Methane dynamics regulated by microbial community response to permafrost thaw. 4,5,16 . The net effect is that the high methane-emitting fen contributes 7 55 times the greenhouse impact per unit area as the palsa. This thaw progression is also associated 56 with an increase in overall organic matter lability, including a decrease in C:N and an increase in 57 humification rates 9 . We hypothesized, consistent with previous studies of in situ bog and fen 58 systems [17][18][19] , that thaw progression also facilitates a shift from hydrogenotrophic to acetoclastic 59 CH 4 production. 60We used the distinct isotopic signatures of different microbial CH 4 production and 61 consumption pathways to directly relate changes in CH 4 dynamics across the thaw gradient to 62 underlying changes in the microbial community. Methane produced by hydrogenotrophic 63 methanogens generally has lower  13 C and higher D ( 13 C = -110 to -60‰ and D = -250 to -64 170‰) relative to that produced by acetoclastic methanogens ( 13 C = -60 to -50‰ and D = -400 65 to -250‰) 19,20 . If methanotrophic microbes then oxidize CH 4 , lighter molecules are 66 preferentially consumed, leaving the remaining CH 4 13 C-and D-enriched relative to the original 67 CH 4 pool (see expected patterns in Extended Data Fig 1) 19 . Greater fractionation is associated with hydrogenotrophic methanogenesis, and was 85 found in the thawing Sphagnum site (average  C = 1.053 ± 0.002). Significantly less 86 fractionation (p=0.002) associated with more acetoclastic production or with consumption by 87 oxidation was found in the fully thawed Eriophorum porewater (average  C = 1.046 ± 0.001). 88Here, increases in acetoclastic production, not oxidation, best explain isotopic shifts because 89 lower  C and higher  13 C-CH 4 are accompanied by significantly lower D-CH 4 (Extended Data 90 Fig. 1, p< 0.001) 19 . This is consistent with the pattern of isotopes in CH 4 emissions as well as 91 incubations of Stordalen peat 9 and studies showing bog-to-fen shifts from hydrogenotrophic to 92 acetoclastic methanogenesis [17][18][19] . 93The CH 4 flux and isotope results provide compelling but indirect evidence for changes in 94 CH 4 -cycling microbial communities with permafrost thaw. These microbiological changes could 95 be shifts in activity of particular community members or changes in community composition. We 96 examined the role of community composition through 16S rRNA gene amplicon sequencing. All 97 known methanogens belong to a small number of archaeal lineages within the Euryarchaeota 23 . 98As expected, the shift from CH 4 -neutral intact permafrost palsa to CH 4 -emitting wetland 99 corresponded to a substantial increase in the relative abundance of methanogenic archaeal 100 lineages (Fig. 1c, Extended Data Table 2,3). In the aerobic palsa and surface Sphagnum habitats, 101 methanogens were found in low relative abundance (average <0.6%), while the anaerobic 102 environments of the Eriophorum and deeper (below the water table) Sphagnum habitats harbored 10...

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Most free-living planktonic cells are streamlined and in spite of their limitations in functional flexibility, their vast populations have radiated into a wide range of aquatic habitats. Here we compared the metabolic potential of subgroups in the Alphaproteobacteria lineage SAR11 adapted to marine and freshwater habitats. Our results suggest that the successful leap from marine to freshwaters in SAR11 was accompanied by a loss of several carbon degradation pathways and a rewiring of the central metabolism. Examples for these are C1 and methylated compounds degradation pathways, the Entner–Doudouroff pathway, the glyoxylate shunt and anapleuretic carbon fixation being absent from the freshwater genomes. Evolutionary reconstructions further suggest that the metabolic modules making up these important freshwater metabolic traits were already present in the gene pool of ancestral marine SAR11 populations. The loss of the glyoxylate shunt had already occurred in the common ancestor of the freshwater subgroup and its closest marine relatives, suggesting that the adaptation to freshwater was a gradual process. Furthermore, our results indicate rapid evolution of TRAP transporters in the freshwater clade involved in the uptake of low molecular weight carboxylic acids. We propose that such gradual tuning of metabolic pathways and transporters toward locally available organic substrates is linked to the formation of subgroups within the SAR11 clade and that this process was critical for the freshwater clade to find and fix an adaptive phenotype.

Biogenic production and release of methane (CH ) from thawing permafrost has the potential to be a strong source of radiative forcing. We investigated changes in the active layer microbial community of three sites representative of distinct permafrost thaw stages at a palsa mire in northern Sweden. The palsa site (intact permafrost and low radiative forcing signature) had a phylogenetically clustered community dominated by Acidobacteria and Proteobacteria. The bog (thawing permafrost and low radiative forcing signature) had lower alpha diversity and midrange phylogenetic clustering, characteristic of ecosystem disturbance affecting habitat filtering. Hydrogenotrophic methanogens and Acidobacteria dominated the bog shifting from palsa-like to fen-like at the waterline. The fen (no underlying permafrost, high radiative forcing signature) had the highest alpha, beta and phylogenetic diversity, was dominated by Proteobacteria and Euryarchaeota and was significantly enriched in methanogens. The Mire microbial network was modular with module cores consisting of clusters of Acidobacteria, Euryarchaeota or Xanthomonodales. Loss of underlying permafrost with associated hydrological shifts correlated to changes in microbial composition, alpha, beta and phylogenetic diversity associated with a higher radiative forcing signature. These results support the complex role of microbial interactions in mediating carbon budget changes and climate feedback in response to climate forcing.

SummaryBiogenic production and release of methane (CH4) from thawing permafrost has the potential to be a strong source of radiative forcing. We investigated changes in the active layer microbial community of three sites representative of distinct permafrost thaw stages at a palsa mire in northern Sweden. The palsa sites with intact permafrost, and low radiative forcing signature had a phylogenetically clustered community dominated by Acidobacteria and Proteobacteria. The bog with thawing permafrost and low radiative forcing signature was dominated by hydrogenotrophic methanogens and Acidobacteria, had lower alpha diversity, and midrange phylogenetic clustering, characteristic of ecosystem disturbance affecting habitat filtering, shifting from palsa-like to fen-like at the waterline. The fen had no underlying permafrost, and the highest alpha, beta and phylogenetic diversity, was dominated by Proteobacteria and Euryarchaeota, and was significantly enriched in methanogens. The mire microbial network was modular with module cores consisting of clusters of Acidobacteria, Euryarchaeota, or Xanthomonodales. Loss of underlying permafrost with associated hydrological shifts correlated to changes in microbial composition, alpha, beta, and phylogenetic diversity associated with a higher radiative forcing signature. These results support the complex role of microbial interactions in mediating carbon budget changes and climate feedback in response to climate forcing.

While fastidious microbes can be abundant and ubiquitous in their natural communities, many fail to grow axenically in laboratories due to auxotrophies or other dependencies. To overcome auxotrophies, these microbes rely on their surrounding cohort. A cohort may consist of kin (ecotypes) or more distantly related organisms (community) with the cooperation being reciprocal or nonreciprocal and expensive (Black Queen hypothesis) or costless (by-product). These metabolic partnerships (whether at single species population or community level) enable dominance by and coexistence of these lineages in nature. Here we examine the relevance of these cooperation models to explain the abundance and ubiquity of the dominant fastidious bacterioplankton of a dimictic mesotrophic freshwater lake. Using both culture-dependent (dilution mixed cultures) and culture-independent (small subunit [SSU] rRNA gene time series and environmental metagenomics) methods, we independently identified the primary cohorts of actinobacterial genera “Candidatus Planktophila” (acI-A) and “Candidatus Nanopelagicus” (acI-B) and the proteobacterial genus “Candidatus Fonsibacter” (LD12). While “Ca. Planktophila” and “Ca. Fonsibacter” had no correlation in their natural habitat, they have the potential to be complementary in laboratory settings. We also investigated the bifunctional catalase-peroxidase enzyme KatG (a common good which “Ca. Planktophila” is dependent upon) and its most likely providers in the lake. Further, we found that while ecotype and community cooperation combined may explain “Ca. Planktophila” population abundance, the success of “Ca. Nanopelagicus” and “Ca. Fonsibacter” is better explained as a community by-product. Ecotype differentiation of “Ca. Fonsibacter” as a means of escaping predation was supported but not for overcoming auxotrophies. IMPORTANCE This study examines evolutionary and ecological relationships of three of the most ubiquitous and abundant freshwater bacterial genera: “Ca. Planktophila” (acI-A), “Ca. Nanopelagicus” (acI-B), and “Ca. Fonsibacter” (LD12). Due to high abundance, these genera might have a significant influence on nutrient cycling in freshwaters worldwide, and this study adds a layer of understanding to how seemingly competing clades of bacteria can coexist by having different cooperation strategies. Our synthesis ties together network and ecological theory with empirical evidence and lays out a framework for how the functioning of populations within complex microbial communities can be studied.

Functional attributes of microbial communities are difficult to study, and most current techniques rely on DNA-and rRNA-based profiling of taxa and genes, including microarrays containing sequences of known microorganisms. To quantify gene expression in environmental samples in a culture-independent manner, we constructed an environmental functional gene microarray (E-FGA) consisting of 13,056 mRNA-enriched anonymous microbial clones from diverse microbial communities to profile microbial gene transcripts. A new normalization method using internal spot standards was devised to overcome spotting and hybridization bias, enabling direct comparisons of microarrays. To evaluate potential applications of this metatranscriptomic approach for studying microbes in environmental samples, we tested the E-FGA by profiling the microbial activity of agricultural soils with a low or high flux of N 2 O. A total of 109 genes displayed expression that differed significantly between soils with low and high N 2 O emissions. We conclude that mRNA-based approaches such as the one presented here may complement existing techniques for assessing functional attributes of microbial communities.

Modern microbial and ecosystem sciences require diverse interdisciplinary teams that are often challenged in “speaking” to one another due to different languages and data product types. Here we introduce the IsoGenie Database (IsoGenieDB; https://isogenie-db.asc.ohio-state.edu/), a de novo developed data management and exploration platform, as a solution to this challenge of accurately representing and integrating heterogenous environmental and microbial data across ecosystem scales. The IsoGenieDB is a public and private data infrastructure designed to store and query data generated by the IsoGenie Project, a ~10 year DOE-funded project focused on discovering ecosystem climate feedbacks in a thawing permafrost landscape. The IsoGenieDB provides (i) a platform for IsoGenie Project members to explore the project’s interdisciplinary datasets across scales through the inherent relationships among data entities, (ii) a framework to consolidate and harmonize the datasets needed by the team’s modelers, and (iii) a public venue that leverages the same spatially explicit, disciplinarily integrated data structure to share published datasets. The IsoGenieDB is also being expanded to cover the NASA-funded Archaea to Atmosphere (A2A) project, which scales the findings of IsoGenie to a broader suite of Arctic peatlands, via the umbrella A2A Database (A2A-DB). The IsoGenieDB’s expandability and flexible architecture allow it to serve as an example ecosystems database.