Abstract
Climate change may reduce biodiversity leading to a reduction in ecosystem productivity. Despite numerous reports of a strong correlation of microbial diversity and ecosystem productivity, little is known about the warming effects on plant associated microbes. Here we explore the impact of experimental warming on the microbial and nitrogen-fixing (diazotroph) community associated with the widespread and ecologically relevant Sphagnum genus in a field warming experiment. To quantify changes in the abundance, diversity, and community composition of Sphagnum microbiomes with warming we utilized qPCR and Illumina sequencing of the 16S SSU rRNA and nifH gene. Microbial and diazotroph community richness and Shannon diversity decreased with warming (p<0.05). The diazotroph communities shifted from diverse communities to domination by primarily Nostocaceae (25% in control samples to 99% in elevated temperature samples). In addition, the nitrogen fixation activity measured with the acetylene reduction assay (ARA) decreased with warming treatment. This suggests the negative correlation of temperature and microbial diversity corresponds to a reduction in functional potential within the diazotroph community. The results indicate that climate warming may alter the community structure and function in peat moss microbiomes, with implications for impacts to host fitness and ecosystem productivity, and carbon uptake potential of peatlands.
Introduction
Climate change represents a large threat to the function and stability of ecosystems, potentially leading to altered abundance range shifts (Parmesan, 2006), and species extinction (Parmesan, 2006; Bestion et al., 2015) that ultimately result in decreased biodiversity. Despite years of research on the importance of diversity in driving the productivity and function in numerous ecosystems (Tilman et al., 2012; Liang et al., 2016; Kolton et al., 2017; Laforest-Lapointe et al., 2017), the relationship of warming and biodiversity remains unclear in many ecosystems. The majority of research on biodiversity and warming has focused mainly on multicellular eukaryotic organisms with little attention to the prokaryotes associated with them, but recent work has highlighted the key role that microbial biodiversity may play in determining the ecological response of ecosystems to warming (Bardgett & Putten, 2014; Bestion et al., 2017).
Plant-microbe symbioses are widespread and ecologically important host-microbe associations. Plant-associated microbiomes have direct roles in ecosystem functioning through effects on carbon (Lu et al., 2006; Knief et al., 2012) and nitrogen cycles (Vile et al., 2014; Moyes et al., 2016). Plant microbial communities are structured by biotic factors (Bragina et al., 2012; Berg et al., 2014; Edwards et al., 2015) and abiotic factors (Bulgarelli et al., 2012; Lundberg et al., 2012; Carrell & Frank, 2014; Edwards et al., 2015) and have been found to be susceptible to environmental perturbations such as drought (Santos-Medellín et al., 2017), nitrogen deposition (Gschwendtner et al., 2016), and salinity (Yang et al., 2016). Plant microbiomes also affect host plant health and productivity (Berendsen et al., 2012; Chaparro et al., 2012; Berg et al., 2014), with more productive and healthy plants supporting greater microbial diversity (van der Heijden et al., 2008; Berendsen et al., 2012; Bever et al., 2013; Agler et al., 2016; Delgado-Baquerizo et al., 2016; Kolton et al., 2017). Despite the importance of microbes to plant function and ecosystem processes, and the sensitivity of plant-microbial symbioses to environmental disturbances, the response of plant associated-microbial diversity to climate warming is not well understood.
Sphagnum mosses play a large role in the global carbon cycle and are considered to be particularly vulnerable to climate change (McGuire et al., 2009; Turetsky et al., 2012). These bryophytes are inhabited by diverse microbes (Opelt et al., 2007; Kostka et al., 2016) with direct roles in the carbon cycle through methane oxidation (Raghoebarsing et al., 2005; Kip et al., 2010; Bragina et al., 2013a), as well as other important ecosystem functions (Kostka et al., 2016) such as nitrogen fixation (Bragina et al., 2011, 2013b, 2014; Vile et al., 2014; Warren et al., 2017)(Bragina et al., 2011, 2013a, 2014; Vile et al., 2014; Warren et al., 2017) that enables plant growth under nitrogen-limited conditions characteristic of the bogs where these mosses are found. Warming experiments have demonstrated that elevated temperature causes a reduction of Sphagnum biomass (Turetsky et al., 2012). Moreover a recent study demonstrated elevated temperature may have both negative and positive impacts on Sphagnum microbial functional groups, which may destabilize carbon cycling in peatlands (Jassey et al., 2013), but the effect of temperature on the community composition and diversity of Sphagnum microbiomes remains unknown.
In this study, we investigated the impact of experimental warming on the microbial community associated with Sphagnum. The objective of this study was to quantify changes in the abundance, diversity, and community composition of Sphagnum microbiomes with increased temperatures in the Spruce and Peatland Responses Under Changing Environments (SPRUCE) experiment (Hanson et al., 2017) which provided in situ field warming treatments from ambient to +9°C at the S1-Bog of the Marcell Experimental Forest in northern Minnesota (Kolka et al., 2011). The study focused on the nitrogen-fixing (diazotroph) functional guild that enables plant growth under the extreme nutrient-limited conditions characteristic of ombrotrophic bog ecosystems (Limpens & Heijmans, 2008; Larmola et al., 2014; Vile et al., 2014).
Materials and Methods
Experimental site and warming experiment
The SPRUCE experiment at the S1 bog on the Marcell Experimental Forest (Hanson et al., 2017) employs a whole-ecosystem warming approach to produce nominal warming treatments of +0, +2.25, +4.5, +6.75 and +9 °C for a Picea mariana – Sphagnum spp. raised bog ecosystem. The experiment includes ten 12-m diameter plots with open-top enclosures (enclosed) and two ambient 12-m diameter plots without enclosures (non-enclosed). Briefly, the warming methodology combining air warming with deep-peat-heating from mild electrical resistance heaters to produce target warming levels superimposed over the natural diurnal and seasonal variability (Hanson et al. 2017). The experiment is located in the S1-Bog on the Marcell Experimental Forest (Kolka et al, 2011). The S1 Bog is an acidic and nutrient-deficient ombrotrophic Sphagnum-dominated peatland bog (surface pH≤4.0). The average means of annual precipitation and air temperature are 768 mm and 3.3°C respectively (Sebestyen et al., 2011).
Sampling
To characterize the Sphagnum microbiome responses to warming, individual Sphagnum stems were randomly collected within each plot in June 2016 following continuous whole-ecosystem warming initiated in August of 2015. Samples were overnight shipped on ice to Oak Ridge National laboratory. Upon arrival, a subset of samples was shipped on ice overnight to Georgia Institute of Technology for ARA and the remaining plants were immediately pulverized with sterile mortar and pestle in liquid nitrogen for DNA extraction.
DNA extraction, PCR and DNA sequencing
To characterize the abundance and community composition of Sphagnum microbiomes, DNA was extracted from 50 mg of each pulverized Sphagnum sample using a MoBio PowerPlant Plant Kit (MoBio, Carlsbad, CA, USA). Extracted DNA was frozen and shipped on dry ice to Georgia Institute of Technology for amplification and sequencing.
The diversity and composition of Sphagnum associated microbial communities was determined by applying a high-throughput sequencing-based protocol that targets PCR-generated amplicons from the V4 variable regions of the 16S rRNA gene using the primer set 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) as previously described (Wilson et al., 2016; Kolton et al., 2017). The diversity and composition of diazotrophic communities were assessed by targeting nifH (encoding the nitrogenase reductase subunit) as a molecular marker for nitrogen-fixing microorganisms. Primers IGK3 (5’-GCIWTHTAYGGIAARGGIGGIATHGGIAA-3’) and DVV (5’-TIGCRAAICCICCRCAIACIACRTC-3’) were employed to generate 396 bp PCR products (Gaby & Buckley, 2014). The 16A SSU rRNA and nifH amplicons were barcoded with unique 10-base barcodes (Fluidigm Corporation), and sequenced on an Illumina MiSeq2000 platform at the Georgia Institute of Technology following standard protocols (Caporaso et al., 2012; http://www.earthmicrobiome.org/emp-standard-protocols/16s/; Gilbert et al., 2010; Gaby et al., 2017, submitted).
Sequence processing and analysis
First, Illumina-generated 16S SSU rRNA and nifH gene amplicon sequences were paired with PEAR (Zhang et al., 2014) and primers were trimmed with the software Mothur v1.36.1 (Schloss et al., 2009). Resulting sequences were quality filtered using a Phred quality score Q30 and Q25 for 16S SSU and nifH respectively using the standard QIIME 1.9.1 pipeline (Caporaso et al., 2010). Sequences were clustered into operational taxonomic units (OTUs) by using UCLUST algorithm with a threshold of 97% identity. Representative sequences were aligned using PyNAST (Caporaso et al., 2010) against the Greengenes core set for 16S SSU and against nifH gene alingment (DeSantis et al., 2006; Gaby & Buckley, 2014). Taxonomies of these high-quality sequences were annotated to the Greengenes database (release 13_8) (DeSantis et al., 2006) or a manually curated nifH database (Gaby & Buckley, 2014) using the RDP classifier (Wang et al., 2007) with a minimum confidence threshold of 50%. The 16S SSU rRNA sequences classified as “chloroplast” or “mitochondria” were removed from the alignment. An approximately maximum-likelihood tree was constructed from the aligned of bacterial representative sequences, using FastTree (Price et al., 2009). Prior to conducting diversity analyses, OTUs were rarefied to 3500 reads per sample for 16S SSU rRNA amplicons and 1500 reads per sample for nifH amplicons. The OTU-based alpha diversity was calculated based on the total number of phylotype (observed richness) and on Shannon’s diversity index (H′). Faith’s phylogenetic diversity (PD) was calculated to assess phylogenetic based alpha diversity. The OTU-based beta diversity indices were estimated based on Bray–Curtis distances.
The Illumina-generated 16S SSU rRNA and nifH gene amplicon sequences have been deposited in the BioProject database, (ncbi.nlm.nih.gov/bioproject) under accession PRJNA407792 and PRJNA407800 respectively.
Quantitative PCR amplification
All quantitative polymerase chain reaction assays were performed in triplicates using the StepOnePlus platform (Applied Biosystems, USA) and PowerUp SYBR Green Master Mix (Applied Biosystems, USA). Absolute quantification of 16S SSU rRNA and nifH genes were conducted with primer pairs 331F (5’-CCTACGGGAGGCAGCAGT-3’)/518R (5’-ATTACCGCGGCTGCTG-3’) and PolF (5’-TGCGAYCCSAARGCBGACTC-3’) /PolR (5’-ATSGCCATCATYTCRCCGGA-3’) respectively (Muyzer & Waal, 1993; Poly et al., 2001). The 16S SSU rRNA quantification reaction was carried out in 20 μl containing 7.8 μl of PCR grade water, 0.1 μl of each primer (final concentration 0.5 μM), 10 μl of PowerUp SYBR Green Master Mix (Applied Biosystems, USA) and 2 μl of sample DNA. The cycling program included 2 min at 50 °C, 2 min at 95 °C, followed by 40 cycles of 95 °C for 15 s, 55 °C for 15 s and 72 °C for 1 min. The nifH gene quantification reaction was carried out in 20 μl containing 6.8 μl of PCR grade water, 0.6 μl of each primer (final concentration 0.3 μM), 10 μl of PowerUp SYBR Green Master Mix (Applied Biosystems, USA) and 2 μl of sample DNA. The cycling program included 2 min at 50 °C, 2 min at 95 °C, followed by 45 cycles of 95 °C for 15 s, 63 °C for 1 min. Amplification specificity was studied by melting curve analysis of the PCR products, performed by ramping the temperature to 95 °C for 15 s and back to 60 °C for 1 min, followed by increases of 0.15 °C s-1 up to 95 °C. Melting curve calculation and determination of Tm values were performed using the polynomial algorithm function of StepOnePlus Software (Applied Biosystems, USA). In all experiments, negative controls containing no template DNA were subjected to the same qPCR procedure to exclude or detect any possible DNA contamination. Standard curves were obtained with serial dilution of standard plasmids containing target Escherichia coli k12 16S rRNA or Azotobacter vinelandii nifH gene fragments as the insert. The abundance of standard plasmid inserts ranged from 2.97 × 103 to 2.97 × 109 (bacterial 16S SSU rRNA gene) or 24.2 to 2.42 × 106 (nifH gene).
Acetylene reduction assay
To determine the effect of warming on nitrogen fixation activity, fresh tissue from the ambient and warming plots exposed to the highest temperatures (+9°C) were interrogated using the acetylene reduction assay (ARA) as previously described (Warren et al., 2017). Briefly, samples of Sphagnum were collected from ambient enclosed and non-enclosed plots and +9°C enclosed plots in triplicate and stored at 4°C until the start of incubations. A 1.0-1.5 g sample of green-only Sphagnum was placed into 35 ml glass serum bottles, stoppered with black butyl stoppers, sealed with an aluminum crimp seal, and 10% headspace was replaced with 10% room air or with 10% C2H2. Controls that were not amended with C2H2 did not produce detectable ethylene. All treatments were incubated for one week in the light at 25°C. A gas chromatograph with flame ionization detector (DRI Instruments, Torrance, CA, USA) equipped with a HayeSep N column was used to quantify ethylene (C2H4). The accumulation of C2H4 was determined twice daily until C2H4 production was linear (∼3 days). Samples were dried at the end of incubations at 80°C for 48 hours to determine dry weight for normalization of ARA rates.
Data analysis
Statistical analysis was conducted in R (R Core Team, 2015). Warming effects on microbiome community composition were assessed with a Spearman Rho test between warming treatments and a heatmap was generated from the relative abundance of distinct OTUs that showed significant differences (p<0.05) and had >0.1% relative abundance in at least a single treatment. General Linear Models (GLMs) were used to evaluate the effects of warming on microbial diversity measurements of enclosed plots. A Mann-Whitney test was used to compare diversity between ambient plots, with or without an enclosure structure. Beta diversity was visualized using non-metric multidimensional scaling ordination (NMDS) from Bray-Curtis similarity distances. Analysis of similarities (ANOSIM) and permutational multivariate analysis of variance (PERMANOVA), each with 999 permutations, were used to determine if beta diversity differed significantly among treatments.
Results
Response of microbiome abundance, community composition, and diversity to warming
The overall microbial abundance as determined by qPCR did not vary by warming treatment (p=0.2; Table 1).
The Sphagnum microbiome communities were dominated by Proteobacteria (62%) and Acidobacteria (17%), with smaller contributions from candidate division WPS-2 (4%), Cyanobacteria (4%), Bacterioidetes, (3%) Verrucomicrobia (2%), and Actinobacteria (1%) with Cyanobacteria varying across warming treatments though not significant (Fig. 1). The Proteobacteria were dominated by the order Rhodospirillales (33%) followed by Caulobaceterales (7%), Xanthomondales (8%), and Burkholderiales (3%). Despite the dominance in major phyla and genera groups across treatment, several OTUs varied significantly across warming treatment (Table S1). Cyanobacteria in the Nostocaceae family, OTU 278041 most similar to Nostoc sp. and OTU 4242238 most similar to Cylindrospermum sp., increased in relative abundance from 0.4 to 4.1% and from 0 to 1%, respectively, across all warming treatments (p=0.04). Warming treatments had a varied effect on Acetobacteraceae with relative abundance decreasing in +2.25°C and +4.5°C treatments but returning to similar abundances in +6.75 and +9°C treatments.
The richness and phylogenetic diversity of Sphagnum microbiomes decreased with warming. Observed richness and Shannon index decreased with warming (p<0.05), while phylogenetic diversity decreased with warming treatment but was only significant at p=0.08 (Fig. 2, Table 2). Sphagnum bacterial communities were structured by warming treatments (p<0.003) with Bray-Curtis distance similarity higher within treatment than between treatments (Fig. S1). Percent similarity for all samples was 52% (standard deviation = 5%) with a range of 31-65% similarity. (R2=0.3, p=0.004).
Response of diazotroph abundance, diversity, community composition and function to warming
The abundance of diazotrophs as determined by qPCR of nifH genes significantly decreased (p=0.004) with increasing temperature (Table 1). All nifH gene profiles were dominated by the phyla Cyanobacteria (60-100%) and Proteobacteria (0.5-40%) with Cyanobacteria increasing in abundance and Proteobacteria decreasing with warming treatments. Abundant members of the Cyanobacteria phylum were comprised of Nostocaceae (25-99%), Rivulariaceae (0-27%), and Chlorogloeopsdidaceae (0-0.7%), with Nostocaceae becoming more dominant with warming (Fig. 1). The Rhizobiales (0.1-35%) and Rhodospirillales (0-4%) were detected in abundance from the Proteobacteria phylum, with relative abundance decreasing across warming treatments. To provide greater resolution into shifts in diazotroph populations, an OTU heatmap was generated from the top 20 OTUs of each treatment (Fig. 1). Notably, ambient warming plots were largely dominated by sequences most similar to the genera Methyloferula (17-40%) and Calothrix (0-32%) which both decreased across warming treatments: +2.25°C (0-25%), +4.5°C (0-7%), +6.75°C (0-6%), and +9°C (0-3%). With increased warming, sequences closely related to the genus Nostoc became more dominant though different Nostoc species dominated across each temperature treatment. Sequences most similar to Nostoc punctiforme dominated the +2.25°C (20-80%), +4.5°C (26-88%), and +6.75°C (46-83%) treatments while +9°C was dominated by Nostoc sp. PCC7524 (0-100%).
Warming reduced the richness and diversity of the diazotroph community (p<0.05, Table 2), although each treatment did not respond equally. When compared to +0°C, diazotroph richness at +2.25°C and + 4.5°C decreased by 30% and 54%, respectively, while richness in the +6.75°C and +9°C plots only decreased by 14% richness (Fig. 2, Table S2). Shannon indices followed a similar pattern with a reduction in diversity of 27% in the +2.25°C plots, 52% in the + 4.5°C plots, 18% in the 6.75°C plots and only 3% in the +9°C plots (Fig. 2). The diazotroph community was structured by temperature treatment in that samples from the same treatment clustered closer to one another than other treatments (R2=0.3546, p=0.041). However, the clustering was not incremental with diazotroph communities from 0°C and 9°C clustering closer to one another than with 6.75°C (Fig. S1).
Nitrogen fixation rates determined by ARA showed considerable variability within warming treatments, with some samples showing no detectable activity while others had rates as high as 172 nmol g-1 hr-1. Average rates of nitrogen fixation decreased by ∼50% from +0°C (47 ±9 nmol g-1 hr-1) to +9°C (21 ± 6 nmol g-1 hr-1), but the decline was only significant at p=0.1, due to variation between replicates (Fig. 3).
Experimental enclosure affect
To test if the presence of the experimental structure had a significant impact on Sphagnum general bacterial and diazotroph community composition and diversity, we measured 16S SSU rRNA and nifH genes of Sphagnum in ambient plots without an enclosure (ambient) and ambient plots with an enclosure warmed at +0°C above outside ambient conditions. We found that the enclosure had no statistical effect on 16S SSU rRNA and nifH gene composition, abundance, diversity, richness or evenness (Figure S2, Tables S1, S2). Temperature did not significantly change community structure for either 16S SSU rRNA (R2=0.02, p=0.4) or nifH genes (R2=0.01, p=0.6).
Discussion
Determining the potential effects of climate drivers such as temperature on Sphagnum microbiomes is an important step toward effectively predicting the response of ecosystem function in ombrotrophic bogs to climate change. Here we demonstrate that temperature strongly influences general microbial and diazotroph community structure and diversity. Additionally, Sphagnum microbiome communities from ambient plots without enclosure were not significantly different in microbiome or diazotroph composition, abundance, or diversity, than Sphagnum microbiome communities in plots with enclosures, indicating that differences between temperature treatments were not an artifact of the experimental warming structure.
Warming effects on overall microbiome communities
The Proteobacteria, Acidobacteria, and Cyanobacteria dominated all samples, and have been found to dominate Sphagnum in other bog systems (Bragina et al., 2014). Despite consistent dominance by the same phyla, overall community structure differed by warming treatments, likely due to variation at a lower taxonomic level. We did see variation in species within bacterial families, possibly as a result of differential temperature optima of bacterial species. Overall, observed richness, diversity and phylogenetic diversity were negatively correlated with temperature. Phylogenetic diversity is a divergence based method that has been described as more powerful than qualitative measurements given the correlation of 16S SSU rRNA similarity and phenotypic similarities in microbial key features such as metabolic capabilities or other functions (Lozupone & Knight, 2008). This would suggest that while we see a reduction in overall phylotype counts, we also see a reduction in metabolic capabilities.
A reduction of microbial diversity may make ecosystems more susceptible to environmental perturbations and when considering additional perturbations such as N deposition or different precipitation patterns, these communities may be even more impacted (Aanderud et al., 2013). Here we found a reduction of richness and diversity in both the general microbial community and diazotroph community. Indeed a reduction of richness and evenness of microbial communities in other ecosystems such as soil or rhizosphere, were associated with a decrease in ecosystem functioning such as nutrient cycling (Philippot et al., 2013; Wagg et al., 2014), plant productivity (Bell et al., 2005; van der Heijden et al., 2008; Lau & Lennon, 2011; Fierer et al., 2013) and plant resilience against pathogen invasion (Jousset et al., 2011; Mendes et al., 2011). Moreover, reduction in microbial diversity is frequently associated with reduced activation of plant defense systems (Mendes et al., 2011, 2013; Berendsen et al., 2012). Additionally, Sphagnum mosses have been found to harbor potential latent plant pathogens and in many organisms disease outbreaks are dependent on the abundance of pathogens and the diversity of microbiomes (Bragina et al., 2011; Elad & Pertot, 2014; Tout et al., 2015). Alternatively, a reduction in diversity could correspond to a loss of pathogenic taxa, which might be beneficial to host plants. Therefore, further study will be needed to determine the specific ecosystem functions that are mediated by the Sphagnum microbiome and impacted by warming.
Warming effects on diazotroph communities
Nitrogen is essential to the growth and maintenance of Sphagnum plants and previous research revealed highly specific and diverse diazotrophs (Bragina et al., 2013a)(Bragina et al., 2013a) are a major source of N in Sphagnum-dominated peatlands (Lindo et al., 2013; Larmola et al., 2014; Vile et al., 2014; Novak et al., 2016). In corroboration of patterns in overall microbiome communities, diazotroph diversity and abundance were negatively correlated with temperature. This suggests that the reduction of microbial diversity may lead to a reduction of functional potential within the diazotroph functional guild. Within the diazotroph community, we found a shift in community composition with elevated temperature leading to a community dominated by primarily by Nostoc and void of diazotrophic methanotrophs. In addition, another filamentous cyanobacterium, Stigonema, was shown to decrease in relative abundance across temperature treatment to below detection in the +9°C treatment. Interestingly, Nostoc has been described as “cheaters” in the feather moss microbiome as it dominated the cyanobacterial community but had low nifH gene expression and thus not providing much nitrogen to the host. Conversely, Stigonema made up less than 29% of the cyanobacterial community but accounted for the majority of nifH gene expression suggesting Stigonema is responsible for the majority of fixed nitrogen (Warshan et al., 2016). Though it is possible an observed reduction in nitrogen fixation may be attributed to the increase in the presence of a “cheater” and/or disruption of supportive metabolic pathways it cannot be concluded from our data that Nostoc is a cheater in our system. Concurrent with an increase in Nostoc relative abundance we found a decrease in diazotroph absolute abundance indicating that Nostoc may not be increasing in abundance but rather other microbial populations, such as the methanotrophs, are dropping out of the community.
Diazotroph function
Nitrogen fixation activity and temperature were negatively correlated which may be due to plant-specific tolerance to water stress and desiccation given that nitrogen fixation associated with moss is influenced by moisture (Zielke et al., 2002; Sorensen et al., 2006; Sorensen & Michelsen, 2011). Additionally, oxygen level, photosynthetic activity (Warren et al., 2017), and phosphorous (Rousk et al., 2017) or nitrogen availability (Kox et al., 2016) have also been found to limit diazotrophy in Sphagnum (Warren et al., 2017). However, we observed a reduction in diazotroph absolute abundance indicating diazotrophs were not inactive but rather undetectable with our methods in elevated temperature treatments. Alternatively, this may be attributed to the diazotroph optimal temperature for nitrogen fixation (Gundale et al., 2012) or a disruption in microbiome composition. The nitrogenase enzyme commonly contains molybdenum (Rousk et al., 2017; Warren et al., 2017) as its cofactor but may contain vanadium or iron in its place (Miller & Eady, 1988). Thus the change across temperatures could be attributed to altered metal availability. With a reduction in nitrogen fixation, Sphagnum may become more reliant on nitrogen provided by non-associative diazotrophs such as bacteria in the pore water or below peat. However, if the Sphagnum associated microbes are susceptible to elevated temperature, diazotrophs in the water may be even more so. Additionally, Sphagnum competition for other sources of nitrogen may disrupt free-living microbial communities causing larger consequences at the ecosystem level.
Though we found a general pattern of a reduction in potential rates of nitrogen fixation, it is important to note that acetylene inhibits the enzyme methane monooxygenase and thus the diazotrophy of methanotrophs. A recent study calibrated ARA with 15N incorporation and found a conversion factor of 3.9 for 15N2-to-ARA in the same bog as our experiment, indicating the presence of diazotrophic methanotrophs that were inhibited by acetylene (Warren et al., 2017). In our study, the use of the conversion factor is inappropriate given the demonstration of an altered diazotroph community. While it is possible we have underestimated diazotroph activity, our observations of decreased nitrogen fixation activity with warming are supported by a decline in diazotroph abundance and the relative abundance of diazotrophic methanotrophs.
With warming induced reduction of diazotroph abundance and function, one might logically expect a decline in peatland ecosystems carbon storage capacity. The considerable accumulation of C as peat results from a long-term excess of Net Primary Productivity (NPP) of plants over peat decomposition. In peatlands a simple mass balance demonstrates N-deposition alone does not account for the N needed to support the observed NPP (Wieder et al., 2010). A recent study demonstrated diazotrophs may account for 12-25 times more N than from atmospheric inputs alone, accenting the important link between diazotrophy and NPP (Vile et al., 2014). Sphagnum has demonstrated differential NPP response to warming (Aerts et al., 2006) but no studies have examined the Sphagnum microbial community and diazotroph responses to warming. Here we present data that suggests warming may disrupt the diazotroph community and function, which ultimately may reduce NPP or the accumulation of peat and therefore may be an important component to include in future Sphagnum and peatland response studies.
Microbial associates play an important role in Sphagnum health and growth as well as bog ecosystem functioning. In this study, we conducted a warming experiment to elucidate the temperature effects on Sphagnum microbiomes. We propose that climate warming may alter microbiome function as a result of decreased biodiversity. The consequences of decreased functional potential are not clear and merits future studies to determine how the alteration of overall microbiome and diazotroph function may scale to the ecosystem level. Such knowledge will provide a more comprehensive understanding of how climate may impact the future function of Sphagnum dominated bog ecosystems.
Acknowledgements
The experiment were maintained as part of the SPRUCE project and supported by the U.S. Department of Energy’s Office of Science, Biological and Environmental Research (DOE BER). Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. Department of Energy under contract DE-AC05-00OR22725. Sample collection, processing and manuscript writing was supported by the Laboratory Directed Research and Development Program of Oak Ridge National Laboratory, managed by UT-Battelle, LLC, for the U. S. Department of Energy. Sequencing was supported by U.S. DOE BER under award numbers DE-SC0007144 and DE-SC0012088.
Footnotes
This manuscript has been authored by UT-Battelle, LLC under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a nonexclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan).