The effects of herbivore grazing on peat vegetation and soil O2 availability
The exclosures (EX) were established 18 years prior to sampling, preventing the access of herbivores to the vegetation (Fig. 1a). During this time, a vascular plant community dominated by Poa arctica (up to 90% coverage) and Cardamine pratensis (up to 20% coverage) had developed. The vascular plants Ranunculus hyperborus, Saxifraga cernua and Saxifraga cespitosa were also present. Eight moss species within the brown moss family Amblystegiaceae were identified, making up the entire moss community within the exclosures (Table 1). At the grazed sites (GR), a total of seven moss species dominated the vegetation (up to 100% coverage), while two vascular plant species were detected (P. arctica and R. hyperborus) (Table 1).
Due to the higher coverage of vascular plants in the exclosures, these sites contained much higher root densities, previously quantified by Sjögersten et al. (2011) to be 12 times higher. The O2 [%] within the first two centimeters of peat was ~ 20% in both the exclosures and grazed sites. The O2 concentration decreased with depth, being ~ 19–20% at 5 cm depth within the exclosures and 13–16% in the grazed sites (Table S1—online resource 2). We observed surface temperatures in the range of 10.4–10.8 °C (EX) and 8.3–14 °C (GR), while at 10 cm soil depth the temperature was approximately 5–6 °C in both sites (Table S1—online resource 2). The water content of the peat soils was lower in the exclosures (mean: 84.5%) compared to the grazed sites (mean: 92.4%) (Table S2—online resource 2). The total soil organic matter percentage of the fresh weight was higher in the exclosures (mean EX: 13.0%; mean GR: 6.5%) (Table S2—online resource 2). The pH of the upper soil layer in both the exclosures and grazed sites was between 7.0 and 7.1.
Soil polysaccharide, sugar and amino acid content vary between exclosures and grazed sites
To identify the relationship between vegetation and soil polysaccharide composition we mapped the polysaccharide content in three different plant types—P. arctica, S. cespitosa and a mixture of Amblystegiaceae mosses. The same polysaccharide identification was done for the peat soil from the exclosures and grazed sites. A correspondence analysis confirmed that all three plant polysaccharide profiles were distinctly different from one another (Figures S1a and S1b—online resource 3). Saxifraga cespitosa was richer in the pectins homogalacturonan, rhamnogalacturonan and arabinan as well as cellulose and the hemicelluloses xyloglucan, mixed linkage glucans (MLG) and xylan. Poa arctica was richer in glucan, arabinoxylan and the glycoprotein AGP (arabinogalactan protein), while the mosses were richer in mannan-containing hemicelluloses, homogalacturonan (HG) with an intermediate degree of methyl esterification (DE) and galactan (Table S4—online resource 2).
Correspondingly we found that cellulose, glucan, xyloglucan, arabinoxylan and rhamnogalacturonan were more abundant in the soils from the exclosures while homogalacturonan was equally abundant at both sites. For arabinan and mannan-containing hemicelluloses, only some tested antibodies had significantly higher abundances in the exclosures (Table 2 and Figure S1c and S1d—online resource 3).
The pore water concentrations of sugars, including glucose, fructose, mannose, xylose and galactose, were significantly higher in the peat soils from the exclosures (Fig. 2a). Glucose was the most abundant sugar in the pore water of the exclosures (mean: 40.3 µM) and the grazed sites (mean: 9.9 µM), followed by fructose (EX mean: 18.3 µM and grazed site mean: 5.4 µM). There was less mannose, xylose and galactose in the peat, but the concentrations were always higher in the exclosures (mean EX: mannose 5.8 µM, xylose 2.2 µM, galactose 0.4 µM) than the grazed sites (mean GR: mannose 0.8 µM, xylose b.d., galactose 0.02 µM).
We also observed significantly higher concentrations of a broad range of amino acids in the exclosures (Fig. 2b and Table S3—online resource 2). Aspartic acid and alanine had the highest concentrations with mean values of 10.5 and 13.2 µM, respectively, in the exclosures, while the concentrations in the grazed sites were much lower (0.9 and 2.3 µM). The amino acids glycine, leucine, proline, serine and threonine ranged from 4.0 to 6.8 µM in the exclosures and 0.6 to 1.3 µM in the grazed sites. Only glutamic acid was found at lower concentrations in the exclosures (mean EX: 0.9 µM) compared to the grazed sites (mean GR: 2.0 µM). The other amino acids were below 1.8 µM (mean values) but always at higher concentrations in the exclosures than the grazed sites.
Microbial activities in the peat soil
To study the link between the vegetation, soil chemistry and the composition and activity of the microbial community we extracted total nucleic acids for the analysis of DNA and RNA from four replicate samples of the upper 2 cm oxic layer of peat soil in the exclosures and four from the grazed sites. As depicted in Fig. 2c we observed higher DNA amounts in the exclosures than the grazed sites (mean: EX 92.5 µg DNA gDW peat−1, GR 58.3 µg DNA gDW peat−1), while the amount of RNA per gram dry weight was equally high at the two sites (mean: EX 71.2 µg RNA gDW peat−1, GR 69.6 µg RNA gDW peat−1) (Fig. 2c and Table S5—online resource 2). Next, we investigated the potential enzyme activities for decomposition of some of the most common plant polysaccharides—cellulose, mannan, xyloglucan, xylan, arabinoxylan, galactan, arabinan and rhamnogalacturonan (Sarkar et al. 2009). This confirmed that the potential for polysaccharide degradation was significantly higher in the exclosures than the grazed sites (Fig. 2d and Table S3—online resource 2).
In order to study the microbial communities and their patterns of gene transcription, we removed ribosomes from the total RNA by ribodepletion and sequenced the remaining RNA as well as the total DNA from four replicates collected in the exclosures, and four in the grazed sites, giving eight metatranscriptomic and eight metagenomic libraries in total. Each of the 16 libraries contained ~ 38–55 million sequence reads with a length of ~ 150 bp. The ribodepleted RNA libraries contained 48–83% non-rRNA sequences, ~ 30% of which could be taxonomically classified and 10–15% of which could be functionally classified (Table S6—online resource 2). The microbial community composition based on taxonomic annotation of mRNA was overall similar in the exclosures and grazed sites, but notable differences were also observed (Figs. 3 and S2—online resource 3). Bacterial genes and transcripts comprised approximately 90% of total mRNA and 92% of total rRNA genes at exclosed sites, compared to approximately 96% of total mRNA and 95% of total rRNA genes at grazed sites. Correspondingly, the abundances of reads assigned to eukaryotic kingdoms (fungi, Protista and Metazoa) were particularly high in the exclosures relative to the grazed sites (Figs. 3 and S3—online resource 3). However, the 16S rRNA gene abundance and transcriptional activity were dominated by Actinobacteria, followed by Alpha-, Beta-, Delta- and Gamma-proteobacteria, Chloroflexi, Acidobacteria, Verrucomicrobia and Bacteriodetes in both grazed sites and exclosures. There were also considerable numbers of reads assigned to Ciliophora, fungi and Nematoda (Figs. 3 and S2—online resource 3). Overall, the most abundant taxa (SSU rRNA gene abundance) were the transcriptionally (mRNA abundance) most active (r = 0.86–0.94; only the taxa displayed in the boxes considered: Fig. 3a and b).
To identify which microbial taxa were responsible for polymer degradation we extracted from the metagenomes and metatranscriptomes genes and transcripts encoding enzymes for polysaccharide and lignin degradation (Table S7—online resource 2). In the exclosures, the microbial transcription for cellulose degradation was highest (0.40%), followed by hemicellulose (0.36%), pectin (0.24%) and lignin (0.16%) (Fig. 4a). In the grazed sites, the transcription for hemicellulose degradation was highest (0.31%), followed by cellulose (0.21%), pectin (0.07%) and lignin (0.04%). Corresponding to the higher abundance of fungi (Figs. 3 and S3—online resource 3), a larger fraction of the genes and transcripts for polymer decomposition were assigned to fungi in the exclosures (18.95%) than the grazed sites (1.38%) (Fig. 4a). The majority of these transcripts were assigned to the fungal phyla Basidiomycota (6.32% EX & 0.60% GR) and Ascomycota (10.56% EX & 0.48% GR). Actinobacteria (30.92% EX & 20.14% GR), Proteobacteria (11.28% EX & 14.51% GR) and Bacteriodetes (8.46% EX & 13.82% GR) were the most transcriptionally active bacterial polysaccharide degraders, while the majority of transcripts for lignin degradation were assigned to Proteobacteria (28.15% EX & 33.03% GR) (Fig. 4b). The taxonomic distribution of genes for polymer degradation was similar at exclosed and grazed sites, with the exception that the transcript to gene ratio was much higher for fungal than bacterial taxa (Fig. 4b).
Taxa that had different transcriptional activities in the exclosures and grazed sites were identified by correspondence analysis (Figure S4a—online resource 3). The transcriptional profiles from the exclosures were separated from the grazed sites along the first axis, explaining 42.7% of the inertia. By their contribution to the first axis inertia we identified the major eukaryotic and prokaryotic orders with different transcriptional activities in the exclosures and grazed sites. Out of the 20 taxa contributing most to inertia in each direction (Figure S4b—online resource 3; and Table S8—online resource 2), 15 taxa had higher numbers of transcripts in either the exclosures or the grazed sites, and an average relative abundance of mRNA transcripts above 0.5%. Among these, three eukaryotic (Agaricales, Helotiales and Entomobryomorpha) and seven bacterial orders (Lactobacillales, Chitinophagales, Burkholderiales, Sphingobacteriales, Nakamurellales, Corynebacteriales, Micrococcales) were more active in the exclosures, whereas five bacterial orders (Methylococcales, Anaerolineales, Solibacterales, Thiotrichales, Desulfobacterales) were more active in the grazed sites. These 15 orders accounted for 7 to 18% of the total number of microbial mRNA transcripts. We then compared the transcriptional activity and relative abundance of SSU rRNA genes for these 15 taxa (Figure S4b—online resource 3), finding similar patterns in the SSU rRNA genes and the mRNA for some of the taxa. Particularly interesting were the much higher relative abundances of the three eukaryotic orders in the exclosures: Agaricales was on average 7.7-fold (SSU rRNA genes) and 71.4-fold (mRNA) higher, Helotiales was 29.8-fold (SSU rRNA genes) and 46.9-fold (mRNA) higher, while Entomobryomorpha was 20.4-fold (SSU rRNA genes) and 11.8-fold (mRNA) more abundant and transcriptionally active (Fig. 5).
The number of polymer degradation transcripts assigned to Helotiales (of fungal transcripts: 21.5% EX, 4.0% GR) and Agaricales (of fungal transcripts: 37.6% EX, 4.3% GR) show that these are the major fungal polysaccharide degraders in the exclosures. Their profiles included transcripts for cellulose, xylans, glucans, mannans, pectins, lignin, chitin and bacterial cell walls (Fig. 5a and Table S9—online resource 2). Helotiales expressed genes for a broader range of substrates than Agaricales (Table S9—online resource 2), but for both fungal orders the relative abundance of transcripts for most polymers were higher in the exclosures (Fig. 5a). Similarly, the transcriptional activity of the arthropod order Entomobryomorpha was much higher in the exclosures (Fig. 5b). It had a narrower substrate range than the fungi, lacking transcripts for xylan, pectin and lignin decomposition (Table S9—online resource 2). However, Entomobryomorpha transcripts for bacterial cell wall, chitin, cellulose and oligosaccharide degradation were present, and consistently more abundant in the exclosures than the grazed sites (Fig. 5b). Finally we assessed whether the above patterns of transcription for polymer degradation were reflected in the transcription for central metabolisms in these three orders. We functionally annotated the Agaricales, Helotiales and Entomobryomorpha transcripts using KEGG (Kyoto encyclopedia of genes and genomes). This revealed that the transcript abundances for central metabolisms were highest in the exclosures for all three taxa (Figure S5—online resource 3), but with considerable variation between samples, especially for the fungi.