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Abstract SummaryGenome-centric analysis of metagenomic samples is a powerful method for understanding the function of microbial communities. Calculating read coverage is a central part of analysis, enabling differential coverage binning for recovery of genomes and estimation of microbial community composition. Coverage is determined by processing read alignments to reference sequences of either contigs or genomes. Per-reference coverage is typically calculated in an ad-hoc manner, with each software package providing its own implementation and specific definition of coverage. Here we present a unified software package CoverM which calculates several coverage statistics for contigs and genomes in an ergonomic and flexible manner. It uses “Mosdepth arrays” for computational efficiency and avoids unnecessary I/O overhead by calculating coverage statistics from streamed read alignment results. Availability and implementationCoverM is free software available at https://github.com/wwood/coverm. CoverM is implemented in Rust, with Python (https://github.com/apcamargo/pycoverm) and Julia (https://github.com/JuliaBinaryWrappers/CoverM_jll.jl) interfaces. 

Abstract The dynamics of methane (CH₄) cycling in high-latitude peatlands through different pathways of methanogenesis and methanotrophy are still poorly understood due to the spatiotemporal complexity of microbial activities and biogeochemical processes. Additionally, long-termin situmeasurements within soil columns are limited and associated with large uncertainties in microbial substrates (e.g. dissolved organic carbon, acetate, hydrogen). To better understand CH₄cycling dynamics, we first applied an advanced biogeochemical model,ecosys, to explicitly simulate methanogenesis, methanotrophy, and CH₄transport in a high-latitude fen (within the Stordalen Mire, northern Sweden). Next, to explore the vertical heterogeneity in CH₄cycling, we applied the PCMCI/PCMCI+ causal detection framework with a bootstrap aggregation method to the modeling results, characterizing causal relationships among regulating factors (e.g. temperature, microbial biomass, soil substrate concentrations) through acetoclastic methanogenesis, hydrogenotrophic methanogenesis, and methanotrophy, across three depth intervals (0–10 cm, 10–20 cm, 20–30 cm). Our results indicate that temperature, microbial biomass, and methanogenesis and methanotrophy substrates exhibit significant vertical variations within the soil column. Soil temperature demonstrates strong causal relationships with both biomass and substrate concentrations at the shallower depth (0–10 cm), while these causal relationships decrease significantly at the deeper depth within the two methanogenesis pathways. In contrast, soil substrate concentrations show significantly greater causal relationships with depth, suggesting the substantial influence of substrates on CH₄cycling. CH₄production is found to peak in August, while CH₄oxidation peaks predominantly in October, showing a lag response between production and oxidation. Overall, this research provides important insights into the causal mechanisms modulating CH₄cycling across different depths, which will improve carbon cycling predictions, and guide the future field measurement strategies. 

Abstract Soil microorganisms are pivotal in the global carbon cycle, but the viruses that affect them and their impact on ecosystems are less understood. In this study, we explored the diversity, dynamics, and ecology of soil viruses through 379 metagenomes collected annually from 2010 to 2017. These samples spanned the seasonally thawed active layer of a permafrost thaw gradient, which included palsa, bog, and fen habitats. We identified 5051 virus operational taxonomic units (vOTUs), doubling the known viruses for this site. These vOTUs were largely ephemeral within habitats, suggesting a turnover at the vOTU level from year to year. While the diversity varied by thaw stage and depth‐related patterns were specific to each habitat, the virus communities did not significantly change over time. The abundance ratios of virus to host at the phylum level did not show consistent trends across the thaw gradient, depth, or time. To assess potential ecosystem impacts, we predicted hostsin silicoand found viruses linked to microbial lineages involved in the carbon cycle, such as methanotrophy and methanogenesis. This included the identification of viruses ofCandidatusMethanoflorens, a significant global methane contributor. We also detected a variety of potential auxiliary metabolic genes, including 24 carbon‐degrading glycoside hydrolases, six of which are uniquely terrestrial. In conclusion, these long‐term observations enhance our understanding of soil viruses in the context of climate‐relevant processes and provide opportunities to explore their role in terrestrial carbon cycling. 

Abstract With rising global temperatures, permafrost carbon stores are vulnerable to microbial degradation. The enzyme latch theory states that polyphenols should accumulate in saturated peatlands due to diminished phenol oxidase activity, inhibiting resident microbes and promoting carbon stabilization. Pairing microbiome and geochemical measurements along a permafrost thaw-induced saturation gradient in Stordalen Mire, a model Arctic peatland, we confirmed a negative relationship between phenol oxidase expression and saturation but failed to support other trends predicted by the enzyme latch. To inventory alternative polyphenol removal strategies, we built CAMPER, a gene annotation tool leveraging polyphenol enzyme knowledge gleaned across microbial ecosystems. Applying CAMPER to genome-resolved metatranscriptomes, we identified genes for diverse polyphenol-active enzymes expressed by various microbial lineages under a range of redox conditions. This shifts the paradigm that polyphenols stabilize carbon in saturated soils and highlights the need to consider both oxic and anoxic polyphenol metabolisms to understand carbon cycling in changing ecosystems. 

ABSTRACT While wetlands are major sources of biogenic methane (CH₄), our understanding of resident microbial metabolism is incomplete, which compromises the prediction of CH₄emissions under ongoing climate change. Here, we employed genome-resolved multi-omics to expand our understanding of methanogenesis in the thawing permafrost peatland of Stordalen Mire in Arctic Sweden. In quadrupling the genomic representation of the site’s methanogens and examining their encoded metabolism, we revealed that nearly 20% of the metagenome-assembled genomes (MAGs) encoded the potential for methylotrophic methanogenesis. Further, 27% of the transcriptionally active methanogens expressed methylotrophic genes; forMethanosarcinalesandMethanobacterialesMAGs, these data indicated the use of methylated oxygen compounds (e.g., methanol), while forMethanomassiliicoccales, they primarily implicated methyl sulfides and methylamines. In addition to methanogenic methylotrophy, >1,700 bacterial MAGs across 19 phyla encoded anaerobic methylotrophic potential, with expression across 12 phyla. Metabolomic analyses revealed the presence of diverse methylated compounds in the Mire, including some known methylotrophic substrates. Active methylotrophy was observed across all stages of a permafrost thaw gradient in Stordalen, with the most frozen non-methanogenic palsa found to host bacterial methylotrophy and the partially thawed bog and fully thawed fen seen to house both methanogenic and bacterial methylotrophic activities. Methanogenesis across increasing permafrost thaw is thus revised from the sole dominance of hydrogenotrophic production and the appearance of acetoclastic at full thaw to consider the co-occurrence of methylotrophy throughout. Collectively, these findings indicate that methanogenic and bacterial methylotrophy may be an important and previously underappreciated component of carbon cycling and emissions in these rapidly changing wetland habitats. IMPORTANCEWetlands are the biggest natural source of atmospheric methane (CH₄) emissions, yet we have an incomplete understanding of the suite of microbial metabolism that results in CH₄formation. Specifically, methanogenesis from methylated compounds is excluded from all ecosystem models used to predict wetland contributions to the global CH₄budget. Though recent studies have shown methylotrophic methanogenesis to be active across wetlands, the broad climatic importance of the metabolism remains critically understudied. Further, some methylotrophic bacteria are known to produce methanogenic by-products like acetate, increasing the complexity of the microbial methylotrophic metabolic network. Prior studies of Stordalen Mire have suggested that methylotrophic methanogenesis is irrelevantin situand have not emphasized the bacterial capacity for metabolism, both of which we countered in this study. The importance of our findings lies in the significant advancement toward unraveling the broader impact of methylotrophs in wetland methanogenesis and, consequently, their contribution to the terrestrial global carbon cycle. 

Abstract Northern peatlands are a globally significant source of methane (CH₄), and emissions are projected to increase due to warming and permafrost loss. Understanding the microbial mechanisms behind patterns in CH₄production in peatlands will be key to predicting annual emissions changes, with stable carbon isotopes (δ¹³C‐CH₄) being a powerful tool for characterizing these drivers. Given that δ¹³C‐CH₄is used in top‐down atmospheric inversion models to partition sources, our ability to model CH₄production pathways and associated δ¹³C‐CH₄values is critical. We sought to characterize the role of environmental conditions, including hydrologic and vegetation patterns associated with permafrost thaw, on δ¹³C‐CH₄values from high‐latitude peatlands. We measured porewater and emitted CH₄stable isotopes, pH, and vegetation composition from five boreal‐Arctic peatlands. Porewater δ¹³C‐CH₄was strongly associated with peatland type, with δ¹³C enriched values obtained from more minerotrophic fens (−61.2 ± 9.1‰) compared to permafrost‐free bogs (−74.1 ± 9.4‰) and raised permafrost bogs (−81.6 ± 11.5‰). Variation in porewater δ¹³C‐CH₄was best explained by sedge cover, CH₄concentration, and the interactive effect of peatland type and pH (r² = 0.50,p < 0.001). Emitted δ¹³C‐CH₄varied greatly but was positively correlated with porewater δ¹³C‐CH₄. We calculated a mixed atmospheric δ¹³C‐CH₄value for northern peatlands of −65.3 ± 7‰ and show that this value is more sensitive to landscape drying than wetting under permafrost thaw scenarios. Our results suggest northern peatland δ¹³C‐CH₄values are likely to shift in the future which has important implications for source partitioning in atmospheric inversion models. 

Abstract Quantifying the temperature sensitivity of methane (CH₄) production is crucial for predicting how wetland ecosystems will respond to climate warming. Typically, the temperature sensitivity (often quantified as a Q₁₀value) is derived from laboratory incubation studies and then used in biogeochemical models. However, studies report wide variation in incubation-inferred Q₁₀values, with a large portion of this variation remaining unexplained. Here we applied observations in a thawing permafrost peatland (Stordalen Mire) and a well-tested process-rich model (ecosys) to interpret incubation observations and investigate controls on inferred CH₄production temperature sensitivity. We developed a field-storage-incubation modeling approach to mimic the full incubation sequence, including field sampling at a particular time in the growing season, refrigerated storage, and laboratory incubation, followed by model evaluation. We found that CH₄production rates during incubation are regulated by substrate availability and active microbial biomass of key microbial functional groups, which are affected by soil storage duration and temperature. Seasonal variation in substrate availability and active microbial biomass of key microbial functional groups led to strong time-of-sampling impacts on CH₄production. CH₄production is higher with less perturbation post-sampling, i.e. shorter storage duration and lower storage temperature. We found a wide range of inferred Q₁₀values (1.2–3.5), which we attribute to incubation temperatures, incubation duration, storage duration, and sampling time. We also show that Q₁₀values of CH₄production are controlled by interacting biological, biochemical, and physical processes, which cause the inferred Q₁₀values to differ substantially from those of the component processes. Terrestrial ecosystem models that use a constant Q₁₀value to represent temperature responses may therefore predict biased soil carbon cycling under future climate scenarios. 

Abstract Northern post-glacial lakes are significant, increasing sources of atmospheric carbon through ebullition (bubbling) of microbially-produced methane (CH₄) from sediments. Ebullitive CH₄flux correlates strongly with temperature, reflecting that solar radiation drives emissions. However, here we show that the slope of the temperature-CH₄flux relationship differs spatially across two post-glacial lakes in Sweden. We compared these CH₄emission patterns with sediment microbial (metagenomic and amplicon), isotopic, and geochemical data. The temperature-associated increase in CH₄emissions was greater in lake middles—where methanogens were more abundant—than edges, and sediment communities were distinct between edges and middles. Microbial abundances, including those of CH₄-cycling microorganisms and syntrophs, were predictive of porewater CH₄concentrations. Results suggest that deeper lake regions, which currently emit less CH₄than shallower edges, could add substantially to CH₄emissions in a warmer Arctic and that CH₄emission predictions may be improved by accounting for spatial variations in sediment microbiota. 

Abstract Changes in the sequence of an organism’s genome, i.e., mutations, are the raw material of evolution. The frequency and location of mutations can be constrained by specific molecular mechanisms, such as diversity-generating retroelements (DGRs). DGRs have been characterized from cultivated bacteria and bacteriophages, and perform error-prone reverse transcription leading to mutations being introduced in specific target genes. DGR loci were also identified in several metagenomes, but the ecological roles and evolutionary drivers of these DGRs remain poorly understood. Here, we analyze a dataset of >30,000 DGRs from public metagenomes, establish six major lineages of DGRs including three primarily encoded by phages and seemingly used to diversify host attachment proteins, and demonstrate that DGRs are broadly active and responsible for >10% of all amino acid changes in some organisms. Overall, these results highlight the constraints under which DGRs evolve, and elucidate several distinct roles these elements play in natural communities. 

Abstract Stordalen Mire is a peatland in the discontinuous permafrost zone in arctic Sweden that exhibits a habitat gradient from permafrost palsa, toSphagnumbog underlain by permafrost, toEriophorum‐dominated fully thawed fen. We used three independent approaches to evaluate the annual, multi‐decadal, and millennial apparent carbon accumulation rates (aCAR) across this gradient: seven years of direct semi‐continuous measurement of CO₂and CH₄exchange, and 21 core profiles for²¹⁰Pb and¹⁴C peat dating. Year‐round chamber measurements indicated net carbon balance of −13 ± 8, −49 ± 15, and −91 ± 43 g C m⁻² y⁻¹for the years 2012–2018 in palsa, bog, and fen, respectively. Methane emission offset 2%, 7%, and 17% of the CO₂uptake rate across this gradient. Recent aCAR indicates higher C accumulation rates in surface peats in the palsa and bog compared to current CO₂fluxes, but these assessments are more similar in the fen. aCAR increased from low millennial‐scale levels (17–29 g C m⁻² y⁻¹) to moderate aCAR of the past century (72–81 g C m⁻² y⁻¹) to higher recent aCAR of 90–147 g C m⁻² y⁻¹. Recent permafrost collapse, greater inundation and vegetation response has made the landscape a stronger CO₂sink, but this CO₂sink is increasingly offset by rising CH₄emissions, dominated by modern carbon as determined by¹⁴C. The higher CH₄emissions result in higher net CO_{2‐equivalent}emissions, indicating that radiative forcing of this mire and similar permafrost ecosystems will exert a warming influence on future climate.