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Wetlands are the largest natural source of atmospheric methane 1 , the second most important greenhouse gas 2 . Methane flux to the atmosphere depends strongly on the climate 3 ; however, by far the largest part of the methane formed in wetland ecosystems is recycled and does not reach the atmosphere 4,5 . The biogeochemical controls on the efficient oxidation of methane are still poorly understood. Here we show that submerged Sphagnum mosses, the dominant plants in some of these habitats, consume methane through symbiosis with partly endophytic methanotrophic bacteria, leading to highly effective in situ methane recycling. Molecular probes revealed the presence of the bacteria in the hyaline cells of the plant and on stem leaves. Incubation with 13 C-methane showed rapid in situ oxidation by these bacteria to carbon dioxide, which was subsequently fixed by Sphagnum, as shown by incorporation of 13 C-methane into plant sterols. In this way, methane acts as a significant (10-15%) carbon source for Sphagnum. The symbiosis explains both the efficient recycling of methane and the high organic carbon burial in these wetland ecosystems.Peat bogs alternate between lawns and pools. Lawns are dominated by species that grow up to several decimetres above the water table. Pools are dominated by aquatic species, such as Sphagnum cuspidatum, that form layers of living plants below the water table. We investigated the methane-oxidizing activity of submerged S. cuspidatum from peat bog pools at different field locations in the Netherlands, and compared it to the activity of S. magellanicum and S. papillosum growing in lawns. The potential methane-oxidizing activity was substantially higher in the submerged mosses (Fig. 1). In control experiments with bog water, methane was not oxidized, indicating that the methanotrophic bacteria were mainly present on or in the living Sphagnum tissue.The identity and location of these methanotrophs was determined in a molecular approach. Total genomic DNA from washed Sphagnum plants was isolated and bacterial 16S ribosomal RNA genes were amplified, cloned into Escherichia coli, sequenced and analysed phylogenetically. One of the 16S rRNA gene sequences of the clone library was affiliated to a cluster of type II methanotrophs that contained acidophilic methanotrophs isolated from Sphagnum bogs, such as Methylocella palustris (identity 93%) 6 and Methylocapsa acidiphila (identity 93%) 7 .The full 16S rRNA gene sequence was used to design two specific oligonucleotide probes for fluorescence in situ hybridization (FISH). FISH was combined with serial sectioning of the stems and the stem leaves of multiple individuals of submerged S. cuspidatum. The methanotrophic bacterium targeted by the probes was the dominant methanotroph in S. cuspidatum sections, accounting for over 75% of

Sphagnum peatlands are important ecosystems in the methane cycle. Methane-oxidizing bacteria in these ecosystems serve as a methane filter and limit methane emissions. Yet little is known about the diversity and identity of the methanotrophs present in and on Sphagnum mosses of peatlands, and only a few isolates are known. The methanotrophic community in Sphagnum mosses, originating from a Dutch peat bog, was investigated using a pmoA microarray. A high biodiversity of both gamma-and alphaproteobacterial methanotrophs was found. With Sphagnum mosses as the inoculum, alpha-and gammaproteobacterial acidophilic methanotrophs were isolated using established and newly designed media. The 16S rRNA, pmoA, pxmA, and mmoX gene sequences showed that the alphaproteobacterial isolates belonged to the Methylocystis and Methylosinus genera. The Methylosinus species isolated are the first acid-tolerant members of this genus. Of the acidophilic gammaproteobacterial strains isolated, strain M5 was affiliated with the Methylomonas genus, and the other strain, M200, may represent a novel genus, most closely related to the genera Methylosoma and Methylovulum. So far, no acidophilic or acid-tolerant methanotrophs in the Gammaproteobacteria class are known. All strains showed the typical features of either type I or II methanotrophs and are, to the best of our knowledge, the first isolated (acidophilic or acid-tolerant) methanotrophs from Sphagnum mosses.Methane is an important greenhouse gas, and its concentration has been rising rapidly since industrial times (31). Methanotrophs are a sink for methane and occur in many different ecosystems like rice paddies, soils, volcanic areas, and peat bogs (17,33,55). Acidic peat bogs are the most extensive type of wetland, occupying about 3% of the total land area and storing an enormous amount of carbon (32). Methanotrophs present in these peatlands can act as a filter for methane, thereby reducing its emissions from these wetlands (56, 57). The biodiversity of methanotrophic communities can be investigated using PCR with primers targeting the 16S rRNA gene or functional genes like the methane monooxygenase genes pmoA, pxmA, and mmoX (18,47,68). To quickly screen the methanotrophic community of an ecosystem, a microarray technique based on the pmoA gene has been developed (8). The primers used in this technique are based on pmoA sequences of currently available cultivated methanotrophs and some uncultivated methanotrophs, and therefore, culture-dependent studies, e.g., isolation of new strains, remain important to expand our knowledge on the microbial methanotrophic communities. Molecular analysis of the endophytic methanotrophic community of Sphagnum mosses is sparse. Recently the abundance of both alpha-and gammaproteobacterial methanotrophs within this community was described (43,44). Other molecular surveys focused on peat lands (soil, mires, etc.) rather than mosses (3,16,22,81).Aerobic methanotrophs occur within the Alpha-and Gammaproteobacteria classes and Verrucomicrobia (17,52,55). ...