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.