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At least 19 recordsLinked to original sources
USGS2008

Stress tolerance in plants via habitat-adapted symbiosis

We demonstrate that native grass species from coastal and geothermal habitats require symbiotic fungal endophytes for salt and heat tolerance, respectively. Symbiotically conferred stress tolerance is a habitat-specific phenomenon with geothermal endophytes conferring heat but not salt tolerance, and coastal endophytes conferring salt but not heat tolerance. The same fungal species isolated from plants in habitats devoid of salt or heat stress did not confer these stress tolerances. Moreover, fungal endophytes from agricultural crops conferred disease resistance and not salt or heat tolerance. We define habitat-specific, symbiotically-conferred stress tolerance as habitat-adapted symbiosis and hypothesize that it is responsible for the establishment of plants in high-stress habitats. The agricultural, coastal and geothermal plant endophytes also colonized tomato (a model eudicot) and conferred disease, salt and heat tolerance, respectively. In addition, the coastal plant endophyte colonized rice (a model monocot) and conferred salt tolerance. These endophytes have a broad host range encompassing both monocots and eudicots. Interestingly, the endophytes also conferred drought tolerance to plants regardless of the habitat of origin. Abiotic stress tolerance correlated either with a decrease in water consumption or reactive oxygen sensitivity/generation but not to increased osmolyte production. The ability of fungal endophytes to confer stress tolerance to plants may provide a novel strategy for mitigating the impacts of global climate change on agricultural and native plant communities.The ISME Journal (2008) 2, 404-416; doi:10.1038/ismej.2007.106; published online 7 February 2008. ?? 2008 International Society for Microbial Ecology All rights reserved.

ISME Journal↗
USGS2012

Diversity of active aerobic methanotrophs along depth profiles of arctic and subarctic lake water column and sediments

Methane (CH 4 ) emitted from high-latitude lakes accounts for 2–6% of the global atmospheric CH 4 budget. Methanotrophs in lake sediments and water columns mitigate the amount of CH 4 that enters the atmosphere, yet their identity and activity in arctic and subarctic lakes are poorly understood. We used stable isotope probing (SIP), quantitative PCR (Q-PCR), pyrosequencing and enrichment cultures to determine the identity and diversity of active aerobic methanotrophs in the water columns and sediments (0–25 cm) from an arctic tundra lake (Lake Qalluuraq) on the north slope of Alaska and a subarctic taiga lake (Lake Killarney) in Alaska's interior. The water column CH 4 oxidation potential for these shallow (~2m deep) lakes was greatest in hypoxic bottom water from the subarctic lake. The type II methanotroph, Methylocystis, was prevalent in enrichment cultures of planktonic methanotrophs from the water columns. In the sediments, type I methanotrophs (Methylobacter, Methylosoma and Methylomonas) at the sediment-water interface (0–1 cm) were most active in assimilating CH 4 , whereas the type I methanotroph Methylobacter and/or type II methanotroph Methylocystis contributed substantially to carbon acquisition in the deeper (15–20 cm) sediments. In addition to methanotrophs, an unexpectedly high abundance of methylotrophs also actively utilized CH 4 -derived carbon. This study provides new insight into the identity and activity of methanotrophs in the sediments and water from high-latitude lakes.

Alaska↗
USGS2014

Carbon flow from volcanic CO 2 into soil microbial communities of a wetland mofette

Effects of extremely high carbon dioxide (CO 2 ) concentrations on soil microbial communities and associated processes are largely unknown. We studied a wetland area affected by spots of subcrustal CO 2 degassing (mofettes) with focus on anaerobic autotrophic methanogenesis and acetogenesis because the pore gas phase was largely hypoxic. Compared with a reference soil, the mofette was more acidic (ΔpH ~0.8), strongly enriched in organic carbon (up to 10 times), and exhibited lower prokaryotic diversity. It was dominated by methanogens and subdivision 1Acidobacteria, which likely thrived under stable hypoxia and acidic pH. Anoxic incubations revealed enhanced formation of acetate and methane (CH 4 ) from hydrogen (H 2 ) and CO 2 consistent with elevated CH 4 and acetate levels in the mofette soil. 13 CO 2 mofette soil incubations showed high label incorporations with ~512   ng 13 C   g   (dry weight (dw)) soil −1   d −1 into the bulk soil and up to 10.7   ng 13 C   g   (dw) soil −1   d −1 into almost all analyzed bacterial lipids. Incorporation of CO 2 -derived carbon into archaeal lipids was much lower and restricted to the first 10   cm of the soil. DNA-SIP analysis revealed that acidophilic methanogens affiliated withMethanoregulaceae and hitherto unknown acetogens appeared to be involved in the chemolithoautotrophic utilization of 13 CO 2 . Subdivision 1 Acidobacteriaceae assimilated 13 CO 2 likely via anaplerotic reactions because Acidobacteriaceae are not known to harbor enzymatic pathways for autotrophic CO 2 assimilation. We conclude that CO 2 -induced geochemical changes promoted anaerobic and acidophilic organisms and altered carbon turnover in affected soils.

The ISME Journal↗
USGS2014

Relationships between protein-encoding gene abundance and corresponding process are commonly assumed yet rarely observed

For any enzyme-catalyzed reaction to occur, the corresponding protein-encoding genes and transcripts are necessary prerequisites. Thus, a positive relationship between the abundance of gene or transcripts and corresponding process rates is often assumed. To test this assumption, we conducted a meta-analysis of the relationships between gene and/or transcript abundances and corresponding process rates. We identified 415 studies that quantified the abundance of genes or transcripts for enzymes involved in carbon or nitrogen cycling. However, in only 59 of these manuscripts did the authors report both gene or transcript abundance and rates of the appropriate process. We found that within studies there was a significant but weak positive relationship between gene abundance and the corresponding process. Correlations were not strengthened by accounting for habitat type, differences among genes or reaction products versus reactants, suggesting that other ecological and methodological factors may affect the strength of this relationship. Our findings highlight the need for fundamental research on the factors that control transcription, translation and enzyme function in natural systems to better link genomic and transcriptomic data to ecosystem processes.

The ISME Journal↗
USGS2017

Acetoclastic Methanosaeta are dominant methanogens in organic-rich Antarctic marine sediments

Despite accounting for the majority of sedimentary methane, the physiology and relative abundance of subsurface methanogens remain poorly understood. We combined intact polar lipid and metagenome techniques to better constrain the presence and functions of methanogens within the highly reducing, organic-rich sediments of Antarctica’s Adélie Basin. The assembly of metagenomic sequence data identified phylogenic and functional marker genes of methanogens and generated the first Methanosaeta sp. genome from a deep subsurface sedimentary environment. Based on structural and isotopic measurements, glycerol dialkyl glycerol tetraethers with diglycosyl phosphatidylglycerol head groups were classified as biomarkers for active methanogens. The stable carbon isotope (δ 13 C) values of these biomarkers and the Methanosaeta partial genome suggest that these organisms are acetoclastic methanogens and represent a relatively small (0.2%) but active population. Metagenomic and lipid analyses suggest that Thaumarchaeota and heterotrophic bacteria co-exist with Methanosaeta and together contribute to increasing concentrations and δ 13 C values of dissolved inorganic carbon with depth. This study presents the first functional insights of deep subsurface Methanosaeta organisms and highlights their role in methane production and overall carbon cycling within sedimentary environments.

ISME Journal↗
USGS2017

Microbial survival strategies in ancient permafrost: insights from metagenomics

In permafrost (perennially frozen ground) microbes survive oligotrophic conditions, sub-zero temperatures, low water availability and high salinity over millennia. Viable life exists in permafrost tens of thousands of years old but we know little about the metabolic and physiological adaptations to the challenges presented by life in frozen ground over geologic time. In this study we asked whether increasing age and the associated stressors drive adaptive changes in community composition and function. We conducted deep metagenomic and 16 S rRNA gene sequencing across a Pleistocene permafrost chronosequence from 19 000 to 33 000 years before present (kyr). We found that age markedly affected community composition and reduced diversity. Reconstruction of paleovegetation from metagenomic sequence suggests vegetation differences in the paleo record are not responsible for shifts in community composition and function. Rather, we observed shifts consistent with long-term survival strategies in extreme cryogenic environments. These include increased reliance on scavenging detrital biomass, horizontal gene transfer, chemotaxis, dormancy, environmental sensing and stress response. Our results identify traits that may enable survival in ancient cryoenvironments with no influx of energy or new materials.

The ISME Journal↗
USGS2019

Global change-driven use of onshore habitat impacts polar bear faecal microbiota

The gut microbiota plays a critical role in host health, yet remains poorly studied in wild species. Polar bears (Ursus maritimus), key indicators of Arctic ecosystem health and environmental change, are currently affected by rapid shifts in habitat that may alter gut homeostasis. Declining sea ice has led to a divide in the southern Beaufort Sea polar bear subpopulation such that an increasing proportion of individuals now inhabit onshore coastal regions during the open-water period (‘onshore bears’) while others continue to exhibit their typical behaviour of remaining on the ice (‘offshore bears’). We propose that bears that have altered their habitat selection in response to climate change will exhibit a distinct gut microbiota diversity and composition, which may ultimately have important consequences for their health. Here, we perform the first assessment of abundance and diversity in the faecal microbiota of wild polar bears using 16S rRNA Illumina technology. We find that bacterial diversity is significantly higher in onshore bears compared to offshore bears. The majority of unique bacterial taxa for onshore bears belonged to the phyla Proteobacteria (a proposed indicator of poor health in adult humans), whereas for offshore bears, Firmicutes (associated with adiposity) dominated. We conclude that climate-driven changes in polar bear land use are associated with distinct microbial communities. In doing so, we present the first case of global change mediated alterations in the gut microbiota of a free-roaming wild animal.

Alaska↗
USGS2020

Skin fungal assemblages of bats vary based on susceptibility to white-nose syndrome

Microbial skin assemblages, including fungal communities, can influence host resistance to infectious diseases. The diversity-invasibility hypothesis predicts that high-diversity communities are less easily invaded than species-poor communities, and thus diverse microbial communities may prevent pathogens from colonizing a host. To explore the hypothesis that host fungal communities mediate resistance to infection by fungal pathogens, we investigated characteristics of bat skin fungal communities as they relate to susceptibility to the emerging disease white-nose syndrome (WNS). Using a culture-based approach, we compared skin fungal assemblage characteristics of 10 bat species that differ in susceptibility to WNS across 10 eastern U.S. states. The fungal assemblages on WNS-susceptible bat species had significantly lower alpha diversity and abundance compared to WNS-resistant species. Overall fungal assemblage structure did not vary based on WNS-susceptibility, but several yeast species were differentially abundant on WNS-resistant bat species. One yeast species inhibited Pseudogymnoascus destructans ( Pd ), the causative agent on WNS, in vitro under certain conditions, suggesting a possible role in host protection. Further exploration of interactions between Pd and constituents of skin fungal assemblages may prove useful for predicting susceptibility of bat populations to WNS and for developing effective mitigation strategies.

ISME Journal↗
USGS2021

Gulf of Mexico blue hole harbors high levels of novel microbial lineages

Exploration of oxygen-depleted marine environments has consistently revealed novel microbial taxa and metabolic capabilities that expand our understanding of microbial evolution and ecology. Marine blue holes are shallow karst formations characterized by low oxygen and high organic matter content. They are logistically challenging to sample, and thus our understanding of their biogeochemistry and microbial ecology is limited. We present a metagenomic and geochemical characterization of Amberjack Hole on the Florida continental shelf (Gulf of Mexico). Dissolved oxygen became depleted at the hole’s rim (32 m water depth), remained low but detectable in an intermediate hypoxic zone (40–75 m), and then increased to a secondary peak before falling below detection in the bottom layer (80–110 m), concomitant with increases in nutrients, dissolved iron, and a series of sequentially more reduced sulfur species. Microbial communities in the bottom layer contained heretofore undocumented levels of the recently discovered phylum Woesearchaeota (up to 58% of the community), along with lineages in the bacterial Candidate Phyla Radiation (CPR). Thirty-one high-quality metagenome-assembled genomes (MAGs) showed extensive biochemical capabilities for sulfur and nitrogen cycling, as well as for resisting and respiring arsenic. One uncharacterized gene associated with a CPR lineage differentiated hypoxic from anoxic zone communities. Overall, microbial communities and geochemical profiles were stable across two sampling dates in the spring and fall of 2019. The blue hole habitat is a natural marine laboratory that provides opportunities for sampling taxa with under-characterized but potentially important roles in redox-stratified microbial processes.

Interational Society of Microbial Ecology (ISME) J↗
USGS2021

Expanding the repertoire of electron acceptors for the anaerobic oxidation of methane in carbonates in the Atlantic and Pacific Ocean

Authigenic carbonates represent a significant microbial sink for methane, yet little is known about the microbiome responsible for the methane removal. We identify carbonate microbiomes distributed over 21 locations hosted by seven different cold seeps in the Pacific and Atlantic Oceans by carrying out a gene-based survey using 16S rRNA- and mcr A gene sequencing coupled with metagenomic analyses. Based on 16S rRNA gene amplicon analyses, these sites were dominated by bacteria affiliated to the Firmicutes, Alpha- and Gammaproteobacteria. ANME-1 and -2 archaeal clades were abundant in the carbonates yet their typical syntrophic partners, sulfate-reducing bacteria, were not significantly present. Based on mcrA amplicon analyses, the Candidatus Methanoperedens clades were also highly abundant. Our metagenome analysis indicated that methane oxidizers affiliated to the ANME-1 and -2, may be capable of performing complete methane- and potentially short-chain alkane oxidation independently using oxidized sulfur and nitrogen compounds as terminal electron acceptors. Gammaproteobacteria are hypothetically capable of utilizing oxidized nitrogen compounds and may be involved in syntrophy with methane-oxidizing archaea. Carbonate structures represent a window for a more diverse utilization of electron acceptors for anaerobic methane oxidation along the Atlantic and Pacific Margin.

ISME Journal↗
USGS2021

Metabolic flexibility of aerobic methanotrophs under anoxic conditions in Arctic lake sediments

Methane (CH 4 ) emissions from Arctic lakes are a large and growing source of greenhouse gas to the atmosphere with critical implications for global climate. Because Arctic lakes are ice covered for much of the year, understanding the metabolic flexibility of methanotrophs under anoxic conditions would aid in characterizing the mechanisms responsible for limiting CH 4 emissions from high-latitude regions. Using sediments from an active CH 4 seep in Lake Qalluuraq, Alaska, we conducted DNA-based stable isotope probing (SIP) in anoxic mesocosms and found that aerobic Gammaproteobacterial methanotrophs dominated in assimilating CH 4 . Aerobic methanotrophs were also detected down to 70 cm deep in sediments at the seep site, where anoxic conditions persist. Metagenomic analyses of the heavy DNA from 13 CH 4 -SIP incubations showed that these aerobic methanotrophs had the capacity to generate intermediates such as methanol, formaldehyde, and formate from CH 4 oxidation and to oxidize formaldehyde in the tetrahydromethanopterin (H 4 MPT)-dependent pathway under anoxic conditions. The high levels of Fe present in sediments, combined with Fe and CH 4 profiles in the persistent CH 4 seep site, suggested that oxidation of CH 4 , or, more specifically, its intermediates such as methanol and formaldehyde might be coupled to iron reduction. Aerobic methanotrophs also possessed genes associated with nitrogen and hydrogen metabolism, which might provide potentially alternative energy conservation options under anoxic conditions. These results expand the known metabolic spectrum of aerobic methanotrophs under anoxic conditions and necessitate the re-assessment of the mechanisms underlying CH 4 oxidation in the Arctic, especially under lakes that experience extended O 2 limitations during ice cover.

Alaska↗
USGS2021

Solutions in microbiome engineering: Prioritizing barriers to organism establishment

Microbiome engineering is increasingly being employed as a solution to challenges in health, agriculture, and climate. Often manipulation involves inoculation of new microbes designed to improve function into a preexisting microbial community. Despite, increased efforts in microbiome engineering inoculants frequently fail to establish and/or confer long-lasting modifications on ecosystem function. We posit that one underlying cause of these shortfalls is the failure to consider barriers to organism establishment. This is a key challenge and focus of macroecology research, specifically invasion biology and restoration ecology. We adopt a framework from invasion biology that summarizes establishment barriers in three categories: (1) propagule pressure, (2) environmental filtering, and (3) biotic interactions factors. We suggest that biotic interactions is the most neglected factor in microbiome engineering research, and we recommend a number of actions to accelerate engineering solutions.

The ISME Journal: Multidisciplinary Journal of Mic↗
USGS2021

Activity-based, genome-resolved metagenomics uncovers key populations and pathways involved in subsurface conversions of coal to methane

Microbial metabolisms and interactions that facilitate subsurface conversions of recalcitrant carbon to methane are poorly understood. We deployed an in situ enrichment device in a subsurface coal seam in the Powder River Basin (PRB), USA, and used BONCAT-FACS-Metagenomics to identify translationally active populations involved in methane generation from a variety of coal-derived aromatic hydrocarbons. From the active fraction, high-quality metagenome-assembled genomes (MAGs) were recovered for the acetoclastic methanogen, Methanothrix paradoxum , and a novel member of the Chlorobi with the potential to generate acetate via the Pta-Ack pathway. Members of the Bacteroides and Geobacter also encoded Pta-Ack and together, all four populations had the putative ability to degrade ethylbenzene, phenylphosphate, phenylethanol, toluene, xylene, and phenol. Metabolic reconstructions, gene analyses, and environmental parameters also indicated that redox fluctuations likely promote facultative energy metabolisms in the coal seam. The active “ Chlorobi PRB” MAG encoded enzymes for fermentation, nitrate reduction, and multiple oxygenases with varying binding affinities for oxygen. “ M. paradoxum PRB” encoded an extradiol dioxygenase for aerobic phenylacetate degradation, which was also present in previously published Methanothrix genomes. These observations outline underlying processes for bio-methane from subbituminous coal by translationally active populations and demonstrate activity-based metagenomics as a powerful strategy in next generation physiology to understand ecologically relevant microbial populations.

Montana, Wyoming↗
USGS2023

Permafrost microbial communities and functional genes are structured by latitudinal and soil geochemical gradients

Permafrost underlies approximately one quarter of Northern Hemisphere terrestrial surfaces and contains 25–50% of the global soil carbon (C) pool. Permafrost soils and the C stocks within are vulnerable to ongoing and future projected climate warming. The biogeography of microbial communities inhabiting permafrost has not been examined beyond a small number of sites focused on local-scale variation. Permafrost is different from other soils. Perennially frozen conditions in permafrost dictate that microbial communities do not turn over quickly, thus possibly providing strong linkages to past environments. Thus, the factors structuring the composition and function of microbial communities may differ from patterns observed in other terrestrial environments. Here, we analyzed 133 permafrost metagenomes from North America, Europe, and Asia. Permafrost biodiversity and taxonomic distribution varied in relation to pH, latitude and soil depth. The distribution of genes differed by latitude, soil depth, age, and pH. Genes that were the most highly variable across all sites were associated with energy metabolism and C-assimilation. Specifically, methanogenesis, fermentation, nitrate reduction, and replenishment of citric acid cycle intermediates. This suggests that adaptations to energy acquisition and substrate availability are among some of the strongest selective pressures shaping permafrost microbial communities. The spatial variation in metabolic potential has primed communities for specific biogeochemical processes as soils thaw due to climate change, which could cause regional- to global- scale variation in C and nitrogen processing and greenhouse gas emissions.

The ISME Journal↗
USGS2024

Transglobal spread of an ecologically relevant sea urchin parasite

Mass mortality of the dominant coral reef herbivore Diadema antillarum in the Caribbean in the early 1980s contributed to a persistent phase shift from coral- to algal-dominated reefs. In 2022, a scuticociliate most closely related to Philaster apodigitiformis caused further mass mortality of D. antillarum across the Caribbean, leading to >95% mortality at affected sites. Mortality was also reported in the related species Diadema setosum in the Mediterranean in 2022, though the causative agent of the Mediterranean outbreak has not yet been determined. In April 2023, mass mortality of Diadema setosum occurred along the Sultanate of Oman's coastline. Urchins displayed signs compatible with scuticociliatosis including abnormal behavior, drooping and loss of spines, followed by tissue necrosis and death. Here we report the detection of an 18S rRNA gene sequence in abnormal urchins from Muscat, Oman, that is identical to the Philaster strain responsible for D. antillarum mass mortality in the Caribbean. We also show that scuticociliatosis signs can be elicited in Diadema setosum by experimental challenge with the cultivated Philaster strain associated with Caribbean scuticociliatosis. These results demonstrate the Philaster sp. associated with D. antillarum mass mortality has rapidly spread to geographically distant coral reefs, compelling global-scale awareness and monitoring for this devastating condition through field surveys, microscopy, and molecular microbiological approaches, and prompting investigation of long-range transmission mechanisms.

ISME Journal↗
USGS2025

Manipulation of the Symbiodiniaceae microbiome confers multigenerational impacts on symbioses and reproductive ecology of its Exaiptasia diaphana host

Symbiodiniaceae-associated microbiota strongly influence cnidarian symbioses. We systematically reduced the bacterial and fungal communities associated with Symbiodiniaceae to study potential effects on the cnidarian holobiont Exaiptasia diaphana (Aiptasia). Clonal anemones were inoculated with xenic Breviolum minutum (SSB01) and microbiome-manipulated cultures after antibacterial or antifungal treatment. The asexual reproduction of pedal laceration allowed for three generations of clonal aposymbiotic Aiptasia to be utilised in this study, from the initial adult generation (G0), to the first (G1), and second (G2) generation. We inoculated small and large G1 Aiptasia with SSB01 algae and monitored onset of symbiosis, rate of algal proliferation, and holobiont characteristics. Sequencing the 16S and 18S rRNA gene regions identified significant differences in the bacterial and fungal communities of the G0 and G1 generations, alongside differences between the size classes of small and large G1 anemones. The microbiome of larger G1 individuals was distinct to the smaller G1 anemones, suggesting a microbiome maturation process. Control Breviolum minutum cultures exhibited a significantly greater proliferation rate in large G1 anemones when compared to antibacterial or antifungal treated cultures, whereas the opposite trend was documented in the small G1 anemones. Although no differences were observed between algal photochemical parameters, or the growth and polyp activity of G1 juveniles, we observed a significant influence in the production of G2 clones between treatments. Overall, we provide strong ecological implications of manipulating Symbiodiniaceae microbiome, not for the algae themselves, but for the maturation of the host Aiptasia, as well as for the cnidarian holobiont over multiple generations.

The ISME Journal↗
USGS2026

Diverse cyanopeptides follow distinct temporal succession patterns in freshwater harmful algal blooms

Toxic cyanobacterial harmful algal blooms (cyanoHABs) threaten freshwater resources globally and are intensifying with increasing eutrophication. Bloom toxicity is strongly influenced by intraspecific variation in the biosynthetic repertoires of toxic cyanobacteria, yet few studies examine the diversity of cyanobacterial cyanopeptides beyond hepatotoxic microcystins . To understand the dynamics and drivers of cyanopeptide diversity in cyanoHABs, we analyzed temporal patterns of cyanobacteria, metabolites, and their biosynthetic gene clusters (BGCs) in western Lake Erie using a 7-year time series (2016–2022) of metagenomic and metabolomic data. Our findings demonstrate that shifts from Microcystis to Dolichospermum occur later in the bloom season, coinciding with lower temperatures. Modules of co-varying BGCs (biosynthesis modules) from these genera were identified with hierarchical clustering, with uncharacterized BGCs among the most abundant. Biosynthesis modules rich in nonribosomal peptide synthetases (NRPS) peaked in early August, coinciding with elevated levels of inorganic nitrogen, warmer temperatures, and high Microcystis abundance. In contrast, modules rich in polyketide synthases (PKS) and ribosomally synthesized and post-translationally modified peptides (RiPPs) peaked following the Microcystis maximum in mid-August. Metabolomic analyses confirmed that metabolites followed shared seasonal patterns with their associated biosynthesis modules, forming three phases characterized by (i) microcystins, (ii) anabaenopeptins and aeruginosins, and (iii) aerucyclamides. These phases co-varied with bottom-up and top-down pressures, with later phases coinciding with increased microbially processed organic nitrogen and reduced detection of grazers. This study demonstrates consistent seasonal patterns of cyanobacterial metabolite succession and co-occurrence beyond microcystins, suggesting tradeoffs between biosynthetic resource demands and ecological controls.

Ohio↗
USGS2026

Deep subsurface organic-rich shale supports abundant, diverse, and novel fungi

As Earth’s principal reservoir of organic carbon and microbial biomass, the deep subsurface hosts microorganisms capable of mobilizing this once-sequestered carbon. Contrary to standard assumptions of eukaryotic scarcity, this study documents abundant fungal communities, ranging from 4.2 × 10 3 to 6.8 × 10 3 fungal cells mL −1 , across a methane-producing organic-rich shale 247–556 meters below the surface. Although fungal:bacterial cell ratios ranged from 1:7028 to 1:713, application of biomass conversion factors developed for oceanic systems yielded a median fungal:bacterial biomass ratio of 1:4.7. 16S rRNA gene amplicons revealed bacterial and archaeal communities mirroring those found in well-characterized extremophilic, carbon-degrading environments, while sequencing of 18S rRNA gene and ITS rRNA spacer amplicons collectively identified a eukaryotic hotspot with 689 fungal OTUs across six phyla. The dominant fungal classes, Agaricomycetes and Dothideomycetes, are well-established degraders of recalcitrant carbon compounds at the surface, suggesting they may similarly contribute to organic matter degradation and ecosystem maintenance in the subsurface. Cultivation and isolation efforts yielded 205 fungal strains, including 13 candidate novel taxa, underscoring the deep subsurface as an underexplored eukaryotic habitat. Stable carbon isotopes indicate methane is predominantly generated via microbial conversion of the fossil carbon, while water isotopes suggest in situ geochemical conditions have been relatively stable since the Late Pleistocene, with subglacial recharge as a plausible mechanism for microbial introduction. Collectively, these findings suggest that fungi are underrecognized contributors to organic matter transformation and functional diversity in the deep biosphere, revealing a critical gap in our understanding of deep subsurface ecosystem processes.

Indiana, Michigan, Ohio↗