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Present-day angiosperm plants produce a plethora of metabolites including pigments that serve for important functions such as photosynthesis, protection against light, attraction of pollinators, and defense against microbes and herbivores. However, little is known about phytochemical constituents of ancient angiosperms, their distribution in the fossil record, their stability in deep time, and diagenesis. Outstanding preservation of ancient angiosperms, including exceptional color preservation, has been reported, but chemical analyses of such valuable specimens are limited by the rarity of the fossil material and the small amounts of potentially preserved metabolites. Here we use highly sensitive targeted liquid chromatography-tandem mass spectrometry in multiple reaction monitoring mode to screen for nanogram quantities of intact ancient phytochemical metabolites and their products in exceptionally well-preserved, about 45-Ma-old leaves from the Eocene Geiseltal fossil Lagerstätte, Germany. We show that diverse chlorophyll derivatives and degradation products as well as polyphenolic pigments are preserved in green to yellow colored angiosperm leaves and the brown coal matrix from Geiseltal. Most interesting is the fossil occurrence of the "unstable" green chlorophyll derivative dihydro-132,173-cyclopheophorbide a-enol, since cyclopheophorbide-enols are otherwise known as unique non-fluorescent chlorophyll catabolites of microorganisms in modern aquatic environments. The monopyrrole hematinic acid is interpreted as a stable product of chlorophyll catabolism via linear tetrapyrroles. Moreover, polyphenolic compounds in the fossil angiosperms are represented by the flavonoid pigments apigenin and luteolin. Our results demonstrate the potential of paleometabolomic-like screening of individual plant fossils to trace the fate of phytochemical constituents and to understand the processes of fossilization at the molecular level.

The history of Earth's atmospheric oxygen is a cornerstone of evolutionary biology. While unequivocal evidence for an increase in atmospheric O2 marks the Great Oxidation Event (GOE) roughly 2.4 billion years ago, evidence underlying proposals for pre-GOE O2 accumulation is debated. Here we have investigated the distribution of genes for oxygen reductases, the enzymes that consume O2 in respiratory chains, across independently generated molecular timescales of prokaryotic evolution. The data indicate that cytochrome bd-oxidases, heme-copper oxidases and alternative oxidases arose in the wake of the GOE ca. 2.4 billion years ago, after which the genes were subjected to abundant lateral gene transfer, a reflection of their utility in redox balance and membrane bioenergetics. The data lead us to propose a straightforward four-stage model for O2 accumulation surrounding the GOE: (i) Negligible O2 existed prior to the GOE. (ii) Cyanobacterial O2 production started at the GOE, yet was capped at 2 % [v/v] atmospheric O2, the threshold at which cyanobacterial nitrogenase is inhibited by O2. (iii) Production of 0.02 atm of O2 (2 % [v/v]) at the GOE buried roughly the entire atmospheric CO2 inventory, causing sudden enrichment of 13C in dissolved inorganic carbon (the Lomagundi 13C anomaly), through RuBisCO isotope discrimination, without atmospheric O2 exceeding 2 % [v/v]. (iv) High atmospheric 12C at the end of the Lomagundi excursion marks the origin of oxygen reductases, their rapid spread via function in respiratory CO2 liberation, and the onset of equilibrium between photosynthetic O2 production and respiratory O2 consumption at 2 % atmospheric O2.

Strong disparity in species richness among organisms is well documented, but heterogeneity in the underlying diversification process is less understood. Using novel probabilistic methods, we investigate clade-specific diversification rate shifts in several species-rich phylogenies, together representing over 300,000 species across the Tree of Life. We find that diversification rate shifts are extremely prevalent across all clades, with more frequent changes in younger clades and an overall excess of upshifts, resulting in an apparent acceleration of net diversification rates. We also reveal that heterogeneity in diversification rates is related to the tempo of diversification itself. While we find support for more prevalent shifts in speciation rates than extinction rates and more upshifts than downshifts, this is partially due to data insufficiency and inference challenges. Our insights are fundamentally enabled by studying numerous large phylogenies with rigorous statistical methods, showing widespread prevalence of diversification rate shifts across the Tree of Life.

Sulfate-reducing bacteria (SRB) drive the process of sulfate reduction in low-temperature sedimentary environments. Through the production of sulfide, they promote the formation of iron-sulfide (Fe-S) minerals when Fe(II) is available. The negative charge of the cell surface of bacteria can promote the binding of Fe(II), leading to the precipitation of Fe-S minerals at the surface of SRB when sulfide is released from cells. We evaluated interactions between Fe-S minerals and the surface of SRB using transmission electron microscopy (TEM) in cultures of Maridesulfovibrio hydrothermalis AM13 grown with 4 mM of Fe(II) over 1 month of incubation. On average, 18% ± 10% of cells were encrusted in cultures collected during the exponential phase. Fe-S mineral deposition occurred at the surface of cells while cells were growing and producing sulfide in the presence of Fe(II), but mineral crusts were removed from most cells shortly after deposition. Cells removed crusts from their surface through the formation of membrane vesicles, which were apparently only produced during growth. Mineralized and non-mineralized membrane vesicles were preserved in mineral aggregates in stationary-phase cultures. On average, 17% ± 7% of cells were encrusted in cultures collected during the stationary phase, indicating that Fe-S minerals precipitated during the exponential phase and removed from the cell surface did not aggregate back onto cells. On the contrary, they formed large aggregates away from cells. When Fe-S mineral precipitation occurred in non-growing cell suspensions that were first exposed to Fe(II) then to sulfide, the proportion of encrusted cells increased to 95% ± 6%, indicating that resting or non-growing cells were not able to remove mineral crusts from their surface. The metabolic status of SRB therefore plays a role in their ability to escape Fe-S mineral entombment.

Observations of morphology are commonly used to evaluate the biogenicity of terrestrial microfossils and could constitute a crucial line of evidence for extraterrestrial life-detection missions in the future. However, evaluating the origin of morphological features in the rock record can be problematic because naturally occurring abiotic structures can resemble biological morphologies, which may lead to false-positive detections of fossilised life. Iron-mineralised chemical gardens have been highlighted as potentially confounding abiotic structures because of their morphological and chemical resemblance to biomineralised filaments. Despite this, the potential for chemical garden structures to be preserved in the fossil record has not been thoroughly investigated. Here, we subjected abiotic iron-mineralised chemical garden structures to artificial maturation using hydrous pyrolysis, in order to evaluate their preservation potential. We found that these abiotic filaments were relatively resistant to degradation caused by maturation when compared with analogous biological material. Additionally, the transformation of ferrihydrite to crystalline iron oxides was found to be relatively inhibited, likely because of the influence of silica. These findings highlight the need for fossilised filamentous material to be distinguished from chemical garden structures before a biological origin can be confidently attributed, particularly when observed in significantly altered rocks.

Mitochondrial introns have a patchy distribution in sponge lineages. Here, we report on the finding of a group-II-intron in Eunapius rarus (Demospongiae, Spongillidae), which constitutes the first report of a mitochondrial intron in freshwater sponges. Group-II-introns are self-splicing ribozymes, and are particularly rare among sponge mitochondrial genomes. The intron contains complete open reading frames (ORFs), including typical intron-encoded proteins (IEPs). Phylogenetic analysis reveals that the intron is more closely related to those found in brown algae, and distant from other sponge group-II-introns, indicating an acquisition of this intron independent from other sponges. Remarkably, the congeneric E. fragilis does not possess this intron in their mitochondrial genome. However, we found pseudogenic copies of the E. rarus group-II-intron in the nuclear genome of E. fragilis, which indicates patterns of group-II-intron presence and their pseudogene transposition into the nuclear genomes in sponges for the first time. Our results show that a group-II-intron must have been present in the last common ancestor of both Eunapius mt genomes, and subsequently lost in E. fragilis, rather than independent acquisition. Consequently, our findings provide an explanation for the patchy distribution of introns in sponges as a result of frequent losses, besides multiple acquisitions.

Challenging to engineer in synthetic glues, wet adhesion is critical for many technical and biomedical applications. Mussels, however, have evolved underwater glues that adhere effectively onto slippery seashore surfaces. Past research on mussel adhesion highlights the importance of the post-translationally modified amino acid 3,4-dihydroxyphenylalanine (DOPA), found in abundance in mussel glue proteins. Yet, DOPA alone is insufficient to match native adhesion in synthetic mimics. Here, we provide evidence that a previously uncharacterized histidine-rich protein (mefp-12) plays a crucial role in the formation, curing, and performance of mussel glue. Biochemical analysis localizes mefp-12 within vesicles of the mussel glue secretory glands, while AI-assisted modeling of its sequence predicts Zn-stabilized coiled coil conformation and several domains resembling zinc-finger motifs. In vitro investigation of a His-rich α-helical peptide from mefp-12 shows Zn- and pH-dependent liquid-liquid phase separation (LLPS), coalescence, and spreading over the substrate. Exposure to seawater pH induces subsequent self-organization of the fluid condensates into solid nanoporous networks resembling the structure of the native mussel glue. Based on these findings we gain a deeper mechanistic understanding of mussel glue formation and function that challenges the dominant DOPA-centric paradigm, providing inspiration for design of bio-inspired wet adhesives.

During the Paleocene-Eocene Thermal Maximum (PETM), there was an increase in global temperatures and emissions of isotopically depleted carbon, resulting in a negative carbon isotope excursion (CIE). This climatic event caused a widespread ocean deoxygenation, leading to substantial biotic turnover. Previous ichnological studies of deep-sea environments have suggested that bioturbating communities perished or diminished considerably during this event. In this study, we present an ichnological analysis of a well-known deep-sea outcrop (Rio Gor section; lower bathyal-upper abyssal depth; 1000-2000 m) from the southern Iberian margin. Contrary to previous studies, at this location, the PETM onset did not result in the extinction of the bioturbating community. In fact, high abundances of trace fossils were recorded during the PETM, indicating favorable paleoenvironmental conditions for the community. We discuss how sedimentary and climatic dynamics played a key role in regulating trace fossil abundance throughout the PETM. The paleogeographical position and deep-water circulation of the area appear to have played a crucial role in preventing low-oxygen deep water masses and the impoverishment of the bioturbating community. Overall, our findings reveal the PETM's positive impact on the bioturbating community at the southern Iberian margin. Given the essential ecological functions of these organisms on the seafloor-such as nutrient recycling and sediment mixing-we emphasize their potential importance in future warmer ocean scenarios.

The Ediacaran-Cambrian boundary, which precedes one of the most significant biotic diversification events in Earth's history, is associated with a global negative carbon isotope excursion termed the BAsal Cambrian carbon isotope Excursion (BACE). Late Ediacaran and early Cambrian changes in shallow marine oxygenation have been proposed to relate to the BACE as well as metazoan extinction and radiation. However, reconstructing paleoredox conditions at the Ediacaran-Cambrian boundary is limited by challenges in correlating carbonate strata due to sparse stratigraphic markers and non-unique chemostratigraphic correlations. These imprecise correlations have led to uncertainty in how redox changes across the BACE should be interpreted in relation to broader regional and global environmental patterns. Here, we present redox reconstructions from southwestern Laurentian carbonate successions that record the BACE, including the limestone-dominated Deep Spring Formation, southwestern USA, and the dolostone-dominated La Ciénega Formation, northern Mexico. We combine local (carbonate-bound iodine, I/(Ca + Mg) and cerium anomaly, Ce/Ce*) and global (carbonate-associated uranium isotopes, δ238Ucarb) redox proxies to investigate marine oxygenation in relation to the BACE. Contrary to previous suggestions that a global ocean oxygenation event coincided with the BACE, we do not observe a shift in δ238Ucarb concurrent with the carbon isotope excursion in either section. The δ238Ucarb values differ between two sections, likely reflecting distinct diagenetic offsets attributed to different diagenetic U reduction, but together provide a minimal constraint on the carbonate δ238U value and suggest a more anoxic ocean compared to today. The local proxy results at both sites suggest widespread low-oxygen surface waters with a transient and localized interval of shallow marine oxygenation at one site that coincides with the nadir of the BACE. Persistently low I/(Ca + Mg) ratios, below values observed in today's oxygenated oceans, suggest a broadly redox-stratified surface ocean. Negative Ce anomalies in the La Ciénega Formation were recorded during the BACE nadir, suggesting a short-lived interval of local oxygenation within otherwise low-oxygen conditions. In sum, we do not find evidence for major, widespread oxygenation coincident with the BACE, but a continuation of low-oxygen conditions punctuated by a short-lived oxygenation event in the shallow oceans. These brief fluctuations in oxygen levels, in turn, may have played a role in the onset of behavioral complexity among bilaterian invertebrates during this critical transition.

Tecticornia is the most species-rich genus within the tribe Salicornieae. These halophytes are distributed across the Australian continent along coastlines and inland salt lake shores, playing a key ecological role in these hostile habitats. However, species delimitation within the genus remains controversial and little is known about infrageneric phylogenetic relationships. Therefore, the primary aim of this study was to infer the evolutionary history of Tecticornia and to genetically assess the reliability of current species concepts. We sampled multiple accessions per taxon from nearly all currently recognized species in Australia. We used a target enrichment approach with two bait sets: Angiosperms353 and a custom Salicornieae bait set (Salibaits). Analyses were performed using HybPiper, and we addressed paralogy using a tree-based approach. In addition, we tested the potential influence of missing data and/or missing gene trees on the topology of the final phylogenetic tree. Despite extensive gene tree discordance and the presence of short branches, the customized Salibaits set consistently produced better-resolved trees than the Angiosperms353 bait set. Missing data were found to have a negligible effect on the final tree inference. These data highlight that there is genetic support for lineages in line with observed morphological variation, suggesting markedly more taxon diversity than is currently circumscribed. While we have shown there is genetic evidence to support the characterization of several new species awaiting formal description, it is clear that further molecular and morphological investigation is required to resolve continent-wide species aggregates, each comprising multiple novel taxa. The target enrichment method effectively addressed the challenges of species delimitation in Tecticornia posed by reduced morphology and high ecological plasticity. We have shown that while there are several complexes constituting variants widely distributed across the Australian continent, some well-defined taxa have highly restricted distributions, which may represent conservation priorities.

We present a genome assembly from an individual Lycopodina hypogea (carnivorous sponge; Porifera; Demospongiae; Poecilosclerida; Cladorhizidae). The genome sequence has a total length of 235.10 megabases. Most of the assembly (98.85%) is scaffolded into 15 chromosomal pseudomolecules. The mitochondrial genome has also been assembled, with a length of 31.1 kilobases. Gene annotation of this assembly by Ensembl identified 16 317 protein-coding genes. From the metagenome data we recovered 39 bins, of which 27 were high-quality MAGs, including four fully circularised genomes. The MAGs included archaea and bacteria involved in nitrification and sulfate-reduction as well as known sponge symbionts affiliated with Gammaproteobacteria ( Candidatus Spongiihabitans, Porisulfidus) and Acidimicrobiales ( Candidatus Poriferisodalaceae), among others.

Phylogenetic comparative methods are a major tool for evaluating macroevolutionary hypotheses. Methods based on the mean-reverting stochastic Ornstein-Uhlenbeck process allow for modelling adaptation on a phenotypic adaptive landscape that itself evolves and where fitness peaks depend on measured characteristics of the external environment and/or other organismal traits. Here, we give an overview of the conceptual framework for the many implementations of these methods and discuss how we might interpret estimated parameters. We emphasize that the ability to model a changing adaptive landscape sets these methods apart from other approaches and discuss why this aspect captures long-term trait evolution more realistically. Recent multivariate extensions of these methods provide a powerful framework for testing evolutionary hypotheses but are also more complicated to use and interpret. We provide some guidance on their usage and put recent literature on the topic in biological rather than mathematical terms. We further show how these methods provide a starting point for modelling reciprocal selection (i.e., coevolution) between interacting lineages. We then briefly review some critiques of the methodologies. Finally, we provide some ideas for future developments that we think will be useful to evolutionary biologists.

Multicellular cable bacteria are capable of transferring electrons over centimeter distances through an internal array of conductive fibers. These long, filamentous bacteria function as a living electrochemical cell, performing sulfide oxidation at one end and oxygen reduction at the other end. To investigate how O2 reduction is linked to the long-distance electron transport along the conductive fibers, we performed a detailed electrochemical characterization of native filaments as well as extracted "fiber skeletons" without membranes or cytoplasm. Our data show that fibers skeletons only perform longitudinal electron transport and are not electrochemically active towards oxygen. Still, native cable bacterium filaments are capable of high oxygen reduction rates, thus indicating that dedicated enzyme systems in the periplasm or inner membrane are responsible for O2 reduction. A chemical inhibition assay on native cable bacterium filaments indicates that cytochromes are involved in electron transfer from the conductive fibers towards O2. Together, our data provide empirical support for a model in which diffusible c-type cytochromes mediate electron transport through the periplasm, shuttling electrons between separate respiratory complexes and the conductive fiber network. As such, our study resolves a crucial aspect of the unique electrogenic metabolism in cable bacteria, and clarifies the application potential of the highly conductive fibers in Bio-electrochemical System technologies.

Continuous characters have received comparatively little attention in Bayesian phylogenetic estimation. This is predominantly because they cannot be modeled by a standard phylogenetic Q-matrix approach due to their non-discrete nature. In this paper, we explore the use of continuous traits under two Brownian motion models to estimate a phylogenetic tree for Dicynodontia, a well-studied group of early synapsids (stem mammals) in which both discrete and continuous characters have been extensively used in parsimony-based tree reconstruction. We examine the differences in phylogenetic signal between a continuous trait partition, a discrete trait partition, and a joint analysis with both types of characters. We find that continuous and discrete traits contribute substantially different signal to the analysis, even when other parts of the model (clock and tree) are held constant. Tree topologies resulting from the new analyses differ strongly from the established phylogeny for dicynodonts, highlighting continued difficulty in incorporating truly continuous data in a Bayesian phylogenetic framework.

Inferring how rates of speciation and extinction vary across lineages has proven to be a difficult statistical problem. Here we describe a stochastic-diversification model-called the birth-death-shift (BDS) process-in which diversification rates may vary across both extant and extinct and unsampled lineages. We estimate the parameters of this model in a Bayesian statistical framework from phylogenies of exclusively extant lineages. We perform simulation studies to validate the implementation of our method and to characterize its statistical behavior. We also perform analyses of an empirical primates dataset, which reveal that estimates of branch-specific diversification rates are robust to the assumed prior distribution on the number of diversification-rate shifts. Our implementation of the BDS model in RevBayes provides biologists with a flexible approach for estimating branch-specific diversification rates under a statistically coherent model.

Microorganisms have profoundly shaped Earth's biological and geological history, from the origins of oxygenic photosynthesis to present-day global biogeochemical cycles. Metagenomics-through its ability to recover genomic information directly from environmental samples-has revolutionized our understanding of microbial evolution by uncovering unbeknownst lineages, revealing functional adaptations, and reshaping our view of the Tree of Life. By bypassing the need for cultivation, shotgun metagenomics and metabarcoding approaches have enabled researchers to investigate microbial diversity, ecology, and evolutionary processes across aquatic, terrestrial, extreme, and host-associated environments. This review highlights recent advances in evolutionary biology driven by metagenomics, including studies on deep evolutionary branching events, microbial adaptation to extreme environments, the evolution of host-associated microbiomes, and the emergence and spread of pathogens and antimicrobial resistance. The integration of ancient DNA has expanded our ability to reconstruct past ecosystems and disease dynamics, offering insights into long-term microbial evolution. In parallel, studies of microbial domestication and urban settings reveal how human practices have shaped microbial genomes over millennia. Despite significant progress, key challenges remain-including improving bioinformatic tools for degraded ancient DNA, resolving deep phylogenetic relationships, identifying adaptive variants, and linking genomic shifts to ecosystem-level processes. The future of microbial evolutionary research will depend on combining longitudinal metagenomic data, experimental evolution, functional assays, and predictive modeling to better understand microbial responses to climate change and anthropogenic pressures. Together, these approaches will deepen our understanding of microbial evolution and its consequences for life on Earth-past, present, and future.

BACKGROUND: Cable bacteria are filamentous, sulphur-oxidizing microorganisms of the Desulfobulbaceae family that conduct electrons over centimetre-scale distances, coupling sulphide oxidation in deeper sediments to oxygen reduction near the surface. Geochemical evidence demonstrates high rates of aerobic sulphide oxidation in sediments inhabited by cable bacteria. Still, the underlying physiological and molecular basis of this electrogenic sulphur metabolism remains unresolved. Previous genomic analysis proposed that cable bacteria oxidize sulphide by reversing the canonical dissimilatory sulphite reduction (Dsr) pathway. RESULTS: We evaluated the sulphur metabolism of cable bacteria and related Desulfobulbales through comparative genomics, using an expanded set of 31 quality-filtered cable bacteria genomes, including 7 closed assemblies. We showed that cable bacteria encode a complete Dsr pathway, including the previously missing dsrD and dsrT genes, as well as a novel gene cluster with DsrOP homologues. All Dsr genes were classified as reductive-type, and phylogenetic analyses indicated a close affiliation with those of other Desulfobulbaceae (sulphate-reducing and/or sulphur-disproportionating bacteria). In addition, several other previously unrecognized sulphur-metabolism genes were identified in both cable bacteria and closely related Desulfobulbales, including a novel subtype of sulphide:quinone oxidoreductase (SQR), a putative rhodanese–persulphide dioxygenase fusion (Rho–PDO), and a YTD gene cluster (consisting of five genes) previously proposed to be characteristic of sulphur-disproportionation lineages. Structural predictions indicate that three uncharacterized YTD-encoded proteins assemble into a DsrEFH-like double heterotrimer, albeit with highly divergent, non-orthologous sequences. Finally, we integrated publicly available transcriptomic and proteomic data to confirm the in vivo expression of these genes, with expression patterns mirroring those of Desulfolithobacter dissulfuricans and Desulfurivibrio alkaliphilus. CONCLUSION: Cable bacteria show minimal genetic divergence and little differential expression in their sulphur-metabolism genes compared to related organisms. Together, our findings challenge the idea that sulphide oxidation occurs via a reversed Dsr pathway. We propose a unique sulphur metabolism model for cable bacteria, in which a canonical reductive/disproportionating sulphur-metabolism repertoire (similar to Desulfolithobacter dissulfuricans and Desulfurivibrio alkaliphilus) is coupled to net sulphide oxidation through long-distance electron transport. Key steps include sulphide oxidation to polysulphide by SQR, putative conversion to sulphite via Rho–PDO and/or proteins encoded in the YTD cluster, and subsequent disproportionation through the Dsr pathway, where sulphide re-enters the cycle. Net sulphide oxidation and sulphate production arise because electrons are efficiently drained via long-distance electron transport, effectively coupling the metabolism to oxygen reduction.

Climate warming threatens Arctic permafrost with seasonal cycles of freezing and thawing. Arctic soil microorganisms regulate carbon stocks and greenhouse gas exchanges with the atmosphere, yet their precise seasonal growth and dormancy dynamics, and their responses to permafrost thaw, are not well understood. We thawed frozen Svalbard active layer soil and traced microbial growth using DNA quantitative stable isotope probing with H218O. We observed temporal growth patterns resulting in distinct early (21-day) and late-stage (98-day) growing microbial populations. In particular, Acidobacteriota, Actinobacteriota, Bacteroidota, Proteobacteria, and predatory and epibiont bacterial taxa (such as those affiliated to Bdellovibrionota and Patescibacteria) were identified in the soil active layer as clades that were growing following thawing. Methane concentrations in our microcosms remained low, yet pmoA genes were 18O-labeled, indicating growth of aerobic methane-oxidizing bacteria. Approximately half of the microbial taxa detected did not grow, suggesting that Arctic soils constitute sizeable reservoirs of dormant microorganisms. Our results reveal complex and temporal microbial dormancy, growth, death, predation, and parasitism dynamics in seasonally changing Arctic soils. These processes likely regulate the exchange and storage of soil carbon across the increasingly vulnerable Arctic region.IMPORTANCEMicroorganisms play key roles in transforming soil carbon into greenhouse gases. As Arctic soils warm as a result of climate change, greater depths and expanses of permanently frozen soil are experiencing seasonal thaw. Despite the importance of active soil microorganisms in transforming soil carbon, the seasonal freezing and thawing of Arctic soils and associated dormancy and re-activation of microbial populations are not well constrained. Here, we thawed and incubated active layer (i.e., seasonally thawing) Arctic soil with a stable isotope to directly label the DNA of growing soil microorganisms. We found that half of the microbial diversity did not grow after thaw and that some groups, including the Bacteroidota and predatory bacteria, grew disproportionately. The growing microbial community shifted over time, and bacteria capable of oxidizing methane grew more after prolonged thaw. These findings highlight that dormancy, predation, and variable growth dynamics are important factors determining ecological and biogeochemical processes in thawing Arctic soil.

It has been reported that compost amendment improves atmospheric CH4 uptake of agricultural soils. However, microbes involved as well as the underlying mechanisms responsible for the observed effect remain unclear. Here we identified active methane-oxidizing bacteria (MOB) at (circum-) atmospheric CH4 concentrations in agricultural soils amended with green compost, and investigated three complementary hypotheses: (i) atmospheric CH4 consumption is driven by highly activated, flush-feeding MOB; (ii) stimulation of internal CH4 production which fuels flush-feeding methanotrophic activity; and (iii) increased availability of H2 that can serve as additional energy source for mixotrophic methanotrophy. First, we showed that MOB previously activated by exposure to high CH4 concentrations can subsequently oxidize atmospheric CH4 via the flush-feeding lifestyle. Second, no internal CH4 production in soil was observed following compost amendment, likely due to lack of suitable substrates for methanogenesis. Third, provision of elevated H2 concentrations did not affect the concurrent atmospheric CH4 oxidation. Phospholipid fatty acid-stable isotope probing revealed that four distinct MOB groups were active at (circum-) atmospheric CH4 concentrations in agricultural soils and green compost: Methylocaldum sp., Methylosinus sporium, Methylocystis sp./Methylosinus trichosporium, and USCα. These findings enhance our understanding of methanotroph ecology and can be used to craft more effective strategies of creating "climate-smart" soils.