Predominance of methanogens over methanotrophs contributes 1 to high methane emissions in rewetted fens 2

The rewetting of drained peatlands alters peat geochemistry and often leads to sustained 27 elevated methane emission. Although this methane is produced entirely by microbial activity, the 28 distribution and abundance of methane-cycling microbes in rewetted peatlands, especially in fens, 29 is rarely described. In this study, we compare the community composition and abundance of 30 methane-cycling microbes in relation to peat porewater geochemistry in two rewetted fens in 31 northeastern Germany, a coastal brackish fen and a freshwater riparian fen, with known high 32 methane fluxes. We utilized 16S rDNA high-throughput sequencing and quantitative polymerase 33 Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-184 Manuscript under review for journal Biogeosciences Discussion started: 26 April 2018 c © Author(s) 2018. CC BY 4.0 License.


Introduction
Rewetting is a technique commonly employed to restore ecological and biogeochemical functioning of drained fens.However, while rewetting may reduce carbon dioxide (CO2) emissions (Wilson et al. 2016), it often increases methane (CH4) emissions in peatlands that remain mostly inundated following rewetting.The factors that contribute to the magnitude and duration of this increase are still uncertain (Joosten et al. 2015, Abdalla et al. 2016).On a 100-year time scale CH4 has a global warming potential 28 times stronger than CO2 (Myhre et al. 2013); thus, increased CH4 emissions could potentially offset the benefit of decreased CO2 emissions (Jurasinski et al. 2016).Although a recent increase in rewetting projects in Germany and other European nations has prompted a number of studies of methane cycling in rewetted peatlands (e.g., Jerman et al. 2009, Hahn-Schöfl et al. 2011, Urbanová et al. 2013, Hahn et al. 2015, Vanselow-Algan et al. 2015, Zak et al. 2015, Emsens et al. 2016), the post-rewetting distribution and abundance of methane-cycling microbes in rewetted fens has seldom been examined (but see Juottonen et al. 2012, Urbanová et al. 2013).
Peat CH4 production and release is governed by a complex array of interrelated factors including climate, water level, plant community, nutrient status, site geochemistry, and the activity of microbes (i.e.bacteria and archaea) that use organic carbon as energy source (Segers 1998, Abdalla et al. 2016).To date, the vast majority of studies in rewetted fens have focused on quantifying CH4 Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-184Manuscript under review for journal Biogeosciences Discussion started: 26 April 2018 c Author(s) 2018.CC BY 4.0 License.emission rates in association with environmental variables such as water level, plant community, and aspects of site geochemistry (Abdalla et al. 2016).Site geochemistry indeed plays an important role for methanogenic communities, as methanogenesis is suppressed in presence of thermodynamically more favorable terminal electron acceptors (TEAs, Blodau 2011).Due to a smaller pool of more favorable electron acceptors and high availability of carbon substrates, organic-rich soils such as peat rapidly establish methanogenic conditions when anoxic (Segers 1998, Keller and Bridgham 2007, Knorr and Blodau 2009).Despite their decisive role as producers (i.e.methanogens) and consumers (i.e.methanotrophs) of CH4 (Conrad 1996), only a few studies have combined a characterization of the CH4-cycling microbial community, site geochemistry, and observed patterns of CH4 production.Existing studies have been conducted in oligotrophic and mesotrophic boreal fens (e.g., Juottonen et al. 2005, Yrjälä et al. 2011, Juottonen et al. 2012), alpine fens (e.g., Liebner et al. 2012, Urbanová et al. 2013, Cheema et al. 2015, Franchini et al. 2015), subarctic fens (Liebner et al. 2015), and incubation experiments (e.g., Jerman et al. 2009, Knorr and Blodau 2009, Urbanová et al. 2011, Emsens et al. 2016).Several studies on CH4-cycling microbial communities have been conducted in minerotrophic temperate fens (e.g., Cadillo-Quiroz et al. 2008, Liu et al. 2011, Sun et al. 2012, Zhou et al. 2017), but these sites were not subject to drainage or rewetting.To our knowledge, only one study has directly compared in situ abundances of methanogens and methanotrophs in drained versus rewetted fens (Juottonen et al. 2012).The studied sites, however, were nutrient-poor fens with acidic conditions.
While studies of nutrient-poor and mesotrophic boreal fens have documented post-rewetting CH4 emissions comparable to or lower than at pristine sites (Komulainen et al. 1998, Tuittila et al. 2000, Juottonen et al 2012), studies of temperate nutrient-rich fens have reported post-flooding CH4 emissions dramatically exceeding emissions in pristine fens (e.g., Augustin andChojnicki 2008, Hahn et al. 2015).These high emissions typically occur together with a significant dieback in vegetation, a mobilization of nutrients and electron acceptors in the upper peat layer, and increased availability of dissolved organic matter (Zak andGelbrecht 2007, Hahn-Schöfl et al. 2011, Hahn Biogeosciences Discuss., et al. 2015, Jurasinski et al. 2016). Vanselow-Algan et al. (2015) have shown that such high CH4 fluxes may continue for decades following rewetting even in bogs.Because of their potential to remain significant CH4 sources on decadal timescales, there is an urgent need to characterize CH4cycling microbial communities and geochemical conditions in rewetted minerotrophic fens.
Therefore, in this study, we examined microbial community composition and abundance in relation to post-flooding geochemical conditions in two rewetted fens in northeastern Germany.In both fens, CH4 emissions increased dramatically after rewetting (Augustin and Chojnicki 2008, Hahn-Schöfl et al. 2011, Hahn et al. 2015, Jurasinski et al. 2016).Average annual CH4 emissions have decreased in both fens since the initial peak (Franz et al. 2016, Jurasinski et al. 2016).
We expected patterns in microbial community composition would reflect the geochemical conditions of the two sites and hypothesized a high abundance of methanogens relative to methanotrophs in both fens.We also expected acetoclastic methanogens, which typically thrive in nutrient-rich fens (Kelly et al. 1992, Galand 2005), to dominate the methanogenic community in both fens.

Study sites
The nature reserve "Heiligensee and Hütelmoor" ('Hütelmoor' in the following, approx.540 ha, 54°12'36.66"N, 12°10'34.28"E), is a coastal, mainly minerotrophic fen complex in Mecklenburg-Vorpommern (NE Germany) that is separated from the Baltic Sea by a narrow (~100 m and less) dune dike (Fig. 1a and b).The climate is temperate in the transition zone between maritime and continental with an average annual temperature of 9.1 °C and an average annual precipitation of 645 mm (data derived from grid product of the German Weather Service, reference climate period: open water bodies that are colonized by Ceratophyllum demersum in summer (Koch et al. 2017).
Intense draining and land amelioration practices began in the 1970s, which lowered the water level to 1.6 m below ground surface and caused aerobic decomposition and concomitant degradation of the peat (Voigtländer et al. 1996).The upper peat layer varies in depth between 0.6 and 3 m and is highly degraded, reaching up to H10 on the von Post humification scale (Hahn et al. 2015).
Active draining ended in 1992, but dry conditions during summertime kept the water table well below ground surface (Schönfeld-Bockholt et al. 2005, Koebsch et al. 2013) until concerns of prolonged aerobic peat decomposition prompted the installation of a weir in 2009 at the outflow of the catchment (Weisner and Schernewski 2013).After installation of the weir, the site was fully flooded year-round with an average water level of 0.6 m, and annual average CH4 flux increased ~186-fold from 0.0014 ± 0.0006 kg CH4 m -2 a -1 to 0.26 ± 0.06 kg CH4 m -2 a -1 (Hahn et al. 2015).
The study site polder Zarnekow ('Zarnekow' in the following, approx.500 ha, 53°52'31.10"N, 12°53'19.60"E) is situated in the valley of the River Peene in Mecklenburg-Vorpommern (NE Germany, Fig. 1a and c).The climate is slightly more continental compared to the Hütelmoor, with a mean annual precipitation of 544 mm and a mean annual temperature of 8.7 °C (German Weather Service, meteorological station Teterow, 24 km southwest of the study site; reference period 1981-2010).The fen can be classified as a river valley mire system consisting of spring mires, wider percolation mires, and flood mires along the River Peene.Drainage and low-intensity agricultural use began in the eighteenth century when land-use changed to pastures and grassland.This was intensified by active pumping in the mid-1970s.Due to land subsidence of several decimeters, after rewetting (October 2004) water table depth increased to 0.1-0.5 m above peat surface.The upper horizon is highly decomposed (0-0.3 m), followed by moderately decomposed peat to a  (Steffenhagen et al. 2012).Following flooding, CH4 flux rates increased to ~0.21 kg m -2 a -1 (Augustin and Chojnicki 2008).No pre-rewetting CH4 flux data were available for the Zarnekow site but published CH4 flux rates of representative drained fens from the same region have been shown to be negligible (Augustin et al. 1998).

Collection of peat cores and porewater samples
Peat and porewater samples were collected at four different locations in Hütelmoor (October 2014) and at five locations in Zarnekow (July 2015) and spanned a distance of 1,200 m and 250 m, respectively, to cover the whole lateral extension at each site (Fig. 1b and c).Peat cores were collected with a Perspex liner (ID: 60 mm, Hütelmoor) and a peat auger (Zarnekow).In order to minimize oxygen contamination, the outer layer of the peat core was omitted.Subsamples for molecular analysis were immediately packed in 50 ml sterile Falcon tubes and stored at -80 °C until further processing.
Pore waters in Hütelmoor were collected with a stainless-steel push-point sampler attached to a plastic syringe to recover the samples from 10 cm depth intervals.Samples were immediately filtered with 0.45 µm membrane disposable syringe filters.Pore waters in Zarnekow were sampled with permanently installed dialysis samplers consisting of slotted polypropylene (PP) pipes (length: 636 mm, ID: 34 mm) surrounded with 0.22 µm polyethersulfone membrane.The PP pipes were fixed at distinct peat depths (surface level, 20 and 40 cm depth) and connected with PP tubes (4x6 mm IDxAD).Water samples were drawn out from the dialysis sampler pipes with a syringe through the PP tube.was then converted into a dissolved CH4 concentration using the temperature-corrected solubility coefficient (Wilhelm et al. 1977).Isotopic composition of dissolved CH4 for Hütelmoor was analyzed using the gas chromatography-combustion-technique (GC-C) and the gas chromatography-high-temperature-conversion-technique (GC-HTC).The gas was directly injected in a Gas Chromatograph Agilent 7890A, methane was quantitatively converted to CO2 and the δ 13 C values were then measured with the isotope-ratio-mass-spectrometer MAT-253 (Thermo Finnigan, Germany).The δ 13 C of dissolved methane in Zarnekow was analyzed using a laser-based isotope analyzer equipped with a small sample isotope module for analyses of discrete gas samples (cavity ring down spectroscopy CRDS; Picarro G2201-I, Santa Clara, CA, USA).
Calibration was carried out before, during and after analyses using certified standards of known isotopic composition (obtained from Isometric Instruments, Victoria, BC, Canada, and from Westfalen AG, Münster, Germany).Reproducibility of results was typically +/-1 ‰.In the presence of high concentrations of hydrogen sulfide interfering with laser-based isotope analysis, samples were treated with iron(III) sulfate to oxidize and/or precipitate sulfide.For both sites, sulfate and nitrate concentrations were analyzed by ion chromatography (IC, Thermo Fisher Scientific Dionex) using an Ion Pac AS-9-HC 4 column, partly after dilution of the sample.
Dissolved metal concentrations were analyzed by ICP-OES (iCAP 6300 DUO, Thermo Fisher Scientific).Accuracy and precision were routinely checked with a certified CASS standard as previously described (Kowalski et al. 2012).

Gene amplification and phylogenetic analysis
Genomic DNA was extracted from 0.2-0.3g of duplicates of peat soil per sample using an EurX Illumina sequencing was performed by GATC Biotech AG using 300 bp paired-end mode and a 20% PhiX Control v3 library to counteract the effects of low-diversity sequence libraries.Raw data was demultiplexed using an own script based on CutAdapt (Martin 2011).Ambiguous nucleotides at sequence ends were trimmed and a 10% mismatch was allowed for primer identification, whereas barcode sequences needed to be present without any mismatches and with a minimum Phred-Score of Q25 for each nucleotide.After sorting, overlapping paired-end reads were merged using PEAR [Q25, p 0.0001, v20] (Zhang et al. 2014).The orientation of the merged sequences was standardized according to the barcode information obtained from demultiplexing.
Low-quality reads were removed using Trimmomatic [SE,LEADING Q25,TRAILING Q25,SLIDINGWINDOW 5:25;MINLEN 200] (Bolger et al. 2014).Chimeric sequences were removed using USEARCH 6.1 and the QIIME-script identify_chimeric_seqs.py (Caporaso et al. 2010).Preprocessed sequences were taxonomically assigned to operational taxonomic units (OTUs) at a nucleotide sequence identity of 97% using QIIME's pick_open_reference_otus.(Quast et al. 2013).The resulting OTU table was filtered for singletons, OTUs assigned to chloroplasts or mitochondria, and for low-abundance OTUs (below 0.2% within each sample).Archaeal and bacterial samples were processed separately while only OTUs that were assigned to the respective domain were considered for further analysis.The 16S rRNA gene sequence data have been deposited at NCBI under the Bioproject PRJNA356778.

qPCR analysis
Quantitative polymerase chain reaction (qPCR) for the determination of methanotrophic and methanogenic functional gene copy numbers and overall bacterial 16S rRNA gene copy numbers was performed via SybrGreen assays on a Bio-Rad CFX instrument (Bio-Rad, Munich, Germany) with slight modifications after Liebner et al. (2015).The functional methanotrophic pmoA gene was amplified with the primer combination A189F/Mb661 (Kolb et al. 2003) suitable for detecting all aerobic methanotrophic Proteobacteria.Annealing was done at 55 °C after a 7-cycle-step touchdown starting at 62 °C.The functional methanogenic mcrA gene was amplified with the mlas/mcrA-rev primer pair (Steinberg and Regan 2009)  was always done at 80 °C to avoid quantification of primer dimers.The specificity of each run was verified through melt-curve analysis and gel electrophoresis.Only runs with efficiencies between 82 and 105% were used for further analysis.Measurements were performed in triplicates.
We determined the ratio of methanogens to methanotrophs based on gene abundances of mcrA and pmoA.The marker gene for the soluble monooxygenase, mmoX, was neglected due to the absence of Methylocella in the sequencing data (Fig. 3).

Data visualization and statistical analysis
All data visualization and statistical analysis were done in R (R Core Team).The taxonomic relative abundances across samples were visualized through bubble plots with the R package ggplot2 (Wickham 2009).Differences in microbial community composition were visualized with 2-dimensional non-metric multidimensional scaling (NMDS) based on Bray-Curtis distances.The NMDS ordinations were constructed using R package vegan (Oksanen et al. 2017).An environmental fit was performed on the ordinations to determine the measured geochemical parameters that may influence community composition.The geochemical data were fitted to the ordinations as vectors with a significance of p < 0.05.Depth profiles were constructed with the porewater geochemical data, as well as with the microbial abundances, to elucidate depthwise trends and assess whether differences in microbial community and abundances among the two fens are related to differences in their respective geochemistry.

Community composition of bacteria and archaea
Bacterial sequences could be affiliated into a total of 30 bacterial phyla (Fig. 2).Among them, Proteobacteria, Acidobacteria, Actinobacteria, Chloroflexi, Nitrospirae and Bacteroidetes were present in all samples.With mean relative abundance of 48%, Proteobacteria was the most abundant phylum.Some taxa (e.g., Verrucomicrobia, Atribacteria (OP9), and AD3) were present only in Hütelmoor.Variation in community composition was larger in Hütelmoor samples than in Zarnekow.Within Proteobacteria, the alpha subdivision was the most dominant group, having contributed 26.7% to all the libraries on average (Fig. 3).The family Hyphomicrobiaceae dominated the Alphaproteobacteria, and was distributed evenly across samples, but missing in the surface and bottom peat layers in Hütelmoor core (HC) 2. In addition, methanotrophs were clearly in low abundance across all samples.Of the few methanotrophs that were detected, type II methanotrophs (mainly Methylocystaceae) outcompeted type I methanotrophs (mainly Methylococcaceae) in the community, while members of the genus Methylocella were absent (Fig. 3).
Within the archaeal community, Bathyarchaeota were mostly dominating over Euryarchaeota (Fig. 4).The MCG group (mainly the order of pGrfC26) in Bathyarchaeota prevailed across all samples but was especially abundant in HC 2 samples.In addition to Bathyarchaeota, methanogenic archaea were important, and on average contributed 30.6% to the whole archaeal community.
Among the methanogens, acetoclastic methanogens were more abundant in most of the samples and Methanosaetaceae (24.8%) were the major component.They were present in most samples and much more dominant than Methanosarcinaceae (2.0%).Hydrogenotrophic methanogens, such as Methanomassiliicoccaceae (1.6%), Methanoregulaceae (1.2%) and Methanocellaceae (0.6%), albeit low in abundance, were detected in many samples.Hütelmoor samples displayed greater variability in archaeal community composition compared to Zarnekow samples.The putative anaerobic methanotrophs of the ANME-2D (Raghoebarsing et al. 2006) clade occurred in patchy abundance with dominance in single spots of both sites.In HC 1 they represented a mean relative abundance of 40.9% of total archaeal reads but were almost absent in all other Hütelmoor cores.
In Zarnekow core (ZC) 3, ANME-2D represented up to approximately 30% of all archaea but were otherwise low in abundance.

Environmental characteristics and site geochemistry
The two rewetted fens varied substantially in their environmental characteristics (e.g., proximity to the sea) and porewater geochemistry (Fig. 5, Tables 1 and 2).Electrical conductivity was more Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-184Manuscript under review for journal Biogeosciences Discussion started: 26 April 2018 c Author(s) 2018.CC BY 4.0 License.than three times higher in Hütelmoor than in Zarnekow, averaging 5.3 and 1.5 mS cm -1 , respectively.Mean pH was approximately neutral (6.5 to 7) in the upper peat profile and comparable in both fens until a depth of about 30 cm where pH was ~6 in the Hütelmoor.
Concentrations of the TEAs nitrate and sulfate were lower in Zarnekow and near zero in the pore water at all depths, while nitrate and sulfate were abundant in the upper and lower peat profile in Hütelmoor at ~1.5 to 3.0 mM and ~4 to 20 mM, respectively (Fig. 5).Iron concentrations were higher in the Hütelmoor pore water, while manganese concentrations were higher in Zarnekow pore water.Dissolved oxygen concentrations in the upper peat profile (i.e.0 to 25 cm depths) were much higher in Hütelmoor than in Zarnekow (Fig. 5).Here DO concentrations averaged ~0.250 mM until a depth of 15 cm at which they dropped sharply, reaching concentrations slightly below 0.050 mM at 25 cm.In Zarnekow, DO concentrations did not exceed 0.1 mM and varied little with depth.Regarding geochemical conditions, HC 1 was distinct from all other Hütelmoor cores and more similar to Zarnekow cores.In HC 1 -the core taken nearest to potential freshwater sources (Fig. 1b) -pore water EC and DO concentrations were lower while pH was slightly higher than all other Hütelmoor cores.Moreover, this was the only Hütelmoor core where nitrate concentrations were undetectable (Fig. 5).Dissolved CH4 concentrations were high, varied within and among fens and were slightly higher in Zarnekow pore water.Stable isotope ratios of ∂ 13 C-CH4 (Fig. 5) in the upper peat (approx.−59‰) suggest a predominance of acetoclastic methanogenesis, with a shift to hydrogenotrophic methanogenesis around −65‰ in the lower peat profile.Also, shifts toward less negative ∂ 13 C-CH4 values in the upper peat layer, as in HC 1 and HC 2, could indicate partial oxidation of CH4 occurred (Chasar et al. 2000).

Environmental drivers of microbial community composition
Bacterial and archaeal population at both peatland sites showed distinct clustering (Fig. 6) with similarly high intra-and inter-site variations but greater overall variation in community composition in the Hütelmoor.Community composition varied much more strongly in HC 2 than in any other core (grey dashed-line polygon in Fig. 6).Bacterial communities in HC 1 were more Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-184Manuscript under review for journal Biogeosciences Discussion started: 26 April 2018 c Author(s) 2018.CC BY 4.0 License.similar to communities in all Zarnekow cores than in other Hütelmoor cores (Fig. 6a).The archaeal community in HC 1 was more similar to Zarnekow cores as well (Fig. 6b).Overall, the influence of depth on microbial community was evident, especially in the Hütelmoor where the differences were more pronounced.Environmental fit vectors suggest pH, oxygen and alternative TEA availability as important factors influencing microbial community composition.The EC vector suggests the importance of brackish conditions in shaping microbial communities in the Hütelmoor (Fig. 6a -c).

Total microbial and functional gene abundances
Quantitative PCR results show that in both fens, mcrA abundance is up to two orders of magnitude greater than pmoA abundance (Fig. 7, Tables 1 and 2).Gene copy numbers of mcrA are overall higher and spatially more stable in Zarnekow than in Hütelmoor.Total microbial abundance declined with depth more strongly in Hütelmoor than in Zarnekow (Fig. 7).There was a pronounced decrease in microbial abundances at 20 cm depth in the Hütelmoor.For example, 16S rRNA gene and pmoA gene copy numbers in deeper samples (below 20 cm depth) are one order of magnitude lower than in upper samples on average, while the mcrA gene abundance are approximately two orders of magnitude lower.Hütelmoor samples also exhibited larger heterogeneity in terms of abundances than Zarnekow samples.

Fen geochemistry and relations to microbial community composition
The rewetting of drained fens promotes elevated CH4 production and emission, which can potentially offset carbon sink benefits.Very few studies have attempted to link microbial community dynamics and site geochemistry with observed patterns in CH4 production and/or emission in rewetted fens while such data are crucial for predicting long-term changes to CH4 cycling (Galand et al. 2002, Yrjälä et al. 2011, Juottonen et al. 2012).In this study, we show that CH4-cycling microbial community composition is related to patterns in site geochemistry in two Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-184Manuscript under review for journal Biogeosciences Discussion started: 26 April 2018 c Author(s) 2018.CC BY 4.0 License.rewetted fens with high CH4 emissions, high methanogen abundances, and low methanotroph abundances.Our results suggest that high methanogen abundances concurrent with low methanotroph abundances contribute to increased CH4 production and the resulting high emissions in rewetted peatlands with readily available substrate.Thus, we present microbial evidence for sustained elevated CH4 emissions in mostly inundated rewetted temperate fens.
The environmental conditions and associated geochemistry of the two rewetted fens were largely different.Depth profiles of porewater geochemical parameters show the fens differed in EC throughout the entire peat profile, while pH and concentrations of alternative TEAs differed at certain depths.In general, concentrations of TEAs oxygen, sulfate, nitrate, and iron were higher in the Hütelmoor.In Zarnekow, geochemical conditions varied little across the fen and along the peat depth profiles (Fig. 5).As expected, the geochemical heterogeneity was reflected in microbial community structure in both sites, suggesting the importance of environmental characteristics and associated geochemical conditions as drivers of microbial community composition (Figs. 2,3,4,6).The NMDS ordinations (Fig. 6) show significant variation in archaeal and bacterial community composition in the coastal brackish fen, and much less variation in the freshwater riparian fen.
Environmental fit vectors (Fig. 6) suggest that salinity (indicated by the EC vector), pH, oxygen and alternative TEA availability are the most important measured factors influencing microbial communities in the two fens.Patterns in microbial community composition have previously been linked to salinity (e.g., Chambers et al. 2016), pH (e.g., Yrjälä et al. 2011), and TEA availability in peatlands (e.g., He et al. 2015).
Comparing the geochemical depth profiles (Fig. 5) with the relative abundance of bacteria and archaea (Figs. 3 and 4) provides a more complete picture of the relationships between microbial communities and site geochemistry, particularly with respect to TEA utilization.While the porewater depth profiles suggest there is little nitrate available for microbial use in HC 1, the relative abundance plot for Archaea showed that this core was dominated by ANME-2D.ANME-2D were recently discovered to be anaerobic methanotrophs that oxidize CH4 performing reverse Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-184Manuscript under review for journal Biogeosciences Discussion started: 26 April 2018 c Author(s) 2018.CC BY 4.0 License.methanogenesis using nitrate as an electron acceptor (Haroon et al. 2013).However, ANME-2D has also been implicated in the iron-mediated anaerobic oxidation of methane (Ettwig et al. 2016), and the HC 1 site showed slightly higher total iron concentrations.The relevance of ANME-2D as CH4 oxidizers in terrestrial habitats is still not clear.Rewetting converts the fens into widely anaerobic conditions, thus providing conditions suitable for the establishment of anaerobic oxidation of methane, but this has yet to be demonstrated in fens.The patchy occurrence and locally high abundance of ANME-2D both in Hütelmoor and in Zarnekow suggests an ecological relevance of this group.Shifts toward a less negative d 13 C-CH4 signature in the upper peat profile, especially in HC 1 where ANME-2D was abundant, may indicate partial oxidation occurred, but we could only speculate whether or not they are actively involved in CH4 oxidation.
Although TEA input may be higher in the Hütelmoor, here, methanogenic conditions also predominate.This finding contrasts the measured oxygen concentrations in the upper peat profile, however seasonal analysis of oxygen concentrations in both sites suggests highly fluctuating oxygen regimes both spatially and temporary (data not shown).Such non-uniform distribution of redox processes has already been described elsewhere, in particular for methanogenesis (Hoehler et al. 2001, Knorr et al. 2009).It is possible that oxygen levels in both fens are highly dynamic allowing for both aerobic and anaerobic carbon turnover processes.Further, oxygen may not necessarily be available within aggregates in which anaerobic pathways predominate.Anaerobic conditions are also reflected by the extensive and stable occurrence of the strictly anaerobic syntrophs (e.g., Syntrophobacteraceae, Syntrophaceae) in most samples, even in the top centimeters.This suggests that syntrophic degradation of organic material is taking place in the uppermost layer and the fermented substances are easily available for methanogens.Recent studies from wetlands also show that methanogenesis can occur in aerobic layers, driven mainly by Methanosaeta (Narrowe et al. 2017, Wagner 2017), which were detected in a high abundance in this study (Fig. 4).As geochemistry and microbial community composition differ among the sites in this study, it is thus notable that a similarly high abundance of methanogens, and low abundance Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-184Manuscript under review for journal Biogeosciences Discussion started: 26 April 2018 c Author(s) 2018.CC BY 4.0 License. of methanotrophs was detected in both fens.The dominance of methanogens implies that readily available substrates and favorable geochemical conditions promote high anaerobic carbon turnover despite seasonally fluctuating oxygen concentrations in the upper peat layer.

Microbial evidence for high CH4 emissions
Methanogens (mainly Methanosaetaceae) dominated nearly all of the various niches detected in this study, while methanotrophs were highly under-represented in both sites (Figs. 3 and 4).
Functional and ribosomal gene copy numbers not only show a high ratio of methanogen to methanotroph abundance (Fig. 7) irrespective of site and time of sampling, but also a small contribution of methanotrophs to total bacterial population in both sites.Methanotrophs constitute only ~0.06% of the total bacterial population in the Hütelmoor and ~0.05% at Zarnekow.It should be noted that in this study we measured only gene abundances and not transcript abundances, so that the pool both of active methanogens and methanotrophs was likely smaller than the numbers presented here (Freitag and Prosser 2009, Freitag et al. 2010, Cheema et al. 2015, Franchini et al. 2015).Also, as we were unable to obtain microbial samples from before rewetting, a direct comparison of microbial abundances was not possible.Compared to pristine fens, however, we detected a relatively low abundance of methanotrophs.Liebner et al. (2015), for example, found methanotrophs represented 0.5% of the total bacterial community in a pristine, subarctic transitional bog/fen palsa, while mcrA and pmoA abundances were nearly identical.In a pristine Swiss alpine fen, Liebner et al. (2012) found methanotrophs generally outnumbered methanogens by an order of magnitude.Cheema et al. (2015) and Franchini et al. (2015) reported mcrA abundances higher than pmoA abundances by only one order of magnitude in a separate Swiss alpine fen.In the rewetted fens in our study, mcrA gene abundance was up to two orders of magnitude higher than pmoA abundance (Fig. 7).As most methanotrophs live along the oxicanoxic boundary of the peat surface and plant roots therein (Le Mer and Roger 2001), the low methanotroph abundances in both fens could be explained by disturbances to this boundary zone and associated geochemical pathways following inundation.In rewetted fens, a massive plant Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-184Manuscript under review for journal Biogeosciences Discussion started: 26 April 2018 c Author(s) 2018.CC BY 4.0 License.dieback has been observed along with strong changes in surface peat geochemistry (Hahn-Schöfl et al. 2011, Hahn et al. 2015).The anoxic conditions at the peat surface caused by inundation may have disturbed existing methanotrophic niches, and further, hindered the establishment of new ones, as oxygen availability is the most important factor governing the activity of most methanotrophs (Le Mer andRoger 2001, Hernandez et al. 2015).
Comparable studies have so far been conducted in nutrient-poor or mesotrophic fens where postrewetting CH4 emissions, though higher than pre-rewetting, did not exceed those of similar pristine sites (e.g., Yrjälä et al. 2011, Juottonen et al. 2005, Juottonen et al. 2012).Nevertheless, there is mounting evidence linking CH4-cycling microbe abundances to CH4 dynamics in rewetted fens.Juottonen et al. (2012), for example, compared pmoA gene abundances in three natural and three rewetted fens and found them to be lower in rewetted sites.The same study also measured a lower abundance of mcrA genes in rewetted sites, which was attributed to a lack of available labile carbon compounds.In peatlands, and especially fens, litter and root exudates from vascular plants can stimulate CH4 emissions (Megonigal et al. 2005, Bridgham et al. 2013, Agethen and Knorr 2018), and excess labile substrate has been proposed as one reason for dramatic increases in CH4 emissions in rewetted fens (Hahn-Schöfl et al. 2011).Future studies should compare pre-and postrewetting microbial abundances along with changes in CH4 emissions, plant communities, and peat geochemistry to better assess the effect rewetting has on the CH4-cycling microbial community.

Conclusion
Despite a recent increase in the number of rewetting projects in Northern Europe, few studies have characterized CH4-cycling microbes in restored peatlands, especially fens.In this study, we show that rewetted fens differing in geochemical conditions and microbial community composition have https://doi.org/10.5194/bg-2018-184Manuscript under review for journal Biogeosciences Discussion started: 26 April 2018 c Author(s) 2018.CC BY 4.0 License.
py script and the Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-184Manuscript under review for journal Biogeosciences Discussion started: 26 April 2018 c Author(s) 2018.CC BY 4.0 License.GreenGenes database 13.05 (McDonald et al. 2012) as reference.The taxonomic assignment of representative sequences was further checked for correct taxonomical classification by phylogenetic tree calculations in the ARB environment referenced against the SILVA database (https://www.arb-silva.de)version 119 with annealing at 57 °C.The bacterial 16S rRNA gene was quantified with the primers Eub341F/Eub534R according to Degelmann et al. (2010) with annealing at 58 °C.Different DNA template concentrations were tested prior to the qPCR runs to determine optimal template concentration without inhibitions through co-extracts.The 25 µl reactions contained 12.5 µl of iTaq universal Sybr Green supermix (Bio-Rad, Munich, Germany), 0.25 µM concentrations of the primers, and 5 µl of DNA template.Data acquisition Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-184Manuscript under review for journal Biogeosciences Discussion started: 26 April 2018 c Author(s) 2018.CC BY 4.0 License.

Table 1 :
Environmental conditions, geochemical conditions, and microbial abundances in peat cores from the Hütelmoor, a coastal minerotrophic fen 884 in northeastern Germany.Environmental conditions are described by pH and EC (electrical conductivity).Geochemical parameters shown are dissolved 885 methane (CH4) concentrations, the isotopic signature of methane-bound carbon (∂ 13 C-CH4), and concentrations of terminal electron acceptors which 886 are denoted with their respective chemical abbreviations.Microbial abundances here represent the mean value of averaged subsamples for each depth Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-184Manuscript under review for journal Biogeosciences Discussion started: 26 April 2018 c Author(s) 2018.CC BY 4.0 License.