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At least 235 records · Page 13

Intra‐species genetic variability drives carbon metabolism and symbiotic host interactions in the ectomycorrhizal fungus Pisolithus microcarpus

Summary Ectomycorrhizal (ECM) fungi are integral to boreal and temperate forest ecosystem functioning and nutrient cycling. ECM fungi, however, originate from diverse saprotrophic lineages and the impacts of genetic variation across species, and especially within a given ECM species, on function and interactions with the environment is not well understood. Here, we explore the extent of intra‐species variation between four isolates of the ECM fungus Pisolithus microcarpus , in terms of gene regulation, carbon metabolism and growth, and interactions with a host, Eucalyptus grandis . We demonstrate that, while a core response to the host is maintained by all of the isolates tested, they have distinct patterns of gene expression and carbon metabolism, resulting in the differential expression of isolate‐specific response pathways in the host plant. Together, these results highlight the importance of using a wider range of individuals within a species to understand the broader ecological roles of ECM fungi and their host interactions.

Plett, Krista L.↗

Metal concentration and leaf spatial distribution assessed by synchrotron µXRF in Brazilian nickel hyperaccumulators

Background and Aims: The limited number of known hyperaccumulator species in tropical regions, including Brazil, has hampered the development of nickel (Ni) agromining. In addition, the mechanisms underlying metal accumulation and distribution in these species remain poorly understood, despite their pivotal role in identifying species with economic potential. Furthermore, this study assessed the accumulation potential and foliar spatial distribution of metals in three Brazilian hyperaccumulator species (Pfaffia sarcophylla, Justicia lanstyakii, and Lippia lupulina) growing in one of the world’s largest mafic–ultramafic complexes. Methods Concentrations of Ni and other metals in leaves were determined using portable X-ray fluorescence (pXRF) and inductively coupled plasma optical emission spectroscopy (ICP-OES). Synchrotron-based X-ray microfluorescence (SR-µXRF) was employed to map the spatial distribution of metals across intact leaves. Results: The results revealed leaf Ni concentrations ranging from 320 to 1,950 mg kg⁻ 1 in P. sarcophylla, 1,640 to 6,810 mg kg⁻ 1 in L. lupulina, and 1,990 to 4,900 mg kg⁻ 1 in J. lanstyakii. Nickel, Mn, and Co exhibited similar distribution patterns, concentrating mainly in the leaf margins and veins across all species, while Ca, K, and Mg co-localization suggests regulatory mechanisms for adaptation. Conclusion: Although these plants exhibited Ni hyperaccumulation, our findings suggest limited commercial potential for agromining. Further research on genetic variability and nutrient assimilation, particularly in P. sarcophylla, is recommended to elucidate the mechanisms underlying Ni accumulation and intraspecific variability.

36 MATERIALS SCIENCE↗

Metagenomic mapping of cyanobacteria and potential cyanotoxin producing taxa in large rivers of the United States

Abstract Cyanobacteria and cyanotoxin producing cyanobacterial blooms are a trending focus of current research. Many studies focus on bloom events in lentic environments such as lakes or ponds. Comparatively few studies have explored lotic environments and fewer still have examined the cyanobacterial communities and potential cyanotoxin producers during ambient, non-bloom conditions. Here we used a metagenomics-based approach to profile non-bloom microbial communities and cyanobacteria in 12 major U.S. rivers at multiple time points during the summer months of 2019. Our data show that U.S. rivers possess microbial communities that are taxonomically rich, yet largely consistent across geographic location and time. Within these communities, cyanobacteria often comprise significant portions and frequently include multiple species with known cyanotoxin producing strains. We further characterized these potential cyanotoxin producing taxa by deep sequencing amplicons of the microcystin E ( mcyE ) gene. We found that rivers containing the highest levels of potential cyanotoxin producing cyanobacteria consistently possess taxa with the genetic potential for cyanotoxin production and that, among these taxa, the predominant genus of origin for the mcyE gene is Microcystis . Combined, these data provide a unique perspective on cyanobacteria and potential cyanotoxin producing taxa that exist in large rivers across the U.S. and can be used to better understand the ambient conditions that may precede bloom events in lotic freshwater ecosystems.

59 BASIC BIOLOGICAL SCIENCES↗

High-throughput protein characterization by complementation using DNA barcoded fragment libraries

Abstract Our ability to predict, control, or design biological function is fundamentally limited by poorly annotated gene function. This can be particularly challenging in non-model systems. Accordingly, there is motivation for new high-throughput methods for accurate functional annotation. Here, we used co mplementation of aux otrophs and DNA barcode seq uencing (Coaux-Seq) to enable high-throughput characterization of protein function. Fragment libraries from eleven genetically diverse bacteria were tested in twenty different auxotrophic strains of Escherichia coli to identify genes that complement missing biochemical activity. We recovered 41% of expected hits, with effectiveness ranging per source genome, and observed success even with distant E. coli relatives like Bacillus subtilis and Bacteroides thetaiotaomicron . Coaux-Seq provided the first experimental validation for 53 proteins, of which 11 are less than 40% identical to an experimentally characterized protein. Among the unexpected function identified was a sulfate uptake transporter, an O-succinylhomoserine sulfhydrylase for methionine synthesis, and an aminotransferase. We also identified instances of cross-feeding wherein protein overexpression and nearby non-auxotrophic strains enabled growth. Altogether, Coaux-Seq’s utility is demonstrated, with future applications in ecology, health, and engineering.

59 BASIC BIOLOGICAL SCIENCES↗

Ecology of methyl-coenzyme M reductase encoding Thermoproteota

The recent demonstration that members of at least three classes of archaea affiliated with the Thermoproteota superphylum are involved in the production of the climate-active gas methane has sparked discussions about how well we understand the diversity of methanogens. Here, in this work, we show that members of all three of these lineages, as well as several other, yet uncultured and physiologically uncharacterized groups within the Thermoproteota that encode the key enzyme of anaerobic methane cycling, methyl-coenzyme M reductase (MCR), are widely distributed in anoxic ecosystems. We postulate that the taxonomic, metabolic, and ecological diversity of methanogenic and MCR-encoding Thermoproteota are poorly understood, and that the contribution of methylotrophic and thermoproteotal methanogenesis to methane production is largely unknown. We hypothesize that thermoproteotal methanogens could contribute, potentially substantially, to methane emissions in many anoxic environments that harbor methylated precursors, including wetlands, sediments, peat, rice paddies, wastewater sludge, and geothermal systems. We highlight the necessity to experimentally test the (eco)physiology of these widely distributed archaea using both culture-dependent ( in vitro ) and culture-independent ( in situ ) approaches to assess their potential contribution to methane emissions. Last, we stress the importance of remaining agnostic about the physiology of MCR-encoding Thermoproteota in the absence of experimental data because most of these archaea also carry the genetic potential to grow non-methanogenically.

MCR↗

Century-long timelines of herbarium genomes predict plant stomatal response to climate change

Abstract Dissecting plant responses to the environment is key to understanding whether and how plants adapt to anthropogenic climate change. Stomata, plants’ pores for gas exchange, are expected to decrease in density following increased CO 2 concentrations, a trend already observed in multiple plant species. However, it is unclear whether such responses are based on genetic changes and evolutionary adaptation. Here we make use of extensive knowledge of 43 genes in the stomatal development pathway and newly generated genome information of 191 Arabidopsis thaliana historical herbarium specimens collected over 193 years to directly link genetic variation with climate change. While we find that the essential transcription factors SPCH, MUTE and FAMA, central to stomatal development, are under strong evolutionary constraints, several regulators of stomatal development show signs of local adaptation in contemporary samples from different geographic regions. We then develop a functional score based on known effects of gene knock-out on stomatal development that recovers a classic pattern of stomatal density decrease over the past centuries, suggesting a genetic component contributing to this change. This approach combining historical genomics with functional experimental knowledge could allow further investigations of how different, even in historical samples unmeasurable, cellular plant phenotypes may have already responded to climate change through adaptive evolution.

Environmental Sciences & Ecology↗

Multicellular magnetotactic bacteria are genetically heterogeneous consortia with metabolically differentiated cells

Consortia of multicellular magnetotactic bacteria (MMB) are currently the only known example of bacteria without a unicellular stage in their life cycle. Because of their recalcitrance to cultivation, most previous studies of MMB have been limited to microscopic observations. To study the biology of these unique organisms in more detail, we use multiple culture-independent approaches to analyze the genomics and physiology of MMB consortia at single-cell resolution. We separately sequenced the metagenomes of 22 individual MMB consortia, representing 8 new species, and quantified the genetic diversity within each MMB consortium. This revealed that, counter to conventional views, cells within MMB consortia are not clonal. Single consortia metagenomes were then used to reconstruct the species-specific metabolic potential and infer the physiological capabilities of MMB. To validate genomic predictions, we performed stable isotope probing (SIP) experiments and interrogated MMB consortia using fluorescence in situ hybridization (FISH) combined with nanoscale secondary ion mass spectrometry (NanoSIMS). By coupling FISH with bioorthogonal noncanonical amino acid tagging (BONCAT), we explored their in situ activity as well as variation of protein synthesis within cells. We demonstrate that MMB consortia are mixotrophic sulfate reducers and that they exhibit metabolic differentiation between individual cells, suggesting that MMB consortia are more complex than previously thought. These findings expand our understanding of MMB diversity, ecology, genomics, and physiology, as well as offer insights into the mechanisms underpinning the multicellular nature of their unique lifestyle.

59 BASIC BIOLOGICAL SCIENCES↗

Adaptive modification of antiviral defense systems in microbial community under Cr-induced stress

Background The prokaryotic antiviral defense systems are crucial for mediating prokaryote-virus interactions that influence microbiome functioning and evolutionary dynamics. Despite the prevalence and significance of prokaryotic antiviral defense systems, their responses to abiotic stress and ecological consequences remain poorly understood in soil ecosystems. We established microcosm systems with varying concentrations of hexavalent chromium (Cr(VI)) to investigate the adaptive modifications of prokaryotic antiviral defense systems under abiotic stress. Results Utilizing hybrid metagenomic assembly with long-read and short-read sequencing, we discovered that anti- viral defense systems were more diverse and prevalent in heavily polluted soils, which was corroborated by meta-analyses of public datasets from various heavy metal-contaminated sites. As the Cr(VI) concentration increased, prokaryotes with defense systems favoring prokaryote-virus mutualism gradually supplanted those with defense systems incurring high adaptive costs. Additionally, as Cr(VI) concentrations increased, enriched antiviral defense systems exhibited synchronization with microbial heavy metal resistance genes. Furthermore, the proportion of antiviral defense systems carried by mobile genetic elements (MGEs), including plasmids and viruses, increased by approximately 43% and 39%, respectively, with rising Cr concentrations. This trend is conducive to strengthening the dissemination and sharing of defense resources within microbial communities. Conclusions Overall, our study reveals the adaptive modification of prokaryotic antiviral defense systems in soil ecosystems under abiotic stress, as well as their positive contributions to establishing prokaryote-virus mutualism and the evolution of microbial heavy metal resistance. These findings advance our understanding of microbial adaptation in stressful environments and may inspire novel approaches for microbiome manipulation and bioremediation.

59 BASIC BIOLOGICAL SCIENCES↗

Identification and characterization of a skin microbiome on Caenorhabditis elegans suggests environmental microbes confer cuticle protection

ABSTRACT In the wild, C. elegans are emersed in environments teeming with a veritable menagerie of microorganisms. The C. elegans cuticular surface serves as a barrier and first point of contact with their microbial environments. In this study, we identify microbes from C. elegans natural habitats that associate with its cuticle, constituting a simple “skin microbiome.” We rear our animals on a modified CeMbio, mCeMbio, a consortium of ecologically relevant microbes. We first combine standard microbiological methods with an adapted micro skin-swabbing tool to describe the skin-resident bacteria on the C. elegans surface. Furthermore, we conduct 16S rRNA gene sequencing studies to identify relative shifts in the proportion of mCeMbio bacteria upon surface-sterilization, implying distinct skin- and gut-microbiomes. We find that some strains of bacteria, including Enterobacter sp. JUb101 , are primarily found on the nematode skin, while others like Stenotrophomonas indicatrix JUb19 and Ochrobactrum vermis MYb71 are predominantly found in the animal’s gut. Finally, we show that this skin microbiome promotes host cuticle integrity in harsh environments. Together, we identify a skin microbiome for the well-studied nematode model and propose its value in conferring host fitness advantages in naturalized contexts. IMPORTANCE The genetic model organism C. elegans has recently emerged as a tool for understanding host–microbiome interactions. Nearly all of these studies either focus on pathogenic or gut-resident microbes. Little is known about the existence of native, nonpathogenic skin microbes or their function. We demonstrate that members of a modified C. elegans model microbiome, mCeMbio, can adhere to the animal's cuticle and confer protection from noxious environments. We combine a novel micro-swab tool, the first 16S microbial sequencing data from relatively unperturbed C. elegans , and physiological assays to demonstrate microbially mediated protection of the skin. This work serves as a foundation to explore wild C. elegans skin microbiomes and use C. elegans as a model for skin research.

16S RNA↗

Repatriated Desert Bighorn Sheep Population on the Nevada National Security Site

Ecological studies have been conducted on the Nevada National Security Site (NNSS) since the 1960s. Desert bighorn sheep (Ovis canadensis nelsoni) were considered rare visitors on the NNSS, with only 9 recorded observations between 1963 and 2009, all of which were males. Females and young were not documented definitively until winter 2011, when several were killed by a radio-collared female mountain lion (Puma concolor). Following these observations, we initiated a study of desert bighorn sheep on the NNSS to better understand their movements/interactions with other populations, prevalence of disease, population size, origin, radionuclide burdens and potential radiological dose to humans that may consume harvested animals away from the NNSS. We captured and radio-collared 6 sheep (2 females, 4 males) in November 2015, and 15 (7 females, 8 males) in November 2016. We sampled blood for genetic and disease testing and collected nasal swabs for respiratory disease testing. Sheep from the NNSS spent most of their time around Shoshone Mountain, Fortymile Canyon, and Yucca Mountain but also moved to Bare Mountain, Thirsty Canyon, and Black Mountain. Females greatly expanded their core and overall home ranges during spring, whereas males expanded their home ranges during summer. Of 18 sheep sampled for disease, 12 showed an immune response to Mycoplasma ovipneumoniae, and 5 had the bacteria present. Genetic testing revealed that the ancestry of NNSS sheep is from the Bare Mountain (1991-1995, within 24 km of our study area), Specter Range (1990-1995, within 32 km of our study area), and Stonewall Mountain (1975-1983, within 72 km of our study area) reintroduced populations. Radionuclide burden in NNSS sheep was minimal with no significant difference from sheep captured on the Nevada Test and Training Range and northern Nevada. One marked adult male was legally harvested off the NNSS north of Bare Mountain. Finally, this recently colonized reproducing population of sheep on the NNSS warrants further monitoring, protection, and inclusion in resource management plans.

60 APPLIED LIFE SCIENCES↗

Functional insights of novel Bathyarchaeia reveal metabolic versatility in their role in peatlands of the Peruvian Amazon

ABSTRACT The decomposition of soil organic carbon within tropical peatlands is influenced by the functional composition of the microbial community. In this study, building upon our previous work, we recovered a total of 28 metagenome-assembled genomes (MAGs) classified as Bathyarchaeia from the tropical peatlands of the Pastaza-Marañón Foreland Basin (PMFB) in the Amazon. Using phylogenomic analyses, we identified nine genus-level clades to have representatives from the PMFB, with four forming a putative novel family (“CandidatusPaludivitaceae”) endemic to peatlands. We focus on theCa. Paludivitaceae MAGs due to the novelty of this group and the limited understanding of their role within tropical peatlands. Functional analysis of these MAGs reveals that this putative family comprises facultative anaerobes, possessing the genetic potential for oxygen, sulfide, or nitrogen oxidation. This metabolic versatility can be coupled to the fermentation of acetoin, propanol, or proline. The other clades outsideCa. Paludivitaceae are putatively capable of acetogenesis andde novoamino acid biosynthesis and encode a high amount of Fe 3+ transporters. Crucially, theCa. Paludivitaceae are predicted to be carboxydotrophic, capable of utilizing CO for energy generation or biomass production. Through this metabolism, they could detoxify the environment from CO, a byproduct of methanogenesis, or produce methanogenic substrates like CO 2 and H 2 . Overall, our results show the complex metabolism and various lineages of Bathyarchaeia within tropical peatlands pointing to the need to further evaluate their role in these ecosystems. IMPORTANCE With the expansion of theCandidatusPaludivitaceae family by the assembly of 28 new metagenome assembled genomes, this study provides novel insights into their metabolic diversity and ecological significance in peatland ecosystems. From a comprehensive phylogenic and functional analysis, we have elucidated their putative unique facultative anaerobic capabilities and CO detoxification potential. This research highlights their crucial role in carbon cycling and greenhouse gas regulation. These findings are essential for resolving the microbial processes affecting peat soil stability, offering new perspectives on the ecological roles of previously underexplored and underrepresented archaeal populations.

Microbiology↗

Growth, total lipid, and omega-3 fatty acid production by Nannochloropsis spp. cultivated with raw plant substrate

Improving productivity and lipid concentration in microalgae is important for the economic success of both biofuel and microalgae coproducts production. Nannochloropsis spp. are marine microalgae currently being grown at large-scale for the production of biofuel and lipid coproducts. In this work, we demonstrate improvements of growth and omega-3 production in Nannochloropsis gaditana CCMP526 and Nannochloropsis oceanica CCAP849/10 with plant substrate addition, a potentially economical option for increasing microalgal productivity. Furthermore, we examine growth in the presence of corn stover, switchgrass, sugarcane bagasse, and yard waste. By examining the microbial ecology of N. gaditana cultures with and without plant substrate and with and without antibiotics, we discovered a potential bacterial interaction in these cultures, but its presence is not necessary for algal growth improvements in the presence of plant substrate. Analysis of plant substrate morphology by scanning electron microscopy (SEM) after cultivation in media with and without N. gaditana shows a degradation of specific plant structural features and a colonization of the plant phloem by N. gaditana. An examination of N. gaditana in a Congo red plate assay indicates potential cellulolytic activity, but a preliminary examination of the potential cellulase transcripts does not reveal differential expression of a candidate in the presence of plant. This evidence demonstrates potential raw plant utilization by a marine microalga for increased productivity and provides a potentially economical option for increasing coproduct concentration at industrial scale without genetic engineering.

59 BASIC BIOLOGICAL SCIENCES↗

Genotype–environment associations across spatial scales reveal the importance of putative adaptive genetic variation in divergence

Abstract Identifying areas of high evolutionary potential is a judicious strategy for developing conservation priorities in the face of environmental change. For wide‐ranging species occupying heterogeneous environments, the evolutionary forces that shape distinct populations can vary spatially. Here, we investigate patterns of genomic variation and genotype–environment associations in the hermit thrush ( Catharus guttatus ), a North American songbird, at broad (across the breeding range) and narrow spatial scales (at a hybrid zone). We begin by building a genoscape or map of genetic variation across the breeding range and find five distinct genetic clusters within the species, with the greatest variation occurring in the western portion of the range. Genotype–environment association analyses indicate higher allelic turnover in the west than in the east, with measures of temperature surfacing as key predictors of putative adaptive genomic variation rangewide. Since broad patterns detected across a species' range represent the aggregate of many locally adapted populations, we investigate whether our broadscale analysis is consistent with a finer scale analysis. We find that top rangewide temperature‐associated loci vary in their clinal patterns (e.g., steep clines vs. fixed allele frequencies) across a hybrid zone in British Columbia, suggesting that the environmental predictors and the associated candidate loci identified in the rangewide analysis are of variable importance in this particular region. However, two candidate loci exhibit strong concordance with the temperature gradient in British Columbia, suggesting a potential role for temperature‐related barriers to gene flow and/or temperature‐driven ecological selection in maintaining putative local adaptation. This study demonstrates how patterns identified at the broad (macrogeographic) scale can be validated by investigating genotype–environment correlations at the local (microgeographic) scale. Furthermore, our results highlight the importance of considering the spatial distribution of putative adaptive variation when assessing population‐level sensitivity to climate change and other stressors.

59 BASIC BIOLOGICAL SCIENCES↗

Genetic learning in rule-based and neural systems

The design of neural networks and fuzzy systems can involve complex, nonlinear, and ill-conditioned optimization problems. Often, traditional optimization schemes are inadequate or inapplicable for such tasks. Genetic Algorithms (GA's) are a class of optimization procedures whose mechanics are based on those of natural genetics. Mathematical arguments show how GAs bring substantial computational leverage to search problems, without requiring the mathematical characteristics often necessary for traditional optimization schemes (e.g., modality, continuity, availability of derivative information, etc.). GA's have proven effective in a variety of search tasks that arise in neural networks and fuzzy systems. This presentation begins by introducing the mechanism and theoretical underpinnings of GA's. GA's are then related to a class of rule-based machine learning systems called learning classifier systems (LCS's). An LCS implements a low-level production-system that uses a GA as its primary rule discovery mechanism. This presentation illustrates how, despite its rule-based framework, an LCS can be thought of as a competitive neural network. Neural network simulator code for an LCS is presented. In this context, the GA is doing more than optimizing and objective function. It is searching for an ecology of hidden nodes with limited connectivity. The GA attempts to evolve this ecology such that effective neural network performance results. The GA is particularly well adapted to this task, given its naturally-inspired basis. The LCS/neural network analogy extends itself to other, more traditional neural networks. Conclusions to the presentation discuss the implications of using GA's in ecological search problems that arise in neural and fuzzy systems.

Smith, Robert E.↗

Characterizing the gene–environment interaction underlying natural morphological variation in Neurospora crassa conidiophores using high-throughput phenomics and transcriptomics

Abstract Neurospora crassa propagates through dissemination of conidia, which develop through specialized structures called conidiophores. Recent work has identified striking variation in conidiophore morphology, using a wild population collection from Louisiana, United States of America to classify 3 distinct phenotypes: Wild-Type, Wrap, and Bulky. Little is known about the impact of these phenotypes on sporulation or germination later in the N. crassa life cycle, or about the genetic variation that underlies them. In this study, we show that conidiophore morphology likely affects colonization capacity of wild N. crassa isolates through both sporulation distance and germination on different carbon sources. We generated and crossed homokaryotic strains belonging to each phenotypic group to more robustly fit a model for and estimate heritability of the complex trait, conidiophore architecture. Our fitted model suggests at least 3 genes and 2 epistatic interactions contribute to conidiophore phenotype, which has an estimated heritability of 0.47. To uncover genes contributing to these phenotypes, we performed RNA-sequencing on mycelia and conidiophores of strains representing each of the 3 phenotypes. Our results show that the Bulky strain had a distinct transcriptional profile from that of Wild-Type and Wrap, exhibiting differential expression patterns in clock-controlled genes (ccgs), the conidiation-specific gene con-6, and genes implicated in metabolism and communication. Combined, these results present novel ecological impacts of and differential gene expression underlying natural conidiophore morphological variation, a complex trait that has not yet been thoroughly explored.

59 BASIC BIOLOGICAL SCIENCES↗

Exploring phage–host interactions in Burkholderia cepacia complex bacterium to reveal host factors and phage resistance genes using CRISPRi functional genomics and transcriptomics

Complex interactions of bacteriophages with their bacterial hosts determine phage host range and infectivity. While phage defense systems and host factors have been identified in model bacteria, they remain challenging to predict in non-model bacteria. In this paper, we integrate functional genomics and transcriptomics to investigate phage–host interactions, revealing active phage resistance and host factor genes in Burkholderia cenocepacia K56-2. Burkholderia cepacia complex species are commonly found in soil and are opportunistic pathogens in immunocompromised patients. We studied infection of B. cenocepacia K56-2 with Bcep176, a temperate phage isolated from Burkholderia multivorans. A genome-wide dCas9 knockdown library targeting B. cenocepacia K56-2 was constructed, and a pooled infection experiment identified 63 novel genes or operons coding for candidate host factors or phage resistance genes. The activities of a subset of candidate host factor and resistance genes were validated via single-gene knockdowns. Transcriptomics of B. cenocepacia K56-2 during Bcep176 infection revealed that expression of genes coding for host factor and resistance candidates identified in this screen was significantly altered during infection by 4 h post-infection. Identifying which bacterial genes are involved in phage infection is important to understand the ecological niches of B. cenocepacia and its phages, and for designing phage therapies.

Bacterial Pathogenesis↗

Global Archaeal Diversity Revealed Through Massive Data Integration: Uncovering Just Tip of Iceberg

The domain of Archaea has gathered significant interest for its ecological and biotechnological potential and its role in helping us to understand the evolutionary history of Eukaryotes. In comparison to the bacterial domain, the number of adequately described members in Archaea is relatively low, with less than 1000 species described. It is not clear whether this is solely due to the cultivation difficulty of its members or, indeed, the domain is characterized by evolutionary constraints that keep the number of species relatively low. Based on molecular evidence that bypasses the difficulties of formal cultivation and characterization, several novel clades have been proposed, enabling insights into their metabolism and physiology. Given the extent of global sampling and sequencing efforts, it is now possible and meaningful to question the magnitude of global archaeal diversity based on molecular evidence. To do so, we extracted all sequences classified as Archaea from 500 thousand amplicon samples available in public repositories. After processing through our highly conservative pipeline, we named this comprehensive resource the ‘Global Archaea Diversity’ (GAD), which encompassed nearly 3 million molecular species clusters at 97% similarity, and organized it into over 500 thousand genera and nearly 100 thousand families. Saline environments have contributed the most to the novel taxa of this previously unseen diversity. The majority of those 16S rRNA gene sequence fragments were verified by matches in metagenomic datasets from IMG/M. These findings reveal a vast and previously overlooked diversity within the Archaea, offering insights into their ecological roles and evolutionary importance while establishing a foundation for the future study and characterization of this intriguing domain of life.

59 BASIC BIOLOGICAL SCIENCES↗