Pyrosequencing-Based Assessment of the Bacteria Diversity in Surface and Subsurface Peat Layers of a Northern Wetland, with Focus on Poorly Studied Phyla and Candidate Divisions

with Focus on Poorly Studied Phyla and Candidate Divisions. PLoS ONE 8(5): e63994. doi:10.1371/journal.pone.0063994 Pyrosequencing-Based Assessment of the Bacteria Diversity in Surface and Subsurface Peat Layers of a Northern Wetland, with Focus on Poorly Studied Phyla and Candidate Divisions Yulia M. Serkebaeva 0 Yongkyu Kim 0 Werner Liesack 0 Svetlana N. Dedysh 0 Josh Neufeld, University of Waterloo, Canada 0 1 Winogradsky Institute of Microbiology, Russian Academy of Sciences , Moscow , Russia , 2 Max Planck Institute for Terrestrial Microbiology , Marburg , Germany Northern peatlands play a key role in the global carbon and water budget, but the bacterial diversity in these ecosystems remains poorly described. Here, we compared the bacterial community composition in the surface (0-5 cm depth) and subsurface (45-50 cm) peat layers of an acidic (pH 4.0) Sphagnum-dominated wetland, using pyrosequencing of 16 S rRNA genes. The denoised sequences (37,229 reads, average length ,430 bp) were affiliated with 27 bacterial phyla and corresponded to 1,269 operational taxonomic units (OTUs) determined at 97% sequence identity. Abundant OTUs were affiliated with the Acidobacteria (35.562.4% and 39.261.2% of all classified sequences in surface and subsurface peat, respectively), Alphaproteobacteria (15.961.7% and 25.861.4%), Actinobacteria (9.562.0% and 10.760.5%), Verrucomicrobia (8.561.4% and 0.660.2%), Planctomycetes (5.860.4% and 9.760.6%), Deltaproteobacteria (7.160.4% and 4.4%60.3%), and Gammaproteobacteria (6.660.4% and 2.160.1%). The taxonomic patterns of the abundant OTUs were uniform across all the subsamples taken from each peat layer. In contrast, the taxonomic patterns of rare OTUs were different from those of the abundant OTUs and varied greatly among subsamples, in both surface and subsurface peat. In addition to the bacterial taxa listed above, rare OTUs represented the following groups: Armatimonadetes, Bacteroidetes, Chlamydia, Chloroflexi, Cyanobacteria, Elusimicrobia, Fibrobacteres, Firmicutes, Gemmatimonadetes, Spirochaetes, AD3, WS1, WS4, WS5, WYO, OD1, OP3, BRC1, TM6, TM7, WPS-2, and FCPU426. OTU richness was notably higher in the surface layer (882 OTUs) than in the anoxic subsurface peat (483 OTUs), with only 96 OTUs common to both data sets. Most members of poorly studied phyla, such as the Acidobacteria, Verrucomicrobia, Planctomycetes and the candidate division TM6, showed a clear preference for growth in either oxic or anoxic conditions. Apparently, the bacterial communities in surface and subsurface layers of northern peatlands are highly diverse and taxonomically distinct, reflecting the different abiotic conditions in microhabitats within the peat profile. - . These authors contributed equally to this work. Northern wetlands are water-saturated, peat-accumulating ecosystems located between 45 and 70uN. These environments are recognized as a persistent sink for atmosp2h2eryiceaCr2O1.2, Twhiethy carbon accumulation rates of 1030 g C m harbor a total of 200450 Pg of carbon, which represents about one-third of the global soil carbon pool [1]. Northern wetlands are also known as a major source of the greenhouse gas methane (CH4), which is produced in the anoxic peat layers due to decomposition of organic matter [24]. In addition to their importance as a large terrestrial carbon store, these wetlands hold a key role in the global water balance and represent one of the largest reservoirs of freshwater in the Northern Hemisphere. A large proportion of northern wetlands consists of Sphagnumdominated bogs, which occupy about 3% of the Earths terrestrial surface [5], comprising up to 80% of the area in some regions of West Siberia. Bogs are ombrotrophic ecosystems that are decoupled from the groundwater of the surrounding watershed and receive water and nutrients only via atmospheric deposition. These wetlands are highly acidic (pH values typically below 4.0) and nutrient-poor by nature. Peat water usually contains very low concentrations of mineral N, S, and Fe and, therefore, redox transformations of these elements are only of minor importance in ombrotrophic bogs. Degradation of Sphagnum-derived litter is the basis of the microbial food web in these ecosystems (reviewed in [6]). Despite the global importance of Sphagnum-dominated wetlands as both terrestrial carbon store and freshwater reservoir, the microbial diversity in these ecosystems remains largely unexplored [6]. The research to date has primarily focused on several functional guilds such as methanotrophic bacteria [710], methanogenic archaea [1114], sulfate reducers [15], cellulose degraders [16], and nitrogen-fixing microorganisms [17,18]. A few studies assessed the total microbial diversity in acidic northern peatlands, using T-RFLP fingerprinting and/or clone library analysis as well as, more recently, pyrosequencing of 16 S rRNA genes [1926]. These studies showed that ombrotrophic bogs are usually dominated by members of the poorly studied bacterial phyla, such as the Acidobacteria, Planctomycetes, and Verrucomicrobia, which are represented mostly by as-yet-uncultivated organisms with unknown physiologies and metabolic potentials. During the last decade, a number of peat-inhabiting acidobacteria and planctomycetes were obtained in pure culture and characterized (reviewed in [6]). These isolation-based studies provided the first proof for the presence of cellulose-degrading capabilities in some of the acidobacteria and planctomycetes [16,27,28] and highlighted the role of these bacteria as slow-acting decomposers in acidic and cold wetlands. With the exception of Telmatobacter bradus [27], however, all characterized peat-inhabiting acidobacteria and planctomycetes are aerobic bacteria, which can thrive only in the narrow surface zone of the peat bog profile. The occurrence of anammox planctomycetes in ombrotrophic peatlands has not yet been confirmed [29], although these bacteria were detected in a swampy peat soil fed by nitrate-enriched local groundwater [30]. There is even less known about the lifestyles of peat-inhabiting verrucomicrobia. To date, none of these bacteria has been cultivated from acidic peat. One intriguing question is whether the recently discovered acidophilic methanotrophic Verrucomicrobia [31] occur in acidic northern wetlands. Here, we used 454-pyrosequencing to gain deeper insights into the bacterial diversity associated with surface (05 cm depth) and subsurface (4550 cm) peat of an acidic (pH 4.0) Sphagnumdominated ombrotrophic wetland. We hypothesized that oxygen availability is a major factor shaping bacterial community composition in Sphagnum-dominated peat bogs. We also aimed to test how subsampling affects the detectable diversity patterns. Our analyses were specifically focused on poorly understood and elusive groups of bacteria, such as Acidobacteria, Verrucomicrobia, Planctomycetes, and candidate division TM6. Materials and Methods Sampling Site A (...truncated)