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Despite the importance of soil microorganisms for ecosystem services, long‐term surveys of their communities are largely missing. Using metabarcoding, we assessed temporal dynamics of soil bacterial and fungal communities in three land‐use types, i.e., arable land, permanent grassland, and forest, over five years. Soil microbial communities remained relatively stable and differences over time were smaller than those among sites. Temporal variability was highest in arable soils. Indications for consistent shifts in community structure over five years were only detected at one site for bacteria and at two sites for fungi, which provided further support for long‐term stability of soil microbial communities. A sliding window analysis was applied to assess the effect of OTU abundance on community structures. Partial communities with decreasing OTU abundances revealed a gradually decreasing structural similarity with entire communities. This contrasted with the steep decline of OTU abundances, as subsets of rare OTUs (

28 páginas, 6 figuras. -- This is a post-peer-review, pre-copyedit version of an article published in Microbial Ecology. The final authenticated version is available online at: http://dx.doi.org/10.1007/s00248-021-01785-w, Precipitation is one of the major constraints influencing the diversity, structure, and activity of soil microbial communities in desert ecosystems. However, the effect of changes in precipitation on soil microbial communities in arid soil microbiomes remains unresolved. In this study, using 16S rRNA gene high-throughput sequencing and shotgun metagenome sequencing, we explored changes in taxonomic composition and functional potential across two zones in the Namib Desert with contrasting precipitation regime. We found that precipitation regime had no effect on taxonomic and functional alpha-diversity, but that microbial community composition and functional potential (beta-diversity) changed with increased precipitation. For instance, Acidobacteriota and ‘resistance to antibiotics and toxic compounds’ related genes were relatively more abundant in the high-rainfall zone. These changes were largely due to a small set of microbial taxa, some of which were present in low abundance (i.e. members of the rare biosphere). Overall, these results indicate that key climatic factors (i.e. precipitation) shape the taxonomic and functional attributes of the arid soil microbiome. This research provides insight into how changes in precipitation patterns associated with global climate change may impact microbial community structure and function in desert soils., YN was supported through a Free standing and Research and Development Programme Grant funded by the National Research Foundation (NRF) of South Africa. AV was supported by the project ‘CLU-2019–05 – IRNASA/CSIC Unit of Excellence’, funded by the Junta de Castilla y León and co-financed by the European Union (ERDF ‘Europe drives our growth’).

Microbial abundance and community composition in marine sediments have been widely explored. However, high‐resolution vertical changes of benthic microbial diversity and co‐occurrence patterns are poorly described. The ecological contributions of abundant and rare species in sediments also remain largely unknown. Here, by analysing microbial populations at 14 depth layers of 10 subseafloor sediment cores (water depth 1,250–3,530 m) obtained in the South China Sea, we provided the vertical profiles of microbial β‐diversity and co‐occurrence influenced by subcommunities of different abundance. These 134 sediment samples were clustered into four groups according to sediment depth (1–2, 6–10, 30–90 and 190–790 cm) with obvious shifts in microbial community compositions. The vertical succession of microorganisms was consistent with redox zonation and influenced by terrestrial inputs. Partitioning of vertical β‐diversity showed extremely high species replacement between deep layers and the surface layer, indicating selection‐induced loss of rare species and dispersal of dormant cells and spores. By contrast, for horizontal β‐diversity, richness of rare species became increasingly significant in deep sediments. Accompanying this β‐diversity profile were clear changes in the association pattern, with microorganisms being less connected in deeper sediment layers, probably reflecting reduced syntrophic interactions. Rare species accounted for an indispensable proportion in the co‐occurrence network, and tended to form complex “small worlds.” The rare subcommunity also responded differently to various environmental factors compared with the abundant subcommunity. Our findings expand current knowledge on vertical changes of marine benthic microbial diversity and their association patterns, emphasizing the potential roles of rare species.

13 pages, 5 figures, supporting information https://doi.org/10.1021/acs.est.0c07262, Coastal seawaters receive thousands of organic pollutants. However, we have little understanding of the response of microbiomes to this pool of anthropogenic dissolved organic carbon (ADOC). In this study, coastal microbial communities were challenged with ADOC at environmentally relevant concentrations. Experiments were performed at two Mediterranean sites with different impact by pollutants and nutrients: off the Barcelona harbor (“BCN”), and at the Blanes Bay (“BL”). ADOC additions stimulated prokaryotic leucine incorporation rates at both sites, indicating the use of ADOC as growth substrate. The percentage of “membrane-compromised” cells increased with increasing ADOC, indicating concurrent toxic effects of ADOC. Metagenomic analysis of the BCN community challenged with ADOC showed a significant growth of Methylophaga and other gammaproteobacterial taxa belonging to the rare biosphere. Gene expression profiles showed a taxon-dependent response, with significantly enrichments of transcripts from SAR11 and Glaciecola spp. in BCN and BL, respectively. Further, the relative abundance of transposon-related genes (in BCN) and transcripts (in BL) correlated with the number of differentially abundant genes (in BCN) and transcripts (in BLA), suggesting that microbial responses to pollution may be related to pre-exposure to pollutants, with transposons playing a role in adaptation to ADOC. Our results point to a taxon-specific response to low concentrations of ADOC that impact the functionality, structure and plasticity of the communities in coastal seawaters. This work contributes to address the influence of pollutants on microbiomes and their perturbation to ecosystem services and ocean health., This work was supported by the Spanish Ministry of Economy and Competitiveness (MEIC) through projects ISOMICS (CTM2015-65691-R) and REMARCA (CTM2012-34673). E.C.G. was supported by Agència de Gestió d’Ajuts Universtaris i Recerca, Generalitatde Catalunya (FI AGAUR) Scholarship, Generalitat de Catalunya Fellowship Program (Catalan Government, Generalitat de Catalunya) and by a “FORD ESPAÑA-APADRINA LA CIENCIA” contract. The research group of Global Change and Genomic Biogeochemistry is supported by the Catalan Government (2017SGR800). We thank Dr. J. M. Gasol for support with flow cytometry counts. Inorganic nutrient analyses were performed by M. I. Abad in coordination with Dr. E. Berdalet (Head of the Service) at the “Nutrient Analysis Service of the ICM-CSIC. Dr Piña is acknowledged for fruitful discussions., With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI).

The microbial H2-producing (hydrogenogenic) carbon monoxide (CO)-oxidizing activity by the membrane-associated CO dehydrogenase (CODH)/energy-converting hydrogenase (ECH) complex is an important metabolic process in the microbial community. However, the studies on hydrogenogenic carboxydotrophs had to rely on inherently cultivation and isolation methods due to their rare abundance, which was a bottleneck in ecological study. Here, we provided gene-targeted sequencing method for the diversity estimation of thermophilic hydrogenogenic carboxydotrophs. We designed six new degenerate primer pairs which effectively amplified the coding regions of CODH genes forming gene clusters with ECH genes (CODHech genes) in Firmicutes which includes major thermophilic hydrogenogenic carboxydotrophs in terrestrial thermal habitats. Amplicon sequencing by these primers using DNAs from terrestrial hydrothermal sediments and CO-gas-incubated samples specifically detected multiple CODH genes which were identical or phylogenetically related to the CODHech genes in Firmictes. Furthermore, we found that phylogenetically distinct CODHech genes were enriched in CO-gas-incubated samples, suggesting that our primers detected uncultured hydrogenogenic carboxydotrophs as well. The new CODH-targeted primers provided us with a fine-grained (~ 97.9% in nucleotide sequence identity) diversity analysis of thermophilic hydrogenogenic carboxydotrophs by amplicon sequencing and will bolster the ecological study of these microorganisms. Supplementary Information The online version contains supplementary material available at 10.1007/s00792-020-01211-y.

Microbial eukaryotes (protists) contribute substantially to ecological functioning in marine ecosystems, but the relative importance of factors shaping protist diversity, such as environmental selection and dispersal, remains difficult to parse. Water masses of a back-arc basin with hydrothermal activity provide a unique opportunity for studying the effects of dispersal and environmental selection on protist communities. In this study, we used metabarcoding to characterize protist communities in the Okinawa Trough, a back-arc spreading basin containing at least twenty-five active hydrothermal vent fields. Water was sampled from four depths at fourteen stations spanning the length of the Okinawa Trough, including three sites influenced by nearby hydrothermal vent sites. While significant differences in community structure reflecting water depth were present, protist communities were mostly homogeneous horizontally. Protist communities in the bottom waters affected by hydrothermal activity were significantly different from communities in other bottom waters, suggesting that environmental factors can be especially important in shaping community composition under specific conditions. Amplicon sequence variants that were enriched in hydrothermally influenced bottom waters largely derived from cosmopolitan protists that were present, but rare, in other near-bottom samples, thus highlighting the importance of the rare biosphere.

Background: Bacteria in low abundance represent the majority of Earth’s biodiversity and perform vital ecological functions, but little is known about their biogeography nor the ecological processes that drive their community assembly in terrestrial ecosystems. Here, we investigated the community compositions and phylogenies of rare (relative abundance < 0.1%) and abundant (> 1%) soil bacteria along a transect containing three alpine grassland types (meadow, steppe, and desert) on the Tibetan Plateau. Results: Our results revealed similar biogeographical patterns of rare and abundant bacteria, with their community compositions and phylogenies shifting gradually along the transect. The similar patterns, however, were driven by contrasting community assembly processes, with rare subcommunity being more heavily influenced by stochasticity (72%) than abundant (57%). The composition of abundant subcommunity (80%) was better explained by local (including soil and vegetation factors), geospatial and climatic factors than that of rare subcommunity (41%), while the phylogeny of the rare one (36%) was better explained than that of the abundant one (29%). Variation partitioning analysis indicated that pure local factors consistently explained a higher proportion of the community composition than geospatial factors in both rare (12.3% and 8.7%, respectively) and abundant bacteria (18.3% and 14.1%, respectively). In contrast, the phylogeny of rare subcommunity was explained by local and geospatial factors equally (11.5% and 11.9%, respectively), while that of abundant subcommunity was more explained by geospatial (22.1%) than local factors (11.3%). Furthermore, our results revealed a tighter connection between the community phylogeny and composition in rare than in abundant bacteria. Conclusions: Our results revealed consistent biogeographical patterns of rare and abundant bacteria in grassland soils, but their assembly processes were distinct. We further demonstrated that rare subcommunity was less predictable than the abundant subcommunity by environmental and geospatial factors. Rare and abundant bacteria responded differentially to factors, which was attributed to the distinct life strategies. Our study provides novel insights into the assembly processes and biographical patterns of rare and abundant bacteria in terrestrial ecosystems.

Microbial communities are frequently numerically dominated by just a few species. Often, the long “tail” of the rank-abundance plots of microbial communities constitutes the so-called “rare biosphere,” microorganisms that are highly diverse but are typically found in low abundance in these communities. Their presence in microbial communities has only recently become apparent with advances in high-throughput sequencing technologies. Despite their low numbers, they are thought to play important roles in their communities and may function as potential members to keep the communities intact and resilient. Their phylogenetic diversity also means that they are important subjects for better understanding the interplay between microbial diversity and evolution. I propose that more efforts should be put into characterizing these poorly understood and mostly unknown microbial lineages that hold vast potentials for our understanding of microbial diversity, ecology, and evolution of life on this planet.

Candidate Phyla Radiation (CPR) bacteria and nanoarchaea populate most ecosystems but are rarely detected in soil. We concentrated particles of less than 0.2 μm in size from grassland soil, enabling targeted metagenomic analysis of these organisms, which are almost totally unexplored in largely oxic environments such as soil. We recovered a diversity of CPR bacterial and some archaeal sequences but no sequences from other cellular organisms. The sampled sequences include Doudnabacteria (SM2F11) and Pacearchaeota, organisms rarely reported in soil, as well as Saccharibacteria, Parcubacteria, and Microgenomates. CPR and archaea of the phyla Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, and Nanohaloarchaeota (DPANN) were enriched 100- to 1,000-fold compared to that in bulk soil, in which we estimate each of these organisms comprises approximately 1 to 100 cells per gram of soil. Like most CPR and DPANN sequenced to date, we predict these microorganisms live symbiotic anaerobic lifestyles. However, Saccharibacteria, Parcubacteria, and Doudnabacteria genomes sampled here also harbor ubiquinol oxidase operons that may have been acquired from other bacteria, likely during adaptation to aerobic soil environments. We conclude that CPR bacteria and DPANN archaea are part of the rare soil biosphere and harbor unique metabolic platforms that potentially evolved to live symbiotically under relatively oxic conditions. IMPORTANCE Here, we investigated overlooked microbes in soil, Candidate Phyla Radiation (CPR) bacteria and Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, and Nanohaloarchaeota (DPANN) archaea, by size fractionating small particles from soil, an approach typically used for the recovery of viral metagenomes. Concentration of these small cells (

Although previous research demonstrates that skin-associated archaea are rarely detected within human skin microbiome data, exist at relatively low abundance, and are primarily affiliated with the Methanobacteriota and Halobacteriota phyla, other studies suggest that archaea are consistently detected and relatively abundant on human skin, with skin “archaeomes” dominated by putative ammonia oxidizers of the Nitrososphaeria class (Thermoproteota phylum, formerly Thaumarchaeota). Here, we evaluated new and existing 16S rRNA gene sequence data sourced from mammalian skin and skin-associated surfaces and generated with two commonly used universal prokaryotic primer sets to assess archaeal prevalence, relative abundance, and taxonomic distribution. Archaeal 16S rRNA gene sequences were detected in only 17.5% of 1,688 samples by high-throughput sequence data, with most of the archaeon-positive samples associated with nonhuman mammalian skin. Only 5.9% of human-associated skin sample data sets contained sequences affiliated with archaeal 16S rRNA genes. When detected, the relative abundance of sequences affiliated with archaeal amplicon sequence variants (ASVs) was less than 1% for most mammalian skin samples and did not exceed 2% for any samples. Although several computer keyboard microbial profiles were dominated by Nitrososphaeria sequences, all other skin microbiome data sets tested were primarily composed of sequences affiliated with Methanobacteriota and Halobacteriota phyla. Our findings revise downward recent estimates of human skin archaeal distributions and relative abundances, especially those affiliated with the Nitrososphaeria, reflecting a limited and infrequent archaeal presence within the mammalian skin microbiome. IMPORTANCE The current state of research on mammalian skin-associated archaea is limited, with the few papers focusing on potential skin archaeal communities often in disagreement with each other. As such, there is no consensus on the prevalence or taxonomic composition of archaea on mammalian skin. Mammalian skin health is in part influenced by its complex microbiota and consortium of bacteria and potential archaea. Without a clear foundational analysis and characterization of the mammalian skin archaeome, it will be difficult for future research to explore the potential impact of skin-associated archaea on skin health and function. The current work provides a much-needed analysis of the mammalian skin archaeome and contributes to building a foundation from which further discussion and exploration of the skin archaeome might continue.

Assigning a functional role to a microorganism has historically relied on cultivation of isolates or detection of environmental genome-based biomarkers using a posteriori knowledge of function. However, the emerging field of function-driven single-cell genomics aims to expand this paradigm by identifying and capturing individual microbes based on their in situ functions or traits. To identify and characterize yet uncultivated microbial taxa involved in cellulose degradation, we developed and benchmarked a function-driven single-cell screen, which we applied to a microbial community inhabiting the Great Boiling Spring (GBS) Geothermal Field, northwest Nevada. Our approach involved recruiting microbes to fluorescently labeled cellulose particles, and then isolating single microbe-bound particles via fluorescence-activated cell sorting. The microbial community profiles prior to sorting were determined via bulk sample 16S rRNA gene amplicon sequencing. The flow-sorted cellulose-bound microbes were subjected to whole genome amplification and shotgun sequencing, followed by phylogenetic placement. Next, putative cellulase genes were identified, expressed and tested for activity against derivatives of cellulose and xylose. Alongside typical cellulose degraders, including members of the Actinobacteria, Bacteroidetes, and Chloroflexi, we found divergent cellulases encoded in the genome of a recently described candidate phylum from the rare biosphere, Goldbacteria, and validated their cellulase activity. As this genome represents a species-level organism with novel and phylogenetically distinct cellulolytic activity, we propose the name Candidatus ‘Cellulosimonas argentiregionis’. We expect that this function-driven single-cell approach can be extended to a broad range of substrates, linking microbial taxonomy directly to in situ function.

The release of Escherichia coli (E. coli) O157:H7 has been widely found in various environments, but little is known about the probable influence of the transient E. coli O157:H7 invasion on the native microbial community. Here, we investigated the temporal response of two bacterial biospheres (abundant and rare) of two marsh sediments against E. coli O157:H7 during a 60-day incubation. The diversity of both biospheres showed no evident response to O157:H7 invasion. Temporal factor exhibited greater effects on bacterial variation than O157:H7 invasion. We found that O157:H7 invasion led to an increase in the niche breadth of the bacterial community while decreasing the efficiency of bacterial interaction of the abundant taxa. Moreover, the rare biosphere exhibited enhanced stability against O157:H7 invasion compared with the abundant biosphere, acting as the backbone in resisting external disturbance. Furthermore, each subcommunity assembly showed different randomness levels. The stochastic events were relatively more important in constraining the abundant taxa assembly after invasion. Collectively, E. coli O157:H7 exhibited diverse tangible impact on both biospheres, which unearthed differential responses of abundant and rare biosphere against transient microbial invasion.

International audience; Free-living (FL) marine microbial communities differ from those attached to particles (PA). Likewise, biofilms (B) colonizing artificial surfaces, including plastics or ship hulls, hardly resemble their planktonic surroundings. However, few studies have examined the effect of the environment on these lifestyles and on the source of organisms colonizing marine surfaces. Using 16S rRNA gene metabarcoding, we identified specificities of marine prokaryotic community lifestyles (FL, PA or B) sampled in three coastal polluted locations with dissimilar environmental conditions: the North-Western Mediterranean Sea and the Atlantic and Indian Oceans. Biofilms developed over polyvinyl chloride (PVC) were found to be significantly different from FL or PA collected during the immersions. Alpha-diversity increased from FL to PA and to B, illustrating the integrative aspect of the latter, with little proportion of operational taxonomic units shared with the first two. Beta-diversity clustered first the lifestyles and then the sites. FL and PA were more affected by water quality, especially by trace metal contamination, whereas B were as sensitive to trace metals as to nutrients. Although biofilms should be supplied by the planktonic (ultra) rare biosphere, source tracking could only detect small contributions of FL or PA taxa to B communities.

The Arctic Ocean is facing rapid environmental changes with cascading effects on the entire Arctic marine ecosystem. However, we have a limited understanding of the consequences such changes have on bacteria and archaea (prokaryotes) at the base of the marine food web. In this study, we show how the prokaryotic rare biosphere behaves over a range of highly heterogeneous environmental conditions using 16S rRNA gene reads from amplicon and metagenome sequencing data from seawater samples collected during the Norwegian young sea ICE expedition between late winter and early summer. The prokaryotic rare biosphere was analyzed using different approaches: amplicon sequence variants and operational taxonomic units from the 16S rRNA gene amplicons and operational taxonomic units from the 16S rRNA genes of the metagenomes. We found that prokaryotic rare biosphere communities are specific to certain water masses, and that the majority of the rare taxa identified were always rare and disappeared in at least one sample under changing conditions, suggesting their high sensitivity to environmental heterogeneity. In addition, our methodological comparison revealed a good performance of 16S rRNA gene amplicon sequencing in describing rare biosphere patterns, while the metagenome-derived data were better to capture a significant diversity of so-far uncultivated rare taxa. Our analysis on the dynamics of the rare prokaryotic biosphere, by combining different methodological approaches, improves the description of the types of rarity predicted from Community Assembly theory in the Arctic Ocean. UIDB/04423/2020; UIDP/04565/2020; Programa Operacional Regional de Lisboa 007317 info:eu-repo/semantics/publishedVersion

Selenate (SeO4 2- ) reduction in hydrogen (H2 )-fed membrane biofilm reactors (H2 -MBfRs) was studied in combinations with other common electron acceptors. We employed H2 -MBfRs with two distinctly different conditions: R1, with ample electron-donor availability and acceptors SeO4 2- and sulfate (SO4 2- ), and R2, with electron-donor limitation and the presence of electron acceptors SeO4 2- , nitrate (NO3 - ), and SO4 2- . Even though H2 was available to reduce all input SeO4 2- and SO4 2- in R1, SeO4 2- reduction was preferred over SO4 2- reduction. In R2, co-reduction of NO3 - and SeO4 2- occurred, and SO4 2- reduction was mostly suppressed. Biofilms in all MBfRs had high microbial diversity that was influenced by the "rare biosphere" (RB), phylotypes with relative abundance less than 1%. While all MBfR biofilms had abundant members, such as Dechloromonas and Methyloversatilis, the bacterial communities were significantly different between R1 and R2. For R1, abundant genera were Methyloversatilis, Melioribacter, and Propionivibrio; for R2, abundant genera were Dechloromonas, Hydrogenophaga, Cystobacter, Methyloversatilis, and Thauera. Although changes in electron-acceptor or -donor loading altered the phylogenetic structure of the microbial communities, the biofilm communities were resilient in terms of SeO4 2- and NO3 - reductions, because interacting members of the RB had capacity of respiring these electron acceptors. This article is protected by copyright. All rights reserved.

Microbial biodiversity is represented by a variety of genomic landscapes adapted to dissimilar environments on Earth. These genomic landscapes contain functional signatures connected with the community phenotypes. Here, we assess the genomic microbial diversity landscape at a high-resolution level of a polluted river–associated microbiome (Morelos, Mexico), cultured in a medium enriched with anthraquinone Deep Blue 35 dye. We explore the resultant textile dye microbiome to infer links between predicted biodegradative functions, and metagenomic and metabolic potential, especially using the information obtained from individual reconstructed genomes. By using Hi-C proximity-ligation deconvolution method, we deconvoluted 97 genome composites (80% potentially novel species). The main taxonomic determinants were Methanobacterium, Clostridium, and Cupriavidus genera constituting 50, 22, and 11% of the total community profile. Also, we observed a rare biosphere of novel taxa without clear taxonomic standing. Removal of 50% chemical oxygen demand with 23% decolorization was observed after 30 days of dye enrichment. Genes related to catalase-peroxidase, polyphenol oxidase, and laccase enzymes were predicted as associated with textile dye biodegradation phenotype under our study conditions, highlighting the potential of metagenome-wide analysis to predict biodegradative determinants. This study prompts high-resolution screening of individual genomes within textile dye river sediment microbiomes or complex communities under environmental pressures.

The misuse of antibiotics has led several countries to ban their use as prophylactics against bacterial diseases or as growth promoters in livestock and poultry. Phytobiotics (bioactive compounds extracted from plants) are one of the alternatives, due to their antimicrobial activity and its modulation of the gut microbiota and the improvement of productive properties. Garlic and onion extracts, rich in antimicrobial compounds, are of the most promising alternative to antibiotics. We supplemented a garlic- and onion-based product in the diet to laying hens at the beginning of their productive life. The group supplied with this product produced in one month more eggs and with bigger size. This increase in production was accompanied by changes in the bacterial community of the gut. These changes in the microbiota suggest an improvement in food digestibility, as the most important changes produced by these compounds occur in the most distal parts of the gut. The relative abundance of beneficial Lactococcus in the ileum and Lactobacillus in the cecum increased in the experimental group. Both genera are known to have beneficial effects on host. These results are very promising for the use of these compounds in poultry for short periods., Phytobiotics (bioactive compounds extracted from plants) are one of the explored alternatives to antibiotics in poultry and livestock due to their antimicrobial activity and its positive effects on gut microbiota and productive properties. In this study, we supplemented a product based on garlic and onion compounds in the diet to laying hens at the beginning of their productive life (from 16 to 20 weeks post-hatching). The experimental group showed a significant increase in the number of eggs laid and in their size, produced in one month compared to the control. This increase in production was accompanied by microbiota changes in the ileum and cecum by means of high throughput sequencing analyses. These bacterial shifts in the ileum were mainly the result of compositional changes in the rare biosphere (unweighted UniFrac), while in the cecum, treatment affected both majority and minority bacterial groups (weighted and unweighted UniFrac). These changes in the microbiota suggest an improvement in food digestibility. The relative abundance of Lactococcus in the ileum and Lactobacillus in the cecum increased significantly in the experimental group. The relative abundance of these bacterial genera are known to have positive effects on th, Spanish Ministry of Science and Innovation, Consejería de Economía, Innovación, Ciencia y Empleo (Junta de Andalucía), University of Granada-CEI BioTic (Project No. P-BS-37), INTERCONECTA program (Spanish Ministry of Economy and Competitiveness)

Single cell genomics (SCG) can provide reliable context for assembled genome fragments on the level of individual prokaryotic genomes and has rapidly emerged as an essential complement to cultivation-based and metagenomics research approaches. Targeted cell sorting approaches, which enable the selection of specific taxa by fluorescent labeling, compatible with subsequent single cell genomics offers an opportunity to access genetic information from rare biosphere members which would have otherwise stayed hidden as microbial dark matter.

International audience; Rare species are crucial components of the highly diverse soil microbial pool and over-proportionally contribute to the soil functions. However, much remains unknown about their assembling rules. The biogeographic patterns and species aggregations of the rare bacterial biosphere were assessed using 140 soil samples from a gradient of 2000 km across the main tea-producing areas in China. About 96% OTUs with ~40% sequences were classified as rare taxa. The rare bacterial communities were significantly affected by geographical regions and showed distance-decay effects, indicating that the rare bacteria are not cosmopolitan, they displayed a pattern of limited dispersal and were restricted to certain sites. Variation partitioning analysis (VPA) revealed that environmental variation and spatial factors explained 12.5% and 6.4%, respectively, of the variance in rare bacterial community. The Mantel and partial Mantel tests also showed that the environmental factors had stronger (~3 times) impacts than spatial factors. The null model showed that deterministic processes contributed more than stochastic processes in rare bacterial assembly (75% vs. 25%). There is likely an enrichment in ecological functions within the rare biosphere, considering this high contribution of deterministic processes in the assembly. In addition, the assembly of rare taxa was found to be mainly driven by soil pH. Overall, this study revealed that rare bacteria were not cosmopolitan, and their assembly was more driven by deterministic processes. These findings provided a new comprehensive understanding of rare bacterial biogeographic patterns and assembly rules.

14 pages, 4 figures, 1 table.-- This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY), To study the response of bacteria to different size-fractions of naturally occurring dissolved organic matter (DOM), a natural prokaryotic community from North Atlantic mesopelagic waters (1000 m depth) was isolated and grown in (i) 0.1-μm filtered seawater (CONTROL), (ii) the low-molecular-weight (1 kDa) and low-molecular-weight DOM fractions (H + L-DOM), to test the potential effect of ultrafiltration on breaking the DOM size continuum. Prokaryotic abundance and leucine incorporation were consistently higher in the H + L-DOM niche than in the L-DOM and CONTROL treatments, suggesting a different interaction with each DOM fraction and the disruption of the structural DOM continuum by ultrafiltration, respectively. Rhodobacterales (Alphaproteobacteria) and Flavobacteriales (Bacteroidetes) were particularly enriched in L-DOM and closely related to the colored DOM (CDOM) fraction, indicating the tight link between these groups and changes in DOM aromaticity. Conversely, some other taxa that were rare or undetectable in the original bacterial community were enriched in the H + L-DOM treatment (e.g., Alteromonadales belonging to Gammaproteobacteria), highlighting the role of the rare biosphere as a seed bank of diversity against ecosystem disturbance. The relationship between the fluorescence of protein-like CDOM and community composition of populations in the H + L-DOM treatment suggested their preference for labile DOM. Conversely, the communities growing on the L-DOM niche were coupled to humic-like CDOM, which may indicate their ability to degrade more reworked DOM and/or the generation of refractory substrates (as by-products of the respiration processes). Most importantly, L- and/or H + L-DOM treatments stimulated the growth of unique bacterial amplicon sequence variants (ASVs), suggesting the potential of environmental selection (i.e., changes in DOM composition and availability), particularly in the light of climate change scenarios. Taken together, our results suggest that different size-fractions of DOM induced niche-specialization and differentiation of mesopelagic bacterial communities, Funding for sampling and analyses was supported by the projects “Biodiversidade Funcional do Microplancton nas profundidades mariñas de Galicia” (BIO-PROF, Ref. 10MMA604024PR, 2010–2012, Xunta de Galicia), “Fuentes de Materia Orgánica y Diversidad Funcional del Microplancton en las aguas profundas del Atlántico Norte” (MODUPLAN, Ref. CTM 2011-24008, 2012–2015, Plan Nacional I + D + I; https://moduplansarmiento2014.wordpress.com/) to MMV, and “Deep Standard Oceanographic Sections monitoring program RADIALES-PRODUNDOS”, funded by the Instituto Español de Oceanografía (IEO). Additional funding was provided by the Axencia Galega de Innovación (GAIN, Xunta de Galicia) through IEO-GAIN Programme Contracts (Contratos Programa). TR-R was supported by MICINN program “Personal Técnico de Apoyo a la Investigación” (PTA, Ref. PTA2015-10948-I). MN-C was funded by the projects HOTMIX and FERMIO (MINECO, CTM2011–30010–C02–MAR and CTM2014-57334-JIN, respectively), both co-financed with FEDER funds. EG-F was supported by the BIO-PROF and MODUPLAN projects

Our ability to describe the highly diverse pool of low abundance populations present in natural microbial communities is increasing at an unprecedented pace. Yet we currently lack an integrative view of the key taxa, functions and metabolic activity which make-up this communal pool, usually referred to as the ‘rare biosphere’, across the domains of life. In this context, this review examines the microbial rare biosphere in its broader sense, providing an historical perspective on representative studies which enabled to bridge the concept from macroecology to microbial ecology. It then addresses our current knowledge of the prokaryotic rare biosphere, and covers emerging insights into the ecology, taxonomy and evolution of low abundance microeukaryotic, viral and host-associated communities. We also review recent methodological advances and provide a synthetic overview on how the rare biosphere fits into different conceptual models used to explain microbial community assembly mechanisms, composition and function.

DNA-based techniques are increasingly used to assess biodiversity both above- and belowground. Most effort has focussed on bioinformatics and sample collection, whereas less is known about the consequences of mixing collected environmental DNA (eDNA), post-extraction and pre-PCR. We applied varying degrees of pooling to stand-alone eDNA samples collected across a non-native plant invasion density gradient, and compared the fungal communities of pooled and unpooled samples. Pooling soil eDNA decreased observable fungal rarefied richness in our samples, led to phylum-specific shifts in proportional abundance, and increased the sensitivity of detection for the invasive plant’s overall impact on fungal diversity. We demonstrate that pooling fungal eDNA could change the outcome of similar eDNA studies where the aim is to: 1) identify the rare biosphere within a soil community, 2) estimate species richness and proportional abundance, or 3) assess the impact of an invasive plant on soil fungi. Sample pooling might be appropriate when determining larger-scale overarching responses of soil communities, as pooling increased the sensitivity of measurable effects of an invasive plant on soil fungal diversity.

Understanding how microbial communities are structured in coral holobionts is important to estimate local and global impacts and provide efficient environment management strategies. Several studies investigated the relationship between corals and their microbial communities, including the environmental drivers of shifts in this relationship, associated with diseases and coral cover loss. However, these studies are often geographically or taxonomically restricted and usually focused on the most abundant microbial groups, neglecting the rare biosphere, including archaea in the group DPANN and the recently discovered bacterial members of the candidate phyla radiation (CPR). Although it is known that rare microbes can play essential roles in several environments, we still lack understanding about which taxa comprise the rare biosphere of corals' microbiome. Here, we investigated the host-related and technical factors influencing coral microbial community structure and the importance of CPR and DPANN in this context by analyzing more than a hundred coral metagenomes from independent studies worldwide. We show that coral genera are the main biotic factor shaping coral microbial communities. We also detected several CPR and DPANN phyla comprising corals' rare biosphere for the first time and showed that they significantly contribute to shaping coral microbial communities.

Unraveling the succession of microbial communities is a core ecological research topic. Yet few studies have focused on how long-term secondary succession affects the functional profiles and ecological processes of abundant and rare microbial subcommunities. Here, we used amplicon sequencing and GeoChip analysis to explore the ecological functions of abundant and rare biospheres and their correlation with soil multinutrient cycling. Samples for this study were collected from a well-established secondary succession chronosequence that spans >30 years of dryland ecosystem development on the Loess Plateau of China. Although both abundant and rare subcommunities shifted with succession, the changing of beta-diversity of the microbial communities was primarily driven by species replacement of the rare biosphere. Phylogenetic changes of abundant and rare taxa were associated with their functional traits, which dominated the diversity-related selection along all succession ages. Neutral theory analysis indicated that the assemblage of abundant taxa over all successional ages was regulated by dispersal homogenizing and ecological drift. The null model revealed that homogeneous and variable selection were the dominant assembly processes for rare subcommunities compared with abundant species. pH and nitrogen content were the paramount drivers determining the assembly of microbial communities and functional genes, consistent with the importance of environmental filtering. Furthermore, the rare biosphere had a paramount role in the entire ecological network and was the major driver for most soil processes such as C, N, and S cycling. Nonetheless, a significant portion of soil P cycling was regulated by abundant taxa. Collectively, our study provides insight into the mechanisms underlying microbial community assembly and soil microbe-driven functional changes in biogeochemical processes during secondary succession.

It is commonly thought that bacterial distributions show lower spatial variation than for multicellular organisms. In this article, we present evidence that these inferences are artifacts caused by methodological limitations. Through leveraging innovations in sampling design, sequence processing, and diversity analysis, we provide multifaceted evidence that bacterial communities in fact exhibit strong distribution patterns. This is driven by selection due to factors such as local soil characteristics. Altogether, these findings suggest that the processes underpinning diversity patterns are more unified across all domains of life than previously thought, which has broad implications for the understanding and management of soil biodiversity., Bacteria have been inferred to exhibit relatively weak biogeographic patterns. To what extent such findings reflect true biological phenomena or methodological artifacts remains unclear. Here, we addressed this question by analyzing the turnover of soil bacterial communities from three data sets. We applied three methodological innovations: (i) design of a hierarchical sampling scheme to disentangle environmental from spatial factors driving turnover; (ii) resolution of 16S rRNA gene amplicon sequence variants to enable higher-resolution community profiling; and (iii) application of the new metric zeta diversity to analyze multisite turnover and drivers. At fine taxonomic resolution, rapid compositional turnover was observed across multiple spatial scales. Turnover was overwhelmingly driven by deterministic processes and influenced by the rare biosphere. The communities also exhibited strong distance decay patterns and taxon-area relationships, with z values within the interquartile range reported for macroorganisms. These biogeographical patterns were weakened upon applying two standard approaches to process community sequencing data: clustering sequences at 97% identity threshold and/or filtering the rare biosphere (sequences lower than 0.05% relative abundance). Comparable findings were made across local, regional, and global data sets and when using shotgun metagenomic markers. Altogether, these findings suggest that bacteria exhibit strong biogeographic patterns, but these signals can be obscured by methodological limitations. We advocate various innovations, including using zeta diversity, to advance the study of microbial biogeography. IMPORTANCE It is commonly thought that bacterial distributions show lower spatial variation than for multicellular organisms. In this article, we present evidence that these inferences are artifacts caused by methodological limitations. Through leveraging innovations in sampling design, sequence processing, and diversity analysis, we provide multifaceted evidence that bacterial communities in fact exhibit strong distribution patterns. This is driven by selection due to factors such as local soil characteristics. Altogether, these findings suggest that the processes underpinning diversity patterns are more unified across all domains of life than previously thought, which has broad implications for the understanding and management of soil biodiversity.

Candidate Phyla Radiation (CPR) bacteria and nanoarchaea populate most ecosystems, but are rarely detected in soil. We concentrated particles less than 0.2 μm from grassland soil, enabling targeted metagenomic analysis of these organisms, which are almost totally unexplored in soil. We recovered a diversity of CPR bacteria and some nanoarchaea sequences, but no sequences from other cellular organisms. The sampled sequences include Doudnabacteria (SM2F11) and Pacearchaeota, organisms not previously reported in soil, as well as Saccharibacteria, Parcubacteria and Microgenomates. CPR and DPANN (an acronym of the names of the first included archaea phyla) enrichments of 100-1000-fold were achieved compared to bulk soil, in which we estimate these organisms comprise about 1 to 100 cells per gram of soil. Like most CPR and DPANN sequenced to date, we predict these microorganisms live symbiotic, anaerobic lifestyles. However, Saccharibacteria, Parcubacteria, and Doudnabacteria genomes sampled here also encode ubiquinol oxidase operons that may have been acquired from other bacteria, likely during adaptation to aerobic soil environments. We posit that although present at low abundance, CPR bacteria and DPANN archaea could impact overall soil microbial community function by modulating host organism abundances and activity.

Understanding how species interaction and assembly processes structure the abundant and rare bacterial biospheres in soils is crucial for predicting how biodiversity influences ecosystem functioning. Here, we profiled the bacterial communities across a salt marsh ecosystem gradient to investigate the co-occurrence patterns across taxa and the relative influence of ecological processes mediating the assembly of the abundant and rare biospheres in soil. Our results revealed abundant taxa to be ubiquitous across all sites, whereas the distributions of the rare taxa were relatively more site specific. The α-diversity indices and β-diversity of rare subcommunities were significantly higher than those of the abundant subcommunities. Besides, both the taxonomic and functional composition of soil bacterial communities differed significantly between the two biospheres. Furthermore, the influence of stochasticity differed in each subcommunity. In particular, stochastic processes were relatively more important in constraining the assembly of rare taxa. Co-occurrence network analysis revealed that a few abundant taxa occupy central nodes within the networks, possibly indicating crucial roles as keystone taxa. Collectively, these findings suggest that abundant and rare bacterial biospheres have distinct distributions underpinned by a dynamic interplay of ecological processes and taxon co-occurrence patterns. IMPORTANCE Estuarine salt marshes are highly productive ecosystems subjected to regular disturbances by hydrodynamic exchange. However, little is known about how distinct assembly processes and co-occurrence of taxa influence the structure of the abundant and rare bacterial biospheres in these soil systems. This study aims at unravelling these intricacies by studying a typical estuarine salt marsh located in Hangzhou Bay, China. Our study provides important pieces of evidence on the diverse distribution of rare and abundant bacterial biospheres. We show that a few abundant taxa are central nodes in species co-occurrence, potentially playing important roles as keystone species in the system. In addition, we highlight a dynamic interplay of assembly processes structuring these two subcommunities.

Illumina sequencing is a representative tool for understanding the massive diversity of microbial eukaryotes in natural ecosystems. Here, we investigated the eukaryotic community in a pond (salinity of 2–4) on Dokdo (island) in the East Sea, Korea, using Illumina sequencing with primer sets for the V4 and V9 regions of 18S rDNA from 2016 to 2018 for the first time. Totally, 1,413 operational taxonomic units (OTUs) and 915 OTUs were detected using the V9 and V4 primer sets, respectively. Taxonomic analyses of these OTUs revealed that although the V4 primer set failed to describe the extant diversity for some major sub-division groups, the V9 primer set represented their diversity. Moreover, the rare taxa with

Bacterioplanktonic communities, consisting of a few abundant taxa (AT) and many rare taxa (RT), are essential component of riverine ecosystems. Nonetheless, the biogeographic patterns of bacterioplankton and roles of AT and RT in community structuring and functional composition remain uncertain in large rivers. Here, we employ the Yangtze River, which is the third-longest river in the world, as model system. By using high-throughput sequencing and bioinformatics tool of Tax4Fun, the geographical patterns of bacterioplanktonic taxonomic and predicted functional communities are investigated, and the relative importance of abundant and rare subcommunities in community structuring are explored. Results showed a clear spatial variation that the bacterioplanktonic communities of upper, middle and lower reaches of the river are significantly different from each other. Besides, the Three Gorges Dam exhibited impact on the bacterioplankton of upper reach whose community is relatively closer to that of the Poyang Lake. Both the abundant and rare subcommunities showed spatial variation along the river, which is similar to the total bacterioplanktonic community. The rare subcommunity comprised a majority of community diversity with 23.6% of the total sequences and 94.2% of the total OTUs. The rare subcommunity contributes a major part (56.8%) versus abundant subcommunity (16.3%) of the spatial variation of the total community. In addition, the non-RT exhibits more interactions with RT than with themselves, and all of the 33 keystone species are belonged to RT. Hence, the RT is critical for community structuring and assembling. By contrast, no obvious spatial effect was observed for the predicted functional community. The predicted functions of abundant and rare subcommunities are consistent with that of total community, despite their contrasting community composition. In summary, the rare subcommunity show significantly impact on the community structure and assembling, and play an important role in predicted function as ‘seed bank’ in the Yangtze River.

Microbial ecologists have historically used cultivation-based approaches as well as amplicon sequencing and shotgun metagenomics to characterize microbial diversity in soil. However, challenges persist in the study of microbial diversity, including the recalcitrance of the majority of microorganisms to laboratory cultivation and limited sequence assembly from highly complex samples. The uncultivated majority thus remains a reservoir of untapped genetic diversity. To address some of the challenges associated with bulk metagenomics as well as low throughput of single-cell genomics, we applied flow cytometry-enabled mini-metagenomics to capture expanded microbial diversity from forest soil and compare it to soil bulk metagenomics. Our resulting data from this pooled-cell sorting approach combined with bulk metagenomics revealed increased phylogenetic diversity through novel soil taxa and rare biosphere members. In-depth analysis of genomes within the highly represented Bacteroidetes phylum provided insights into conserved and clade-specific patterns of carbon metabolism., Soil ecosystems harbor diverse microorganisms and yet remain only partially characterized as neither single-cell sequencing nor whole-community sequencing offers a complete picture of these complex communities. Thus, the genetic and metabolic potential of this “uncultivated majority” remains underexplored. To address these challenges, we applied a pooled-cell-sorting-based mini-metagenomics approach and compared the results to bulk metagenomics. Informatic binning of these data produced 200 mini-metagenome assembled genomes (sorted-MAGs) and 29 bulk metagenome assembled genomes (MAGs). The sorted and bulk MAGs increased the known phylogenetic diversity of soil taxa by 7.2% with respect to the Joint Genome Institute IMG/M database and showed clade-specific sequence recruitment patterns across diverse terrestrial soil metagenomes. Additionally, sorted-MAGs expanded the rare biosphere not captured through MAGs from bulk sequences, exemplified through phylogenetic and functional analyses of members of the phylum Bacteroidetes. Analysis of 67 Bacteroidetes sorted-MAGs showed conserved patterns of carbon metabolism across four clades. These results indicate that mini-metagenomics enables genome-resolved investigation of predicted metabolism and demonstrates the utility of combining metagenomics methods to tap into the diversity of heterogeneous microbial assemblages. IMPORTANCE Microbial ecologists have historically used cultivation-based approaches as well as amplicon sequencing and shotgun metagenomics to characterize microbial diversity in soil. However, challenges persist in the study of microbial diversity, including the recalcitrance of the majority of microorganisms to laboratory cultivation and limited sequence assembly from highly complex samples. The uncultivated majority thus remains a reservoir of untapped genetic diversity. To address some of the challenges associated with bulk metagenomics as well as low throughput of single-cell genomics, we applied flow cytometry-enabled mini-metagenomics to capture expanded microbial diversity from forest soil and compare it to soil bulk metagenomics. Our resulting data from this pooled-cell sorting approach combined with bulk metagenomics revealed increased phylogenetic diversity through novel soil taxa and rare biosphere members. In-depth analysis of genomes within the highly represented Bacteroidetes phylum provided insights into conserved and clade-specific patterns of carbon metabolism.

Whole microbial communities regularly merge with one another, often in tandem with their environments, in a process called community coalescence. Such events impose substantial changes: abiotic perturbation from environmental blending and biotic perturbation of community merging. We used an aquatic mixing experiment to unravel the effects of these perturbations on the whole microbiome response and on the success of individual taxa when distinct freshwater and marine communities coalesce. We found that an equal mix of freshwater and marine habitats and blended microbiomes resulted in strong convergence of the community structure toward that of the marine microbiome. The enzymatic potential of these blended microbiomes in mixed media also converged toward that of the marine, with strong correlations between the multivariate response patterns of the enzymes and of community structure. Exposing each endmember inocula to an axenic equal mix of their freshwater and marine source waters led to a 96% loss of taxa from our freshwater microbiomes and a 66% loss from our marine microbiomes. When both inocula were added together to this mixed environment, interactions amongst the communities led to a further loss of 29% and 49% of freshwater and marine taxa, respectively. Under both the axenic and competitive scenarios, the diversity lost was somewhat counterbalanced by increased abundance of microbial taxa that were too rare to detect in the initial inocula. Our study emphasizes the importance of the rare biosphere as a critical component of microbial community responses to community coalescence.

Complex microbial communities shape the dynamics of various environments, ranging from the mammalian gastrointestinal tract to the soil. Advances in DNA sequencing technologies and data analysis have provided drastic improvements in microbiome analyses, for example, in taxonomic resolution, false discovery rate control and other properties, over earlier methods. In this Review, we discuss the best practices for performing a microbiome study, including experimental design, choice of molecular analysis technology, methods for data analysis and the integration of multiple omics data sets. We focus on recent findings that suggest that operational taxonomic unit-based analyses should be replaced with new methods that are based on exact sequence variants, methods for integrating metagenomic and metabolomic data, and issues surrounding compositional data analysis, where advances have been particularly rapid. We note that although some of these approaches are new, it is important to keep sight of the classic issues that arise during experimental design and relate to research reproducibility. We describe how keeping these issues in mind allows researchers to obtain more insight from their microbiome data sets.

The dynamic change of microbial community during sludge acclimation from aerobic to anaerobic in a MBR for coking wastewater treatment was revealed by Illumina Miseq sequencing in this study. The diversity of both Bacteria and Archaea showed an increase-decrease trajectory during acclimation, and exhibited the highest at the domestication interim. Ignavibacteria changed from a tiny minority (less than 1%) to the dominant bacterial group (54.0%) along with acclimation. The relative abundance of Betaproteobacteria kept relatively steady, as in this class some species increased coupled with some other species decreased during acclimation. The dominant Archaea shifted from Halobacteria in initial aerobic sludge to Methanobacteria in the acclimated anaerobic sludge. The dominant bacterial and archaeal groups in different acclimation stages were indigenous microorganisms in the initial sludge, though some of them were very rare. This study supported that the species in "rare biosphere" might eventually become dominant in response to environmental change.

Knowledge about the ecological patterns of abundant and rare bacteria in rhizosphere is still lacking. Through a large-scale sampling across the arable area of a medicinal plant Panax notoginseng, we examined the community assembly of abundant and rare rhizobacteria as well as their putative functions and co-occurrence patterns. At local level, the rare taxa exhibited significantly stronger phylogenetic clustering than the abundant taxa. At metacommunity level, both two subcommunities showed significant biogeographic patterns, but they were assembled by different ecological processes. Null model analysis showed that dispersal limitation (84.68%) governed the assembly of abundant subcommunity, whereas homogeneous selection (90.11%) dominated the dynamics of rare subcommunity. Although the putative functions indicated that the rare taxa potentially enriched more functional traits associated with plant-microbe interactions, the overall abundance profiles of putative pathways in two subcommunities exhibited significant collinearity. Additionally, the topological properties of abundant taxa in the co-occurrence network were significantly higher than those of rare taxa. We propose that stochasticity and determinism dominate the assembly of abundant and rare subcommunities, respectively. The abundant taxa may exhibit stronger environmental adaptability, while the rare taxa are more likely the result of rhizosphere effect. These findings also emphasize the importance of integrating phylogeny in ecological researches on plant-associated microbiota.

Bark represents a unique microbial habitat. Revealing the interactions among bark-associated microbes is important for understanding their diversity, stability, and function, and how core microbiome influences the health and production of the host plant. We used amplicon sequencing of bark from the medicinal plant Eucommia ulmoides collected across nine distinct biogeographical regions in China, and comprehensively analyzed the diversity, rare biosphere and core taxa of bark fungi. The co-occurrence network results showed significant differences in the compositions of core mycobiota in E. ulmoides bark between the nine regions. Ecological factors (e.g., temperature and rainfall) were crucial determinants of differences in the unique core mycobiota of E. ulmoides from different regions. The metacommunity-scale network indicated that Cladosporium, Alternaria, and Teratosphaeria were core fungal taxa of E. ulmoides bark. Moreover, some core fungal taxa included rare taxa in particular local communities which, despite their relatively low abundance, may play a significant role in the community structure of E. ulmoides bark.

Benthic microeukaryotes are key ecosystem drivers in marine sandy beaches, an important and dynamic environment; however, little is known about their diversity and biogeography on a large spatial scale. Here, we investigated the community composition and geographical distributions of benthic microeukaryotes using high-throughput sequencing of the 18S rRNA gene and quantified the contributions of environmental factors and spatial separation on the distribution patterns of both rare and abundant taxa. We collected 36 intertidal samples at 12 sandy beaches from four regions that spanned distances from 0.001 to 12,000 km. We found 12,890 operational taxonomic units (OTUs; 97% sequence identity level) including members of all eukaryotic super-groups and several phyla of uncertain position. Arthropoda and Diatomeae dominated the sequence reads in abundance, but Ciliophora and Discoba were the most diverse groups across all samples. About one-third of the OTUs could not be definitively classified at a similarity level of 80%, supporting the view that a large number of rare and minute marine species may have escaped previous characterization. We found generally similar geographical patterns for abundant and rare microeukaryotic sub-communities, and both showed a significant distance-decay similarity trend. Variation partitioning showed that both rare and abundant sub-communities exhibited a slightly stronger response to environmental factors than spatial (distance) factors. However, the abundant sub-community was strongly correlated with variations in spatial, environmental and sediment grain size factors (66% of variance explained), but the rare assemblage was not (16%). This suggests that different or more complex mechanisms generate and maintain diversity in the rare biosphere in this habitat.

Next-generation sequencing is a common method for analysing microbial community diversity and composition. Configuring an appropriate sequence processing strategy within the variety of tools and methods is a nontrivial task and can considerably influence the resulting community characteristics. We analysed the V4 region of 18S rRNA gene sequences of marine samples by 454-pyrosequencing. Along this process, we generated several data sets with QIIME, mothur, and a custom-made pipeline based on DNAStar and the phylogenetic tree-based PhyloAssigner. For all processing strategies, default parameter settings and punctual variations were used. Our results revealed strong differences in total number of operational taxonomic units (OTUs), indicating that sequence preprocessing and clustering had a major impact on protist diversity estimates. However, diversity estimates of the abundant biosphere (abundance of ≥1%) were reproducible for all conducted processing pipeline versions. A qualitative comparison of diatom genera emphasized strong differences between the pipelines in which phylogenetic placement of sequences came closest to light microscopy-based diatom identification. We conclude that diversity studies using different sequence processing strategies are comparable if the focus is on higher taxonomic levels, and if abundance thresholds are used to filter out OTUs of the rare biosphere.

The Blue Lagoon is a shallow geothermal lake which contains sea- and freshwater and is extremely high in silica content. This study has provided the first in-depth molecular survey of its planktic microbial inhabitants and laid the groundwork for future environmental and physiological work. Most MiSeq sequences obtained were affiliated with Alphaproteobacteria (36%), Cyanobacteria (26%), and Gammaproteobacteria (24%). Cyanobacteria were found to be the principal primary producers with 99.8% of the cyanobacterial sequences belonging to the unicellular Cyanobacterium aponinum. Filamentous genera: Halomicronema and Phormidium were very minor and present only outside the bathing area. The calculated diversity was typical of samples from extreme environments. Elevated salinity and high concentration of silicium attract extremophiles that are either highly adapted specialists for certain conditions or generalists with wide tolerance ranges. Relatively few abundant phyla were represented; 95% of observed OTUs consisted of less than ten sequences and only 0.5% had more than 1000 sequences. All rarefaction curves were far from saturation and a higher number of sequences is apparently required to cover the whole community diversity. Although, the diversity of the abundant taxa was limited, the rare biosphere, detectable only with next generation sequencing, increased significantly the diversity.

Este artículo contiene 14 páginas, 4 figuras, 2 tablas., The potential of increased bioerosion by excavating sponges in future environmental scenarios represents a potential threat to coral reef structure and function. Little is known about prokaryotic associations in excavating sponges despite the fact that evidence indicates they contribute to the sponge growth through their heterotrophic metabolism and may even act as microborers. Here, we provide the first detailed description of the microbial community of multiple bioeroding sponges from the Clionaidae family (Cliona varians, C. tumula, C. delitrix, Spheciospongia vesparium, Cervicornia cuspidifera) collected in inshore and offshore coral reefs in the Florida Keys. A total of 6811 prokaryote OTUs identified using 16S rRNA gene sequencing was detected in the samples studied, including ambient water, belonging to 39 bacterial phyla and 3 archaeal phyla. The microbiomes of species harboring Symbiodinium (C. varians, C. tumula, C. cuspidifera) and the azooxanthellate S. vesparium were dominated by Alphaproteobacteria that represented from 83 to 96% of total sequences. These clionaid sponges presented species-specific core microbiomes, with 4 OTUs being shared by all sponge samples, albeit with species-specific enrichments. The microbiomes of C. varians and S. vesparium were stable but showed certain plasticity between offshore and inshore reefs. The distantly related C. delitrix does not harbor Symbiodinium, and had a microbiome dominated by Gammaproteobacteria, which represented 82% of all sequences. Most of the sponge-enriched OTUs are found in low abundance and belong to the ‘rare biosphere’ category, highlighting the potential importance of these microbes in the ecology of the holobiont. Sponge microbiomes may enhance functional redundancy for the sponge holobiont and allow it to respond to shifting environments over much short time scales than evolutionary change would permit. This work establishes the basis for future research to explore how microbial shifts in bioeroding sponges contribute to bioerosion in the face of a changing environment., This work was funded by grants from the National Science Foundation (OCE-1617255 and IOS-1555440 to MH) and in part by the Spanish Government project PopCOmics CTM2017-88080 (MCIU,AEI/FEDER, UE) to XT and the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement 705464 (“SCOOBA”) to OSS.

Current research on the prokaryotic low abundance taxa, the prokaryotic rare biosphere, is growing, leading to a greater understanding of the mechanisms underlying organismal rarity and its relevance in ecology. From this emerging knowledge it is possible to envision innovative approaches in biotechnology applicable to several sectors. Bioremediation and bioprospecting are two of the most promising areas where such approaches could find feasible implementation, involving possible new solutions to the decontamination of polluted sites and to the discovery of novel gene variants and pathways based on the attributes of rare microbial communities. Bioremediation can be improved through the realization that diverse rare species can grow abundant and degrade different pollutants or possibly transfer useful genes. Further, most of the prokaryotic diversity found in virtually all environments belongs in the rare biosphere and remains uncultivatable, suggesting great bioprospecting potential within this vast and understudied genetic pool. This Mini Review argues that knowledge of the ecophysiology of rare prokaryotes can aid the development of future, efficient biotechnology-based processes, products and services. However, this promise may only be fulfilled through improvements in (and optimal blending of) advanced microbial culturing and physiology, metagenomics, genome annotation and editing, and synthetic biology, to name a few areas of relevance. In the future, it will be important to understand how activity profiles relate with abundance, as some rare taxa can remain rare and increase activity, whereas other taxa can grow abundant. The metabolic mechanisms behind those patterns can be useful in designing biotechnological processes. info:eu-repo/semantics/publishedVersion

The Cuatro Cienegas Basin (CCB), located in Coahuila, Mexico, is considered a living laboratory for astrobiology. This site is an analogue for the Gale Crater in Mars because of its gypsum-rich soils. Moreover, the “pozas” at the site are extremely oligotrophic, which is characteristic of ancient oceans chemistry. Despite the oligotrophic conditions, CCB harbors a very rich microbial diversity in the form of stromatolites and microbial mats. Archaea data at CCB has been sparse given that, in general, archaea do not represent more than 1% of the reads in a metagenome. Here, we describe a new hyper-diverse site with an abundant core of Archaea. This site, which we named “archaean domes,” recreates in their interior the atmosphere of ancient Earth and Mars and can serve as a valuable resource for astrobiological research.

Microorganisms in the deep sea play vital roles in marine ecosystems. However, despite great advances brought by high throughput sequencing and metagenomics, only a small portion of microorganisms living in the environment can be cultivated in the laboratory and systematically studied. In this study, an improved high-throughput microfluidic streak plate (MSP) platform was developed to speed up the isolation of microorganisms from deep-sea sediments and evaluated with deep-sea sediments collected from the Southwest Indian Ridge (SWIR). Based on our previously reported MSP method, we improved its isolation efficiency with a semi-automated droplet picker and improved humidity control to enable long-term cultivation with a low-nutrient medium for up to five months according to the slow-growing nature of most deep-sea species. The improved MSP method allows the isolation of microbes by selection and investigation of microbial diversity by high throughput sequencing of the pooled sample cultures. By picking individual droplets and scale-up cultivation, a total of 772 strains that were taxonomically assigned to 70 species were isolated from the deep-sea sediments in the SWIR, including 15 potential novel species. On the other hand, based on 16S rRNA gene amplicon sequencing analysis, the microbial diversity of the SWIR was studied and documented with culture-dependent and independent methods in this study. The superiority of the MSP platform in revealing the rare biosphere was also evaluated based on amplicon sequencing. The results show that droplet-based single-cell cultivation of the MSP has a much higher ability than traditional agar plate cultivation in obtaining microbial species and more than 90% of operational taxonomic units (OTUs) detected in the MSP pool belong to the rare biosphere. Our results indicate the high robustness and efficiency of the improved MSP platform in revealing the environmentally rare biosphere, especially for slow-growing species. Overall, the MSP platform has a superior ability to recover microbial diversity than conventional agar plates and it was found to hold great potential for recovering rare microbial resources from various environments.

We performed an incubation experiment of seawater confined in plastic bottles with samples collected at three depths (15, 60, and 90 m) after retrieval from a single offshore location in the Mediterranean Sea, from a late summer stratified water column. Two samples representative of each depth were collected and stored in opaque bottles after two periods of 7 h. We took advantage of the “bottle effect” to investigate changes in the natural microbial communities (abundant and rare). We recovered 94 metagenome-assembled genomes (MAGs) and 1089 metagenomic viral contigs and examined their abundance using metagenomic recruitment. We detected a significant fast growth of copiotrophic bacteria such as Alteromonas or Erythrobacter throughout the entire water column with different dynamics that we assign to “clonal,” “polyclonal,” or “multispecies” depending on the recruitment pattern. Results also showed a marked ecotype succession in the phototropic picocyanobacteria that were able to grow at all the depths in the absence of light, highlighting the importance of their mixotrophic potential. In addition, “wall-chain-reaction” hypothesis based on the study of phage–host dynamics showed the higher impact of viral predation on archaea in deeper waters, evidencing their prominent role during incubations. Our results provide a step forward in understanding the mechanisms underlying dynamic patterns and ecology of the marine microbiome and the importance of processing the samples immediately after collection to avoid changes in the community structure.

Greenland's Dark Zone is the largest contiguous region of bare terrestrial ice in the Northern Hemisphere and microbial processes play an important role in driving its darkening and thereby amplifying melt and runoff from the ice sheet. However, the dynamics of these microbiota have not been fully identified. Here we present joint 16S rRNA gene and 16S rRNA (cDNA) comparison of input (snow), storage (cryoconite), and output (supraglacial stream water) habitats across the Dark Zone over the melt season. We reveal that all three Dark Zone communities have a preponderance of rare taxa exhibiting high protein synthesis potential (PSP). Furthermore, taxa with high PSP represent highly connected ‘bottlenecks’ within community structure, consistent with their roles as metabolic hubs. Finally, low abundance-high PSP taxa affiliated with Methylobacterium within snow and stream water suggest a novel role for Methylobacterium in the carbon cycle of Greenlandic snowpacks, and importantly, the export of potentially active methylotrophs to the bed of the Greenland Ice Sheet. By comparing the dynamics of bulk and potentially active microbiota in the Dark Zone of the Greenland Ice Sheet we provide novel insights into the mechanisms and impacts of the microbial colonization of this critical region of our melting planet.

Wildfires destabilize biocrust, requiring decades for most biological constituents to regenerate, but bacteria may recover quickly and mitigate the detrimental consequences of burnt soils. To evaluate the short-term recovery of biocrust bacteria, we tracked shifts in bacterial community form and function in Cyanobacteria/lichen-dominated (shrub interspaces) and Cyanobacteria/moss-dominated (beneath Artemisia tridentata) biocrusts one week, two months, and one year following a large-scale burn manipulations in a cold desert (Utah, USA). We found no evidence of the burned bacterial community recovering to a burgeoning biocrust. The foundational biocrust phyla, Cyanobacteria, dominated by Microcoleus viginatus (Microcoleaceae), disappeared from burned soils creating communities void of photosynthetic taxa. One year after the fire, the burned biocrust constituents had eroded away and the bare soils supported the formation of a convergent community of chemoheterotrophic copiotrophs regardless of location. The emergent community was dominated by a previouly rare Planococcus species (family Planococcaceae, Firmicutes) and taxa in the Cellulomonadaceae (Actinobacteria), and Oxalobacteraceae (Betaproteobacteria). Previously burnt soils maintained similar levels of bacterial biomass, alpha diversity, and richness as unburned biocrusts, but supported diffuse, poorly-interconnected communities with 75% fewer species interactions. Nitrogen fixation declined at least 3.5-fold in the burnt soils but ammonium concentrations continued to rise through the year, suggesting that the exhaustion of organic C released from the fire, and not N, may diminish the longevity of the emergent community. Our results demonstrate that biocrust bacteria may recover rapidly after burning, albeit along a different community trajectory, as rare bacteria become dominant, species interconnectedness diminishes, and ecosystem services fail to rebound.

We report on the characterization of a novel genomic assembly (ARYD3) recovered from formation water (17.6% salinity) and crude oil enrichment amended by isolated soy proteins (0.2%), and incubated for 100 days under anaerobic conditions at 50°C. Phylogenetic and phylogenomic analysis demonstrated that the ARYD3 is unaffiliated with all currently described bacterial phyla and candidate phyla, as evident by the low AAI (34.7%), shared gene content (19.4%), and 78.9% 16S rRNA gene sequence similarity to Halothiobacillus neapolitanus, its closest cultured relative. Genomic characterization predicts a slow-growing, non-spore forming, and non-motile Gram-negative rod. Adaptation to high salinity is potentially mediated by the production of the compatible solutes cyclic 2,3-diphosphoglycerate (cDPG), α-glucosylglycerate, as well as the uptake of glycine betaine. Metabolically, the genome encodes primarily aminolytic capabilities for a wide range of amino acids and peptides. Interestingly, evidence of propionate degradation to succinate via methyl-malonyl CoA was identified, suggesting possible capability for syntrophic propionate degradation. Analysis of ARYD3 global distribution patterns identified its occurrence in a very small fraction of Earth Microbiome Project datasets examined (318/27,068), where it consistently represented an extremely rare fraction (maximum 0.28%, average 0.004%) of the overall community. We propose the Candidatus name Mcinerneyibacterium aminivorans gen. nov, sp. nov. for ARYD3T, with the genome serving as the type material for the novel family Mcinerneyibacteriaceae fam. nov., order Mcinerneyibacteriales ord. nov., class Mcinerneyibacteria class nov., and phylum Mcinerneyibacteriota phyl. nov. The type material genome assembly is deposited in GenBank under accession number VSIX00000000.

Ethyl tert-butyl ether (ETBE) is a gasoline additive that became an important aquifer pollutant. The information about natural bacterial consortia with a capacity for complete ETBE degradation is limited. Here we assess the taxonomical composition of bacterial communities and diversity of the ethB gene (involved in ETBE biodegradation) in ETBE-enrichment cultures that were established from a gasoline-polluted aquifer, either from anoxic ETBE-polluted plume water (PW), or from an upstream non-polluted water (UW). We used a 16S rRNA microarray, and 16S rRNA and ethB gene sequencing. Despite the dissimilar initial chemical conditions and microbial composition, ETBE-degrading consortia were obtained from both PW and UW. The composition of ETBE-enrichment cultures was distinct from their initial water samples, reflecting the importance of the rare biosphere as a reservoir of potential ETBE degraders. No convergence was observed between the enrichment cultures originating from UW and PW, which were dominated by Mesorhizobium and Hydrogenophaga, respectively, indicating that distinct consortia with the same functional properties may be present at one site. Conserved ethB genes were evidenced in both PW and UW ETBE-enrichment cultures and in PW water. Our results suggest that the presence of ethB genes rather than the taxonomical composition of in situ bacterial communities indicate the potential for the ETBE degradation at a given site.

Herein we describe the Archaea diversity in a shallow pond in the Cuatro Ciénegas Basin (CCB), Northeast Mexico, with fluctuating hypersaline conditions containing elastic microbial mats that can form small domes where their anoxic inside reminds us of the characteristics of the Archaean Eon, rich in methane and sulfur gases; thus, we named this site the Archaean Domes (AD). These domes only form after heavy rains that are rare in the Chihuahuan desert. CCB is a unique oasis with hundreds of ponds, containing endemic species of animals, plants and highly diverse and unique microbial communities, despite its very biased stoichiometry, due mostly to extreme low phosphorus content (soils, water columns and sediments). This extreme oligotrophy has favored survival of ancestral microorganisms. Whole metagenome sequencing approach was performed for this unusual site in three different seasons to assess the extent of the Archaea biodiversity, with a focus on extremophiles, since members of the Archaea had been underrepresented in different study sites within the oasis. We found a highly diverse Archaea community compassing ∼5% of the metagenomes. The archaeal portion in all three metagenomes maintained its abundance and most of the strains showed to form a resilient core during three seasonal samplings (2016-2017), despite environmental fluctuations. However, relative abundances of all 230 archaeal OTUs (defined using a 97% cutoff) were low enough (