Linking Zetaproteobacterial diversity and substratum type in iron-rich microbial mats from the Lucky Strike hydrothermal field (EMSO-Azores observatory).

Zetaproteobacteria have been reported in different marine and terrestrial environments all over the globe. They play an essential role in marine iron-rich microbial mats, as one of their autotrophic primary producers, oxidizing Fe(II) and producing Fe-oxyhydroxides with different morphologies. Here, we study and compare the Zetaproteobacterial communities of iron-rich microbial mats from six different sites of the Lucky Strike Hydrothermal Field through the use of the Zetaproteobacterial operational taxonomic unit (ZetaOTU) classification. We report for the first time the Zetaproteobacterial core microbiome of these iron-rich microbial mats, which is composed of four ZetaOTUs that are cosmopolitan and essential for the development of the mats. The study of the presence and abundance of different ZetaOTUs among sites reveals two clusters, which are related to the lithology and permeability of the substratum on which they develop. The Zetaproteobacterial communities of cluster 1 are characteristic of poorly permeable substrata, with little evidence of diffuse venting, while those of cluster 2 develop on hydrothermal slabs or deposits that allow the percolation and outflow of diffuse hydrothermal fluids. In addition, two NewZetaOTUs 1 and 2 were identified, which could be characteristic of anthropic iron and unsedimented basalt, respectively. We also report significant correlations between the abundance of certain ZetaOTUs and that of iron oxide morphologies, indicating that their formation could be taxonomically and/or environmentally driven. We identified a new morphology of Fe(III)-oxyhydroxides that we named "corals." Overall, our work contributes to the knowledge of the biogeography of this bacterial class by providing additional data from the Atlantic Ocean, a lesser-studied ocean in terms of Zetaproteobacterial diversity.IMPORTANCEUp until now, Zetaproteobacterial diversity studies have revealed possible links between Zetaproteobacteria taxa, habitats, and niches. Here, we report for the first time the Zetaproteobacterial core microbiome of iron-rich mats from the Lucky Strike Hydrothermal Field (LSHF), as well as two new Zetaproteobacterial operational taxonomic units (NewZetaOTUs) that could be substratum specific. We highlight that the substratum on which iron-rich microbial mats develop, especially because of its permeability to diffuse hydrothermal venting, has an influence on their Zetaproteobacterial communities. Moreover, our work adds to the knowledge of the biogeography of this bacterial class by providing additional data from the hydrothermal vent sites along the Mid-Atlantic Ridge. In addition to the already described iron oxide morphologies, we identify in our iron-rich mats a new morphology that we named corals. Finally, we argue for significant correlations between the relative abundance of certain ZetaOTUs and that of iron oxide morphologies, contributing to the understanding of the drivers of iron oxide production in iron-oxidizing bacteria.

Gypsum Cave Biofilm Communities are Strongly Influenced by Bat- And Arthropod-Related Fungi.

The Gypsum Karst of Sorbas, Almeria, southeast Spain, includes a few caves whose entrances are open and allow the entry and roosting of numerous bats. Caves are characterized by their diversity of gypsum speleothems, such as stalactites, coralloids, gypsum crusts, etc. Colored biofilms can be observed on the walls of most caves, among which the Covadura and C3 caves were studied. The objective was to determine the influence that bat mycobiomes may have on the fungal communities of biofilms. The results indicate that the fungi retrieved from white and yellow biofilms in Covadura Cave (Ascomycota, Mortierellomycota, Basidiomycota) showed a wide diversity, depending on their location, and were highly influenced by the bat population, the guano and the arthropods that thrive in the guano, while C3 Cave was more strongly influenced by soil- and arthropod-related fungi (Ascomycota, Mortierellomycota), due to the absence of roosting bats.

Assessment of fungal spores and spore-like diversity in environmental samples by targeted lysis.

At particular stages during their life cycles, fungi use multiple strategies to form specialized structures to survive unfavorable environmental conditions. These strategies encompass sporulation, as well as cell-wall melanization, multicellular tissue formation or even dimorphism. The resulting structures are not only used to disperse to other environments, but also to survive long periods of time awaiting favorable growth conditions. As a result, these specialized fungal structures are part of the microbial seed bank, which is known to influence the microbial community composition and contribute to the maintenance of diversity. Despite the importance of the microbial seed bank in the environment, methods to study the diversity of fungal structures with improved resistance only target spores dispersing in the air, omitting the high diversity of these structures in terms of morphology and environmental distribution. In this study, we applied a separation method based on cell lysis to enrich lysis-resistant fungal structures (for instance, spores, sclerotia, melanized yeast) to obtain a proxy of the composition of the fungal seed bank. This approach was first evaluated in-vitro in selected species. The results obtained showed that DNA from fungal spores and from yeast was only obtained after the application of the enrichment method, while mycelium was always lysed. After validation, we compared the diversity of the total and lysis-resistant fractions in the polyextreme environment of the Salar de Huasco, a high-altitude athalassohaline wetland in the Chilean Altiplano. Environmental samples were collected from the salt flat and from microbial mats in small surrounding ponds. Both the lake sediments and microbial mats were dominated by Ascomycota and Basidiomycota, however, the diversity and composition of each environment differed at lower taxonomic ranks. Members of the phylum Chytridiomycota were enriched in the lysis-resistant fraction, while members of the phylum Rozellomycota were never detected in this fraction. Moreover, we show that the community composition of the lysis-resistant fraction reflects the diversity of life cycles and survival strategies developed by fungi in the environment. To the best of our knowledge this is the first time that the fungal diversity is explored in the Salar de Huasco. In addition, the method presented here provides a simple and culture independent approach to assess the diversity of fungal lysis-resistant cells in the environment.

First Insights into the Bacterial Diversity of Mount Etna Volcanic Caves.

While microbial communities in limestone caves across the world are relatively understood, knowledge of the microbial composition in lava tubes is lagging behind. These caves are found in volcanic regions worldwide and are typically lined with multicolored microbial mats on their walls and ceilings. The Mount Etna (Sicily, S-Italy) represents one of the most active volcanos in the world. Due to its outstanding biodiversity and geological features, it was declared Natural Heritage of Humanity by the UNESCO in 2013. Despite the presence of more than 200 basaltic lava tubes, the microbial diversity of these hypogean systems has never been investigated so far. Here, we investigated bacterial communities in four lava tubes of Mount Etna volcano. Field emission scanning electron microscopy (FESEM) was carried out for the morphological characterization and detection of microbial features. We documented an abundant presence of microbial cells with different morphotypes including rod-shaped, filamentous, and coccoidal cells with surface appendages, resembling actinobacteria reported in other lava tubes across the world. Based on 16S rRNA gene analysis, the colored microbial mats collected were mostly composed of bacteria belonging to the phyla Actinomycetota, Pseudomonadota, Acidobacteriota, Chloroflexota, and Cyanobacteria. At the genus level, the analysis revealed a dominance of the genus Crossiella, which is actively involved in biomineralization processes, followed by Pseudomonas, Bacillus, Chujaibacter, and Sphingomonas. The presence of these taxa is associated with the carbon, nitrogen, and ammonia cycles, and some are possibly related to the anthropic disturbance of these caves. This study provides the first insight into the microbial diversity of the Etna volcano lava tubes, and expands on previous research on microbiology of volcanic caves across the world.

Unbiased analyses of ITS folding motifs in a taxonomically confusing lineage: Anagnostidinema visiae sp. nov. (cyanobacteria).

Cyanobacteria are diverse prokaryotic, photosynthetic organisms present in nearly every known ecosystem. Recent investigations around the world have recovered vast amounts of novel biodiversity in seldom sampled habitats. One phylogenetically significant character, the secondary folding structures of the 16S-23S ITS rDNA region, has allowed an unprecedented capacity to erect new species. However, two questions arise: Is this feature as informative as is proposed, and how do we best employ these features? Submerged sinkholes with oxygen-poor, sulfur-rich ground water in Lake Huron (USA) contain microbial mats dominated by both oxygenic and anoxygenic cyanobacteria. We sought to document some of this unique cyanobacterial diversity. Using culture-based investigations, we recovered 45 strains, of which 23 were analyzed employing 16S-23S rDNA sequences, ITS folding patterns, ecology, and morphology. With scant morphological discontinuities and nebulous 16S rDNA gene sequence divergence, ITS folding patterns were effective at articulating cryptic biodiversity. However, we would have missed these features had we not folded all the available motifs from the strains, including those with highly similar 16S rDNA gene sequences. If we had relied solely on morphological or 16S rDNA gene data, then we might well have missed the diversity of Anagnostidinema. Thus, in order to avoid conformation basis, which is potentially common when employing ITS structures, we advocate clustering strains based on ITS rDNA region patterns independently and comparing them back to 16S rDNA gene phylogenies. Using a total evidence approach, we erected a new taxon according to the International Code of Nomenclature for Algae, Fungi, and Plants: Anagnostidinema visiae.

Metabolomic Diversity in Microbial Mats Under Different Environmental Conditions: A Tool to Test Microbial Ecosystem Chemical Change.

Microbial mats are microbial communities capable of recycling the essential elements of life and considered to be the oldest evidence of microbial communities on Earth. Due to their uniqueness and limited sampling material, analyzing their metabolomic profile in different seasons or conditions is challenging. In this study, microbial mats from a small pond in the Cuatro Cienegas Basin in Coahuila, Mexico, were collected in wet and dry seasons. In addition to these samples, mesocosm experiments from the wet samples were set. These mats are elastic and rise after heavy rainfall by forming gas domes structures known as "Archean domes", by the outgassing of methanogenic bacteria, archaea, and sulfur bacteria. Extracts from all mats and mesocosms were subjected to untargeted mass spectrometry-based metabolomics and molecular networking analysis. Interestingly, each mat showed high chemical diversity that may be explained by the temporal dynamic processes in which they were sampled.

A cup half full: the first assessment on the distribution, ecology and need for conservation of the threatened Neptune's cup sponge, Cliona patera, in the Gulf of Thailand.

The Neptune's cup sponge is an iconic species found in marine soft sediment habitats in the Indo-West Pacific, with a history of overharvesting and extreme population declines. Access to SCUBA diving surveys has allowed for its rediscovery at Singapore, its type locality; however, with fewer than ten living individuals documented in the twenty-first century, the species is believed to be in need of conservation. Here, we share the results from surveys across the Gulf of Thailand, allowing for a documentation of 29 additional individuals, with information on their ecology and distribution. Of the 29 individuals, extensive damage or mortality caused by marine debris is recorded for six individuals, and one further individual is believed to be lost due to an unknown cause. Documented threats from the Gulf of Thailand differ from those in Singapore; however, low population sizes and poor connectivity remain a significant concern for the survival of the species.

Lithifying and Non-Lithifying Microbial Ecosystems in the Wetlands and Salt Flats of the Central Andes.

The wetlands and salt flats of the Central Andes region are unique extreme environments as they are located in high-altitude saline deserts, largely influenced by volcanic activity. Environmental factors, such as ultraviolet (UV) radiation, arsenic content, high salinity, low dissolved oxygen content, extreme daily temperature fluctuation, and oligotrophic conditions, resemble the early Earth and potentially extraterrestrial conditions. The discovery of modern microbialites and microbial mats in the Central Andes during the past decade has increased the interest in this area as an early Earth analog. In this work, we review the current state of knowledge of Central Andes region environments found within lakes, small ponds or puquios, and salt flats of Argentina, Chile, and Bolivia, many of them harboring a diverse range of microbial communities that we have termed Andean Microbial Ecosystems (AMEs). We have integrated the data recovered from all the known AMEs and compared their biogeochemistry and microbial diversity to achieve a better understanding of them and, consequently, facilitate their protection.

Meta-omics Provides Insights into the Impact of Hydrocarbon Contamination on Microbial Mat Functioning.

Photosynthetic microbial mats are stable, self-supported communities. Due to their coastal localization, these mats are frequently exposed to hydrocarbon contamination and are able to grow on it. To decipher how this contamination disturbs the functioning of microbial mats, we compared two mats: a contaminated mat exposed to chronic petroleum contamination and a reference mat. The taxonomic and metabolic structures of the mats in spring and fall were determined using metagenomic and metatranscriptomic approaches. Extremely high contamination disturbed the seasonal variations of the mat. ABC transporters, two-component systems, and type IV secretion system-related genes were overabundant in the contaminated mats. Xenobiotic degradation metabolism was minor in the metagenomes of both mats, and only the expression of genes involved in polycyclic aromatic hydrocarbon degradation was higher in the contaminated mat. Interestingly, the expression rates of genes involved in hydrocarbon activation decreased during the 1-year study period, concomitant with the decrease in easily degradable hydrocarbons, suggesting a transient effect of hydrocarbon contamination. Alteromonadales and Oceanospirillales hydrocarbonoclastic bacteria appeared to be key in hydrocarbon remediation in the contaminated mat. Overall, the contaminated microbial mat was able to cope with hydrocarbon contamination and displayed an adaptive functioning that modified seasonal behaviour.

Causes and Biophysical Consequences of Cellulose Production by Pseudomonas fluorescens SBW25 at the Air-Liquid Interface.

Cellulose-overproducing wrinkly spreader mutants of Pseudomonas fluorescens SBW25 have been the focus of much investigation, but conditions promoting the production of cellulose in ancestral strain SBW25 and its effects and consequences have escaped in-depth investigation through lack of an in vitro phenotype. Here, using a custom-built device, we reveal that in static broth microcosms, ancestral SBW25 encounters environmental signals at the air-liquid interface that activate, via three diguanylate cyclase-encoding pathways (Wsp, Aws, and Mws), production of cellulose. Secretion of the polymer at the meniscus leads to modification of the environment and growth of numerous microcolonies that extend from the surface. Accumulation of cellulose and associated microbial growth leads to Rayleigh-Taylor instability resulting in bioconvection and rapid transport of water-soluble products over tens of millimeters. Drawing upon data, we built a mathematical model that recapitulates experimental results and captures the interactions between biological, chemical and physical processes.IMPORTANCE This work reveals a hitherto unrecognized behavior that manifests at the air-liquid interface that depends on production of cellulose and hints at undiscovered dimensions to bacterial life at surfaces. Additionally, the study links activation of known diguanylate cyclase-encoding pathways to cellulose expression and to signals encountered at the meniscus. Further significance stems from recognition of the consequences of fluid instabilities arising from surface production of cellulose for transport of water-soluble products over large distances.

Bacterial and archaeal profiling of hypersaline microbial mats and endoevaporites, under natural conditions and methanogenic microcosm experiments.

Bacterial and archaeal community structure of five microbial communities, developing at different salinities in Baja California Sur, Mexico, were characterized by 16S rRNA sequencing. The response of the microbial community to artificial changes in salinity-sulfate concentrations and to addition of trimethylamine was also evaluated in microcosm experiments. Ordination analyses of the microbial community structure showed that microbial composition was distinctive for each hypersaline site. Members of bacteria were dominated by Bacteroidetes and Proteobacteria phyla, while Halobacteria of the Euryarchaeota phylum was the most represented class of archaea for all the environmental samples. At a higher phylogenetic resolution, methanogenic communities were dominated by members of the Methanosarcinales, Methanobacteriales and Methanococcales orders. Incubation experiments showed that putative hydrogenotrophic methanogens of the Methanomicrobiales increased in abundance only under lowest salinity and sulfate concentrations. Trimethylamine addition effectively increased the abundance of methylotrophic members from the Methanosarcinales, but also increased the relative abundance of the Thermoplasmata class, suggesting the potential capability of these microorganisms to use trimethylamine in hypersaline environments. These results contribute to the knowledge of microbial diversity in hypersaline environments from Baja California Sur, Mexico, and expand upon the available information for uncultured methanogenic archaea in these ecosystems.

Bacterial and Archaeal Diversity in Hypersaline Cyanobacterial Mats Along a Transect in the Intertidal Flats of the Sultanate of Oman.

Hypersaline intertidal zones are highly dynamic ecosystems that are exposed to multiple extreme environmental conditions including rapidly and frequently changing parameters (water, nutrients, temperature) as well as highly elevated salinity levels often caused by high temperatures and evaporation rates. Microbial mats in most extreme settings, as found at the coastline of the subtropical-arid Arabian Peninsula, have been relatively less studied compared to their counterparts around the world. We report, here, for the first time on the diversity of the bacterial and archaeal communities of marine microbial mats along an intertidal transect in a wide salt flat with strongly increased salinity employing Illumina MiSeq technology for amplicon sequencing of 16S rRNA gene fragments. Microbial communities were dominated by typical halotolerant to halophilic microorganisms, with clear shifts in community composition, richness, and diversity along the transect. Highly adapted specialists (e.g., Euhalothece, Salinibacter, Nanohaloarchaeota) were mainly found at the most extreme, upper tidal sites and less specialized organisms with wide tolerance ranges (e.g., Lyngbya, Rhodovibrio, Salisaeta, Halobacteria) in intermediate sites of the transect. The dominating taxa in the lower tidal sites were typical members of well-stabilized mats (e.g., Coleofasciculus, Anaerolineaceae, Thaumarchaeota). Up to 40% of the archaeal sequences per sample represented so far unknown phyla. In conclusion, the bacterial richness and diversity increased from upper towards lower tidal sites in line with increasing mat stabilization and functional diversity, opposed to that of cyanobacteria only and archaea, which showed their highest richness and diversity in upper tidal samples.

Microbial community composition and trophic role along a marked salinity gradient in Laguna Puilar, Salar de Atacama, Chile.

The geological, hydrological and microbiological features of the Salar de Atacama, the most extensive evaporitic sedimentary basin in the Atacama Desert of northern Chile, have been extensively studied. In contrast, relatively little attention has been paid to the composition and roles of microbial communities in hypersaline lakes which are a unique feature in the Salar. In the present study biochemical, chemical and molecular biological tools were used to determine the composition and roles of microbial communities in water, microbial mats and sediments along a marked salinity gradient in Laguna Puilar which is located in the "Los Flamencos" National Reserve. The bacterial communities at the sampling sites were dominated by members of the phyla Bacteroidetes, Chloroflexi, Cyanobacteria and Proteobacteria. Stable isotope and fatty acid analyses revealed marked variability in the composition of microbial mats at different sampling sites both horizontally (at different sites) and vertically (in the different layers). The Laguna Puilar was shown to be a microbially dominated ecosystem in which more than 60% of the fatty acids at particular sites are of bacterial origin. Our pioneering studies also suggest that the energy budgets of avian consumers (three flamingo species) and dominant invertebrates (amphipods and gastropods) use minerals as a source of energy and nutrients. Overall, the results of this study support the view that the Salar de Atacama is a heterogeneous and fragile ecosystem where small changes in environmental conditions may alter the balance of microbial communities with possible consequences at different trophic levels.

Microbial mats and their palaeoenvironmental analysis in offshore - shelf facies of the Los Molles Formation (Toarcian - Lower Callovian) in the Chacay Melehue area, Neuquén Basin, Argentina.

This contribution presents the first study focused on the analysis of microbial mats in the Los Molles Formation (Toarcian - Early Callovian), Neuquén Basin, Argentina. This unit mainly represents offshore-to-shelf environments affected by storms and density currents. The Los Molles Formation is one of the oldest source rocks in the Neuquén Basin and constitutes an unconventional shale gas reservoir of great economic importance. The aim of this work was to identify the microbial activity from the description and interpretation of microbially induced sedimentary structures (MISS), to determine the paleoenvironmental and paleoecological conditions under which they formed, and to establish a possible relationship between these structures and the trace fossil Trichichnus. Samples from the levels with MISS were analyzed and described from macroscopic and binocular observations, petrographic microscope thin sections, and SEM samples with EDS analyses. The results showed several levels of microbial mats presenting diverse MISS, including biolaminations and Kinneyia-like wrinkles structures that were described at the macroscopic level. In thin sections, biolaminations, filament-like microstructures with different degrees of development, oriented grains and pyrite were observed. SEM images and EDS analyses showed different types of filaments, coccoids and EPS with high concentrations of carbon. These results revealed that the studied levels fulfill the established biogenicity criteria, guaranteeing that they have a bacterial origin. The abundance of the trace fossil Trichichnus sp. throughout the section and the proximity to some Kinneyia-like wrinkle structures levels suggests that the same organisms may have generated them. Furthermore, they revealed that the Los Molles Formation, at the time of its deposition, experienced paleoecological and paleoenvironmental conditions appropriate for the establishment and development of microbial mats. The extensive levels of microbial mats in the study area suggest that they may have been a source of organic matter for the generation of hydrocarbons from the Los Molles Formation.

Interpreting an Archaean paleoenvironment through 3D imagery of microbialites.

While stromatolites, and to a lesser extent thrombolites, have been extensively studied in order to unravel Precambrian (>539 Ma) biological evolution, studies of clastic-dominated microbially induced sedimentary structures (MISS) are relatively scarce. The lack of a consolidated record of clastic microbialites creates questions about how much (and what) information on depositional and taphonomic settings can be gleaned from these fossils. We used µCT scanning, a non-destructive X-ray-based 3D imaging method, to reconstruct morphologies of ancient MISS and mat textures in two previously described coastal Archaean samples from the ~3.48 Ga Dresser Formation, Pilbara, Western Australia. The aim of this study was to test the ability of µCT scanning to visualize and make 3D measurements that can be used to interpret the biotic-environmental interactions. Fossil MISS including mat laminae with carpet-like textures in one sample and mat rip-up chips in the second sample were investigated. Compiled δ13C and δ34S analyses of specimens from the Dresser Fm. are consistent with a taxonomically diverse community that could be capable of forming such MISS. 3D measurements of fossil microbial mat chips indicate significant biostabilization and suggest formation in flow velocities >25 cm s-1. Given the stratigraphic location of these chips in a low-flow lagoonal layer, we conclude that these chips formed due to tidal influence, as these assumed velocities are consistent with recent modeling of Archaean tides. The success of µCT scanning in documenting these microbialite features validates this technique both as a first step analysis for rare samples prior to the use of more destructive techniques and as a valuable tool for gaining insight into microbialite taphonomy.

Diversity of Microbial Mats in the Makgadikgadi Salt Pans, Botswana.

The Makgadikgadi Salt Pans are the remnants of a mega paleo-lake system in the central Kalahari, Botswana. Today, the Makgadikgadi Basin is an arid to semi-arid area receiving water of meteoric origin during the short, wet season. Large microbial mats, which support primary production, are formed due to desiccation during the dry season. This study aimed to characterise the microbial diversity of the microbial mats and the underlying sediment. The focus was the Ntwetwe Pan, located west of the Makgadikgadi Basin. Metagenomic analyses demonstrated that the mats consisted of a high relative abundance of Cyanobacteriota (synonym Cyanobacteria) (20.50-41.47%), Pseudomonadota (synonym Proteobacteria) (15.71 to 32.18%), and Actinomycetota (synonym Actinobacteria) (8.53-32.56%). In the underlying sediments, Pseudomonadota, Actinomycetota, and Euryarchaeota represented over 70% of the community. Localised fluctuations in water content and pH did not significantly affect the microbial diversity of the sediment or the mats.

Protist communities of microbial mats from the extreme environments of five saline Andean lagoons at high altitudes in the Atacama Desert.

Introduction: Heterotrophic protists colonizing microbial mats have received little attention over the last few years, despite their importance in microbial food webs. A significant challenge originates from the fact that many protists remain uncultivable and their functions remain poorly understood. Methods: Metabarcoding studies of protists in microbial mats across high-altitude lagoons of different salinities (4.3-34 practical salinity units) were carried out to provide insights into their vertical stratification at the millimeter scale. DNA and cDNA were analyzed for selected stations. Results: Sequence variants classified as the amoeboid rhizarian Rhogostoma and the ciliate Euplotes were found to be common members of the heterotrophic protist communities. They were accompanied by diatoms and kinetoplastids. Correlation analyses point to the salinity of the water column as a main driver influencing the structure of the protist communities at the five studied microbial mats. The active part of the protist communities was detected to be higher at lower salinities (<20 practical salinity units). Discussion: We found a restricted overlap of the protist community between the different microbial mats indicating the uniqueness of these different aquatic habitats. On the other hand, the dominating genotypes present in metabarcoding were similar and could be isolated and sequenced in comparative studies (Rhogostoma, Euplotes, Neobodo). Our results provide a snapshot of the unculturable protist diversity thriving the benthic zone of five athalossohaline lagoons across the Andean plateau.

Arctic cyanobacterial mat community diversity decreases with latitude across the Canadian Arctic.

Cyanobacterial mats are commonly reported as hotspots of microbial diversity across polar environments. These thick, multilayered microbial communities provide a refuge from extreme environmental conditions, with many species able to grow and coexist despite the low allochthonous nutrient inputs. The visibly dominant phototrophic biomass is dependent on internal nutrient recycling by heterotrophic organisms within the mats; however, the specific contribution of heterotrophic protists remains little explored. In this study, mat community diversity was examined along a latitudinal gradient (55-83°N), spanning subarctic taiga, tundra, polar desert, and the High Arctic ice shelves. The prokaryotic and eukaryotic communities were targeted, respectively, by V4 16S ribosomal RNA (rRNA) and V9 18S rRNA gene amplicon high-throughput sequencing. Prokaryotic and eukaryotic richness decreased, in tandem with decreasing temperatures and shorter seasons of light availability, from the subarctic to the High Arctic. Taxonomy-based annotation of the protist community revealed diverse phototrophic, mixotrophic, and heterotrophic genera in all mat communities, with fewer parasitic taxa in High Arctic communities. Co-occurrence network analysis identified greater heterogeneity in eukaryotic than prokaryotic community structure among cyanobacterial mats across the Canadian Arctic. Our findings highlight the sensitivity of microbial eukaryotes to environmental gradients across northern high latitudes.

Investigating Microbial Biosignatures in Aeolian Environments Using Micro-X-Ray: Simulation of PIXL Instrument Analyses at Jezero Crater Onboard the Perseverance Mars 2020 Rover.

Assessing the past habitability of Mars and searching for evidence of ancient life at Jezero crater via the Perseverance rover are the key objectives of NASA's Mars 2020 mission. Onboard the rover, PIXL (Planetary Instrument for X-ray Lithochemistry) is one of the best suited instruments to search for microbial biosignatures due to its ability to characterize chemical composition of fine scale textures in geological targets using a nondestructive technique. PIXL is also the first micro-X-ray fluorescence (XRF) spectrometer onboard a Mars rover. Here, we present guidelines for identifying and investigating a microbial biosignature in an aeolian environment using PIXL-analogous micro-XRF (µXRF) analyses. We collected samples from a modern wet aeolian environment at Padre Island, Texas, that contain buried microbial mats, and we analyzed them using µXRF techniques analogous to how PIXL is being operated on Mars. We show via µXRF technique and microscope images the geochemical and textural variations from the surface to ∼40 cm depth. Microbial mats are associated with heavy-mineral lags and show specific textural and geochemical characteristics that make them a distinct biosignature for this environment. Upon burial, they acquire a diffuse texture due to the expansion and contraction of gas-filled voids, and they present a geochemical signature rich in iron and titanium, which is due to the trapping of heavy minerals. We show that these intrinsic characteristics can be detected via µXRF analyses, and that they are distinct from buried abiotic facies such as cross-stratification and adhesion ripple laminations. We also designed and conducted an interactive survey using the Padre Island µXRF data to explore how different users chose to investigate a biosignature-bearing dataset via PIXL-like sampling strategies. We show that investigating biosignatures via PIXL-like analyses is heavily influenced by technical constraints (e.g., the XRF measurement characteristics) and by the variety of approaches chosen by different scientists. Lessons learned for accurately identifying and characterizing this biosignature in the context of rover-mission constraints include defining relative priorities among measurements, favoring a multidisciplinary approach to the decision-making process of XRF measurements selection, and considering abiotic results to support or discard a biosignature interpretation. Our results provide guidelines for PIXL analyses of potential biosignature on Mars.

Characterization of Sulfur-Rich Microbial Organic Matter in Jurassic Carbonates Using Laser-Assisted Mass Spectrometry.

Laser desorption-ionization mass spectrometry (MS) shows great potential for in situ molecular analysis of planetary surfaces and microanalysis of space-returned samples or (micro)fossils. Coupled with pyrolysis gas chromatography-mass spectrometry (Py-GC-MS) in ESA's ExoMars project, this technique could help assess further the origin of sulfur-bearing organic matter (OM) recently detected on Mars. To unravel this potential, we analyzed sulfurized microbial OM from ca. 150 million year-old carbonates with laser desorption-ionization mass spectrometry (single- and two-step: LDI-MS and L2MS), in comparison with time-of-flight secondary-ion mass spectrometry (ToF-SIMS), gas chromatography-mass spectrometry (GC-MS), and Py-GC-MS. We show that LDI-MS and L2MS readily detect sulfur-bearing moieties such as (alkyl)thiophenes and (alkyl)benzothiophenes. The mineral matrix, however, made the identification of sulfur-bearing molecules challenging in our L2MS experiment. The dominance of small aromatic hydrocarbons (≤14 carbons) in the LDI-MS and L2MS of the extracted soluble and insoluble OM and of the bulk rock is consistent with the low thermal maturity of the sediment and contrasts with the predominance of larger polycyclic aromatic structures commonly observed in meteorites with these techniques. We detected inorganic ions, in particular VO+, in demineralized OM that likely originate from geoporphyrins, which derive from chlorophylls during sediment diagenesis. Finally, insoluble OM yielded distinct compositions compared with extracted soluble OM, with a greater abundance of ions of mass-to-charge ratio (m/z) over 175 and additional N-moieties. This highlights the potential of laser-assisted MS to decipher the composition of macromolecular OM, in particular to investigate the preservation of biomacromolecules in microfossils. Studies comparing diverse biogenic and abiogenic OM are needed to further assess the use of this technique to search for biosignatures.