Novel endolithic bacteria of phylum Chloroflexota reveal a myriad of potential survival strategies in the Antarctic desert.

The ice-free McMurdo Dry Valleys of Antarctica are dominated by nutrient-poor mineral soil and rocky outcrops. The principal habitat for microorganisms is within rocks (endolithic). In this environment, microorganisms are provided with protection against sub-zero temperatures, rapid thermal fluctuations, extreme dryness, and ultraviolet and solar radiation. Endolithic communities include lichen, algae, fungi, and a diverse array of bacteria. Chloroflexota is among the most abundant bacterial phyla present in these communities. Among the Chloroflexota are four novel classes of bacteria, here named Candidatus Spiritibacteria class. nov. (=UBA5177), Candidatus Martimicrobia class. nov. (=UBA4733), Candidatus Tarhunnaeia class. nov. (=UBA6077), and Candidatus Uliximicrobia class. nov. (=UBA2235). We retrieved 17 high-quality metagenome-assembled genomes (MAGs) that represent these four classes. Based on genome predictions, all these bacteria are inferred to be aerobic heterotrophs that encode enzymes for the catabolism of diverse sugars. These and other organic substrates are likely derived from lichen, algae, and fungi, as metabolites (including photosynthate), cell wall components, and extracellular matrix components. The majority of MAGs encode the capacity for trace gas oxidation using high-affinity uptake hydrogenases, which could provide energy and metabolic water required for survival and persistence. Furthermore, some MAGs encode the capacity to couple the energy generated from H2 and CO oxidation to support carbon fixation (atmospheric chemosynthesis). All encode mechanisms for the detoxification and efflux of heavy metals. Certain MAGs encode features that indicate possible interactions with other organisms, such as Tc-type toxin complexes, hemolysins, and macroglobulins.IMPORTANCEThe ice-free McMurdo Dry Valleys of Antarctica are the coldest and most hyperarid desert on Earth. It is, therefore, the closest analog to the surface of the planet Mars. Bacteria and other microorganisms survive by inhabiting airspaces within rocks (endolithic). We identify four novel classes of phylum Chloroflexota, and, based on interrogation of 17 metagenome-assembled genomes, we predict specific metabolic and physiological adaptations that facilitate the survival of these bacteria in this harsh environment-including oxidation of trace gases and the utilization of nutrients (including sugars) derived from lichen, algae, and fungi. We propose that such adaptations allow these endolithic bacteria to eke out an existence in this cold and extremely dry habitat.

Geography and environmental pressure are predictive of class-specific radioresistance in black fungi.

Black fungi are among the most resistant organisms to ionizing radiation on Earth. However, our current knowledge is based on studies on a few isolates, while the overall radioresistance limits across this microbial group and the relationship with local environmental conditions remain largely undetermined. To address this knowledge gap, we assessed the survival of 101 strains of black fungi isolated across a worldwide spatial distribution to gamma radiation doses up to 100 kGy. We found that intra and inter-specific taxonomy, UV radiation, and precipitation levels primarily influence the radioresistance in black fungi. Altogether, this study provides insights into the adaptive mechanisms of black fungi to extreme environments and highlights the role of local adaptation in shaping the survival capabilities of these extreme-tolerant organisms.

The poly-extreme tolerant black yeasts are prevalent under high ultraviolet light and climatic seasonality across soils of global biomes.

Black yeasts are among the most stress-tolerant organisms of the planet, thriving under all types of terrestrial habitats and extreme environments. Yet, their global patterns and ecology remain far less studied, limiting our capacity to identify the main environmental drivers of these important organisms across biomes. To fill this knowledge gap, we analysed topsoils from 235 terrestrial ecosystems across and within globally distributed climate groups (i.e. dry, temperate and continental). We found that soils are important repositories of black yeasts, and that ultraviolet light, fine soil texture, and precipitation seasonality are the most consistent environmental factors associated with their diversity across biomes. Finally, we identified Exophiala and Cladophialophora as the most dominant black yeasts genera in soils across the globe. These findings provide novel evidence of global distribution of black yeasts and their key environmental predictors, giving new insights for speculating the evolution and spreading of these extreme-tolerant organisms throughout both natural and human associated extreme environments.

Endolithic Bacterial Diversity in Lichen-Dominated Communities Is Shaped by Sun Exposure in McMurdo Dry Valleys, Antarctica.

The diversity and composition of endolithic bacterial diversity of several locations in McMurdo Dry Valleys (Continental Antarctica) were explored using amplicon sequencing, targeting the V3 and V4 of the 16S region. Despite the increasing interest in edaphic factors that drive bacterial community composition in Antarctic rocky communities, few researchers focused attention on the direct effects of sun exposure on bacterial diversity; we herein reported significant differences in the northern and southern communities. The analysis of ß-diversity showed significant differences among sampled localities. For instance, the most abundant genera found in the north-exposed rocks were Rhodococcus and Blastococcus in Knobhead Mt.; Ktedonobacter and Cyanobacteria Family I Group I in Finger Mt.; Rhodococcus and Endobacter in University Valley; and Segetibacter and Tetrasphaera in Siegfried Peak samples. In south-exposed rocks, instead, the most abundant genera were Escherichia/Shigella and Streptococcus in Knobhead Mt.; Ktedonobacter and Rhodococcus in Finger Mt.; Ktedonobacter and Roseomonas in University Valley; and Blastocatella, Cyanobacteria Family I Group I and Segetibacter in Siegfried Peak. Significant biomarkers, detected by the Linear discriminant analysis Effect Size, were also found among north- and south-exposed communities. Besides, the large number of positive significant co-occurrences may suggest a crucial role of positive associations over competitions under the harsher conditions where these rock-inhabiting microorganisms spread. Although the effect of geographic distances in these extreme environments play a significant role in shaping biodiversity, the study of an edaphic factor, such as solar exposure, adds an important contribution to the mosaic of microbial biodiversity of Antarctic bacterial cryptoendolithic communities.

Endolithic microbial composition in Helliwell Hills, a newly investigated Mars-like area in Antarctica.

The diversity and composition of Antarctic cryptoendolithic microbial communities in the Mars-analogue site of Helliwell Hills (Northern Victoria Land, Continental Antarctica) are investigated, for the first time, applying both culture-dependent and high-throughput sequencing approaches. The study includes all the domains of the tree of life: Eukaryotes, Bacteria and Archaea to give a complete overview of biodiversity and community structure. Furthermore, to explore the geographic distribution of endoliths throughout the Victoria Land (Continental Antarctica), we compared the fungal and bacterial community composition and structure of endolithically colonized rocks, collected in >30 sites in 10 years of Italian Antarctic Expeditions. Compared with the fungi and other eukaryotes, the prokaryotic communities were richer in species, more diverse and highly heterogeneous. Despite the diverse community compositions, shared populations were found and were dominant in all sites. Local diversification was observed and included prokaryotes as members of Alphaproteobacteria and Crenarchaeota (Archaea), the last detected for the first time in these cryptoendolithic communities. Few eukaryotes, namely lichen-forming fungal species as Lecidella grenii, were detected in Helliwell Hills only. These findings suggest that geographic distance and isolation in these remote areas may promote the establishment of peculiar locally diversified microorganisms.

Astrobiology of life on Earth.

Astrobiology is mistakenly regarded by some as a field confined to studies of life beyond Earth. Here, we consider life on Earth through an astrobiological lens. Whereas classical studies of microbiology historically focused on various anthropocentric sub-fields (such as fermented foods or commensals and pathogens of crop plants, livestock and humans), addressing key biological questions via astrobiological approaches can further our understanding of all life on Earth. We highlight potential implications of this approach through the articles in this Environmental Microbiology special issue 'Ecophysiology of Extremophiles'. They report on the microbiology of places/processes including low-temperature environments and chemically diverse saline- and hypersaline habitats; aspects of sulphur metabolism in hypersaline lakes, dysoxic marine waters, and thermal acidic springs; biology of extremophile viruses; the survival of terrestrial extremophiles on the surface of Mars; biological soils crusts and rock-associated microbes of deserts; subsurface and deep biosphere, including a salticle formed within Triassic halite; and interactions of microbes with igneous and sedimentary rocks. These studies, some of which we highlight here, contribute to our understanding of the spatiotemporal reach of Earth'sfunctional biosphere, and the tenacity of terrestrial life. Their findings will help set the stage for future work focused on the constraints for life, and how organisms adapt and evolve to circumvent these constraints.

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.

Melanin is effective in protecting fast and slow growing fungi from various types of ionizing radiation.

Melanin is a ubiquitous pigment with unique physicochemical properties. The resistance of melanized fungi to cosmic and terrestrial ionizing radiation suggests that melanin also plays a pivotal role in radioprotection. In this study, we compared the effects of densely-ionizing deuterons and sparsely-ionizing X-rays on two microscopic fungi capable of melanogenesis. We utilized the fast-growing pathogenic basiodiomycete forming an induced DOPA-melanin, Cryptococcus neoformans (CN); and the slow-growing environmental rock-inhabiting ascomycete synthesizing a constitutive DHN-melanin, Cryomyces antarcticus (CA); melanized and non-melanized counterparts were compared. CA was more resistant to deuterons than CN, and similar resistance was observed for X-rays. Melanin afforded protection against high-dose (1.5 kGy) deuterons for both CN and CA (p-values < 10-4 ). For X-rays (0.3 kGy), melanin protected CA (p-values < 10-4 ) and probably CN. Deuterons increased XTT activity in melanized strains of both species, while the activity in non-melanized cells remained stable or decreased. For ATP levels the reverse occurred: it decreased in melanized strains, but not in non-melanized ones, after deuteron exposure. For both XTT and ATP, which reflect the metabolic activity of the cells, larger and more statistically-significant differences as a function of melanization status occurred in CN. Our data show, for the first time, that melanin protected both fast-growing and slow-growing fungi from high doses of deuterons under physiological conditions. These observations may give clues for creating melanin-based radioprotectors.

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.

Bradymyces gen. nov. (Chaetothyriales, Trichomeriaceae), a new ascomycete genus accommodating poorly differentiated melanized fungi.

Three slow growing, melanized and morphologically poorly differentiated fungal strains were isolated from a hyperaemic focus near the enlarged spleen of a farmed rainbow trout (Oncorhynchus mykiss) and from a rock collected at 3,200 m a. s. l. (Alps, Italy). Two phylogenetic analyses of the combined nuc18S and nuc28S rDNA and ITS rDNA and ß-tubulin sequences showed that these isolates belong to the Trichomeriaceae, a family of the ascomycete order Chaetothyriales containing black yeasts that cause infections in humans and animals. The strains form a well-supported monophyletic clade. The new genus Bradymyces, with two new species, Bradymyces oncorhynchi and Bradymyces alpinus, is proposed based on phylogenetic, ecophysiological and morphological data. It is characterized by the presence of moniliform hyphae, blastic proliferation, endoconidia, multicellular and muriform bodies, and bodies with dark fragmented incrustations on the surface. Bradymyces most closely resembles members of Knufia. The ex-type isolate of B. oncorhynchi CCF 4369(T) ( = CBS 133066(T) = CCFEE 6134(T)) represents the first case of a Trichomeriaceae member isolated from cold-blooded water vertebrates. B. alpinus [ex-type strain CCFEE 5493(T) ( = CBS 138368(T) = CCF 4803(T))] is represented by two isolates from a single locality in the Alps and in contrast to B. oncorhynchi shows overall slower growth parameters and does not grow at 25 °C.

Dryland microbiomes reveal community adaptations to desertification and climate change.

Drylands account for 45% of the Earth's land area, supporting ~40% of the global population. These regions support some of the most extreme environments on Earth, characterized by extreme temperatures, low and variable rainfall, and low soil fertility. In these biomes, microorganisms provide vital ecosystem services and have evolved distinctive adaptation strategies to endure and flourish in the extreme. However, dryland microbiomes and the ecosystem services they provide are under threat due to intensifying desertification and climate change. In this review, we provide a synthesis of our current understanding of microbial life in drylands, emphasizing the remarkable diversity and adaptations of these communities. We then discuss anthropogenic threats, including the influence of climate change on dryland microbiomes and outline current knowledge gaps. Finally, we propose research priorities to address those gaps and safeguard the sustainability of these fragile biomes.

Metagenomics untangles potential adaptations of Antarctic endolithic bacteria at the fringe of habitability.

Survival and growth strategies of Antarctic endolithic microbes residing in Earth's driest and coldest desert remain virtually unknown. From 109 endolithic microbiomes, 4539 metagenome-assembled genomes were generated, 49.3 % of which were novel candidate bacterial species. We present evidence that trace gas oxidation and atmospheric chemosynthesis may be the prevalent strategies supporting metabolic activity and persistence of these ecosystems at the fringe of life and the limits of habitability.

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.

Positive fungal interactions are key drivers in Antarctic endolithic microcosms at the boundaries for life sustainability.

In the ice-free areas of Victoria Land in continental Antarctica, where the conditions reach the limits for life sustainability, highly adapted and extreme-tolerant microbial communities exploit the last habitable niches inside porous rocks (i.e. cryptoendolithic communities). These guilds host the main standing biomass and principal, if not sole, contributors to environmental/biogeochemical cycles, driving ecosystem processes and functionality in these otherwise dead lands. Although knowledge advances on their composition, ecology, genomic and metabolic features, a large-scale perspective of occurring interactions and interconnections within and between endolithic fungal assemblages is still lacking to date. Unravelling the tight relational network among functional guilds in the Antarctic cryptoendolithic communities may represent a main task. Aiming to fill this knowledge gap, we performed a correlation-network analysis based on amplicon-sequencing data of 74 endolithic microbiomes collected throughout Victoria Land. Endolithic communities' compositional pattern was largely dominated by Lichenized fungi group (83.5%), mainly represented by Lecanorales and Lecideales, followed by Saprotrophs (14.2%) and RIF+BY (2.4%) guilds led by Tremellales and Capnodiales respectively. Our findings highlighted that fungal functional guilds' relational spectrum was dominated by cooperative interactions led by lichenised and black fungi, deeply engaged in community trophic sustain and protection, respectively. On the other hand, a few negative correlations found may help in preserving niche boundaries between microbes living in such strict spatial association.

Rock Traits Drive Complex Microbial Communities at the Edge of Life.

Antarctic deserts are among the driest and coldest ecosystems of the planet; there, some microbes survive under these extreme conditions inside porous rocks, forming the so-called endolithic communities. Yet the contribution of distinct rock traits to support complex microbial assemblies remains poorly determined. Here, we combined an extensive Antarctic rock survey with rock microbiome sequencing and ecological networks and found that contrasting combinations of microclimatic and rock traits such as thermal inertia, porosity, iron concentration, and quartz cement can help explain the multiple complex microbial assemblies found in Antarctic rocks. Our work highlights the pivotal role of rocky substrate heterogeneity in sustaining contrasting groups of microorganisms, which is essential to understand life at the edge on Earth and for the search for life on other rocky planets such as Mars.

Seasonality Is the Main Determinant of Microbial Diversity Associated to Snow/Ice around Concordia Station on the Antarctic Polar Plateau.

The French-Italian Concordia Research Station, situated on the Antarctic Polar Plateau at an elevation of 3233 m above sea level, offers a unique opportunity to study the presence and variation of microbes introduced by abiotic or biotic vectors and, consequently, appraise the amplitude of human impact in such a pristine environment. This research built upon a previous work, which explored microbial diversity in the surface snow surrounding the Concordia Research Station. While that study successfully characterized the bacterial assemblage, detecting fungal diversity was hampered by the low DNA content. To address this knowledge gap, in the present study, we optimized the sampling by increasing ice/snow collected to leverage the final DNA yield. The V4 variable region of the 16S rDNA and Internal Transcribed Spacer (ITS1) rDNA was used to evaluate bacterial and fungal diversity. From the sequencing, we obtained 3,352,661 and 4,433,595 reads clustered in 930 and 3182 amplicon sequence variants (ASVs) for fungi and bacteria, respectively. Amplicon sequencing revealed a predominance of Basidiomycota (49%) and Ascomycota (42%) in the fungal component; Bacteroidota (65.8%) is the main representative among the bacterial phyla. Basidiomycetes are almost exclusively represented by yeast-like fungi. Our findings provide the first comprehensive overview of both fungal and bacterial diversity in the Antarctic Polar Plateau's surface snow/ice near Concordia Station and to identify seasonality as the main driver of microbial diversity; we also detected the most sensitive microorganisms to these factors, which could serve as indicators of human impact in this pristine environment and aid in planetary protection for future exploration missions.