in Antarctic soils and species distribution and diversity are still not fully characterized in the continent. Despite the unique features of this environment and the international interest in its preservation, several factors pose severe threats to the conservation of inhabiting ecosystems. In this light, we aimed to provide an overview of the effects on fungal communities of the main changes endangering the soils of the continent. Among these, the increasing human presence, both for touristic and scientific purposes, has led to increased use of fuels for transport and energy supply, which has been linked to an increase in unintentional environmental contamination. It has been reported that several fungal species have evolved cellular processes in response to these soil contamination episodes, which may be exploited for restoring contaminated areas at low temperatures. Additionally, the effects of climate change are another significant threat to Antarctic ecosystems, with the expected merging of previously isolated ecosystems and their homogenization. A possible reduction of biodiversity due to the disappearance of well-adapted, often endemic species, as well as an increase of biodiversity, due to the spreading of non-native, more competitive species have been suggested. Despite some studies describing the specialization of fungal communities and their correlation with environmental parameters, our comprehension of how soil communities may respond to these changes remains limited. The majority of studies attempting to precisely define the effects of climate change, including in situ and laboratory simulations, have mainly focused on the bacterial components of these soils, and further studies are necessary, including the other biotic components.

Recolonization dynamics of marble monuments after cleaning treatments: A nine-year follow-up study.

The prevention and control of biological patinas on outdoor stone monuments represent a demanding challenge for the conservation of cultural heritage also due to some microorganisms, particularly resistant to treatments, such as black meristematic fungi, an eco-physiological group well known for its tolerance to extreme conditions. Even if several methods and eco-friendly products have been proposed as new alternatives, traditional biocides are still far from being completely replaced. Recolonization is a natural process that occurs sooner or later after cleaning. The time that elapses until its occurrence can vary considerably depending on environmental conditions and the used products; unfortunately, the papers describing the effect of treatments over time are rare. This work aims to shed light on the recolonization process of marble surfaces in the ancient monumental cemetery of Bonaria (Cagliari) after nine years from treatments, evaluating the long-term efficiency of two different cleaning methods, namely dimethyl sulfoxide-based gel (DMSO-based gel) and Biotin T (a didecyldimethylammonium chloride-based product-). In this context, the microflora present before treatments and in the following years was assessed by culture-based methods and identified by molecular techniques, with attention on black meristematic fungi, which were used as reference for the most resistant lithobiontic organisms. Different environmental parameters, such as temperature, exposition, dominant winds, and rainfall, were considered, and infrared thermography, portable light microscopy, and image analysis were used. This research evidenced the influence of water availability and lightning in recolonization processes, the transition from the pioneer fungal community versus more resistant black fungal species after Biotin T treatment, and the long-lasting efficiency of the DMSO-based gel. These findings prove that this low-impact method deserves more attention in the conservation of outdoor marble monuments, emphasizing the importance of long-term studies.

Once upon a Time, There Was a Piece of Wood: Present Knowledge and Future Perspectives in Fungal Deterioration of Wooden Cultural Heritage in Terrestrial Ecosystems and Diagnostic Tools.

Wooden Cultural Heritage (WCH) represents a significant portion of the world's historical and artistic heritage, consisting of immovable and movable artefacts. Despite the expertise developed since ancient times to enhance its durability, wooden artefacts are inevitably prone to degradation. Fungi play a pivotal role in the deterioration of WCH in terrestrial ecosystems, accelerating its decay and leading to alterations in color and strength. Reviewing the literature of the last 25 years, we aimed to provide a comprehensive overview of fungal diversity affecting WCH, the biochemical processes involved in wood decay, and the diagnostic tools available for fungal identification and damage evaluation. Climatic conditions influence the occurrence of fungal species in threatened WCH, characterized by a prevalence of wood-rot fungi (e.g., Serpula lacrymans, Coniophora puteana) in architectural heritage in temperate and continental climates and Ascomycota in indoor and harsh environments. More efforts are needed to address the knowledge fragmentation concerning biodiversity, the biology of the fungi involved, and succession in the degradative process, which is frequently centered solely on the main actors. Multidisciplinary collaboration among engineers, restorers, and life sciences scientists is vital for tackling the challenges posed by climate change with increased awareness. Traditional microbiology and culture collections are fundamental in laying solid foundations for a more comprehensive interpretation of big data.

Microplastics in the diet of Hermetia illucens: Implications for development and midgut bacterial and fungal microbiota.

In a world with a population exceeding 8 billion people and continuing to grow, pollution from food and plastic waste is causing long-term issues in ecosystems. Potential solutions may be found by exploiting insect-based bioconversion. In this context, we investigated the impact of polyvinyl chloride microparticles (PVC-MPs) on the development of Hermetia illucens (black soldier fly; BSF) and its midgut bacterial and fungal microbiota. The impact of PVC-MPs was evaluated feeding BSF larvae with a PVC-MPs-supplemented diet. The larvae exposed to different PVC-MPs concentrations (2.5%, 5%, 10% and 20% w/w) developed into adults with no significant increase in pupal mortality. Faster development and smaller pupae were observed when 20% PVC-MPs was provided. The BSF larvae ingest PVC-MPs, resulting in a reduction in MPs size. Larvae exposed to PVC-MPs did not exhibit differences in gut morphology. Regarding the impact of PVC-MPs on the structure of both bacterial and fungal communities, the overall alpha- and beta-diversity did not exhibit significant changes. However, the presence of PVC-MPs significantly affected the relative abundances of Enterobacteriaceae and Paenibacillaceae among the bacteria and of Dipodascaceae and Plectospharellaceae among the fungi (including yeast and filamentous life forms), suggesting that PVC-MP contamination has a taxa-dependent impact. These results indicate that BSF larvae can tolerate PVC-MPs in their diet, supporting the potential use of these insects in organic waste management, even in the presence of high levels of PVC-MP contamination.

The hidden network of biocrust successional stages in the High Arctic: Revealing abiotic and biotic factors shaping microbial and metazoan communities.

Despite the important role that biocrust communities play in maintaining ecosystem structure and functioning in deglaciated barren soil, few studies have been conducted on the dynamics of biotic communities and the impact of physicochemical characteristics in shaping the different successional stages. In this study an integrated approach encompassing physicochemical parameters and molecular taxonomy was used for identifying the indicator taxa and the presence of intra- and inter-kingdom interactions in five different crust/biocrust successional stages: i) physical crust, ii) cyanobacteria-dominated biocrust, iii) cyanobacteria/moss-dominated biocrust, iv) moss-dominated biocrust and v) bryophyte carpet. The phylum Gemmatimonadota was the bacterial indicator taxon in the early stage, promoting both inter- and intra-kingdom interactions, while Cyanobacteria and Nematoda phyla played a pivotal role in formation and dynamics of cyanobacteria-dominated biocrusts. A multitrophic community, characterized by a shift from oligotrophic to copiotrophic bacteria and the presence of saproxylic arthropod and herbivore insects was found in the cyanobacteria/moss-dominated biocrust, while a more complex biota, characterized by an increased fungal abundance (classes Sordariomycetes, Leotiomycetes, and Dothideomycetes, phylum Ascomycota), associated with highly trophic consumer invertebrates (phyla Arthropoda, Rotifera, Tardigrada), was observed in moss-dominated biocrusts. The class Bdelloidea and the family Hypsibiidae (phyla Rotifera and Tardigrada, respectively) were metazoan indicator taxon in bryophyte carpet, suggesting their potential role in shaping structure and function of this late successional stage. Nitrogen and phosphorus were the main physicochemical limiting factors driving the shift among different crust/biocrust successional stages. Identification and characterization of indicator taxa, biological intra- and inter-kingdom interactions and abiotic factors driving the shift among different crust/biocrust successional stages provide a detailed picture on crust/biocrust dynamics, revealing a strong interconnection among micro- and macrobiota systems. These findings enhance our understanding of biocrust ecosystems in High Arctic, providing valuable insights for their conservation and management in response to environmental shifts due to climate change.

Isolation and screening of black fungi as degraders of volatile aromatic hydrocarbons.

Black fungi reported as degraders of volatile aromatic compounds were isolated from hydrocarbon-polluted sites and indoor environments. Several of the species encountered are known opportunistic pathogens or are closely related to pathogenic species causing severe mycoses, among which are neurological infections in immunocompetent individuals. Given the scale of the problem of environmental pollution and the phylogenetic relation of aromate-degrading black fungi with pathogenic siblings, it is of great interest to select strains able to mineralize these substrates efficiently without any risk for public health. Fifty-six black strains were obtained from human-made environments rich in hydrocarbons (gasoline car tanks, washing machine soap dispensers) after enrichment with some phenolic intermediates of toluene and styrene fungal metabolism. Based on ITS sequencing identification, the majority of the obtained isolates were members of the genus Exophiala. Exophiala xenobiotica was found to be the dominant black yeast present in the car gasoline tanks. A higher biodiversity, with three Exophiala species, was found in soap dispensers of washing machines. Strains obtained were screened using a 2,6-dichlorophenol-indophenol (DCPIP) assay, optimized for black fungi, to assess their potential ability to degrade toluene. Seven out of twenty strains tested were able to use toluene as carbon source.

Effects of 5-year experimental warming in the Alpine belt on soil Archaea: Multi-omics approaches and prospects.

We currently lack a predictive understanding of how soil archaeal communities may respond to climate change, particularly in Alpine areas where warming is far exceeding the global average. Here, we characterized the abundance, structure, and function of total (by metagenomics) and active soil archaea (by metatranscriptomics) after 5-year experimental field warming (+1°C) in Italian Alpine grasslands and snowbeds. Our multi-omics approach unveiled an increasing abundance of Archaea during warming in snowbeds, which was negatively correlated with the abundance of fungi (by qPCR) and micronutrients (Ca and Mg), but positively correlated with soil water content. In the snowbeds transcripts, warming resulted in the enrichment of abundances of transcription and nucleotide biosynthesis. Our study provides novel insights into possible changes in soil Archaea composition and function in the climate change scenario.

Draft genome sequencing of Naganishia species isolated from the polar environments.

The draft genomes of five Naganishia strains were sequenced using MinION and annotated using Funannotate pipeline. Phylogenetic and genomic analyses were performed to provide their genetic relationships, diversity, and potential functional capabilities. This approach will aid in understanding their potential to survive under microgravity and their resilience to extreme environments.

LIFE experiment: isolation of cryptoendolithic organisms from Antarctic colonized sandstone exposed to space and simulated Mars conditions on the international space station.

Desiccated Antarctic rocks colonized by cryptoendolithic communities were exposed on the International Space Station (ISS) to space and simulated Mars conditions (LiFE-Lichens and Fungi Experiment). After 1.5 years in space samples were retrieved, rehydrated and spread on different culture media. Colonies of a green alga and a pink-coloured fungus developed on Malt-Agar medium; they were isolated from a sample exposed to simulated Mars conditions beneath a 0.1 % T Suprasil neutral density filter and from a sample exposed to space vacuum without solar radiation exposure, respectively. None of the other flight samples showed any growth after incubation. The two organisms able to grow were identified at genus level by Small SubUnit (SSU) and Internal Transcribed Spacer (ITS) rDNA sequencing as Stichococcus sp. (green alga) and Acarospora sp. (lichenized fungal genus) respectively. The data in the present study provide experimental information on the possibility of eukaryotic life transfer from one planet to another by means of rocks and of survival in Mars environment.