Sphagnum mosses harbour highly specific bacterial diversity during their whole lifecycle

Abstract

Knowledge about Sphagnum-associated microbial communities, their structure and their origin is important to understand and maintain climate-relevant Sphagnum-dominated bog ecosystems. We studied bacterial communities of two cosmopolitan Sphagnum species, which are well adapted to different abiotic parameters (Sphagnum magellanicum, which are strongly acidic and ombrotrophic, and Sphagnum fallax, which are weakly acidic and mesotrophic), in three Alpine bogs in Austria by a multifaceted approach. Great differences between bacterial fingerprints of both Sphagna were found independently from the site. This remarkable specificity was confirmed by a cloning and a deep sequencing approach. Besides the common Alphaproteobacteria, we found a discriminative spectrum of bacteria; although Gammaproteobacteria dominated S. magellanicum, S. fallax was mainly colonised by Verrucomicrobia and Planctomycetes. Using this information for fluorescent in situ hybridisation analyses, corresponding colonisation patterns for Alphaproteobacteria and Planctomycetes were detected. Bacterial colonies were found in high abundances inside the dead big hyalocytes, but they were always connected with the living chlorocytes. Using multivariate statistical analysis, the abiotic factors nutrient richness and pH were identified to modulate the composition of Sphagnum-specific bacterial communities. Interestingly, we found that the immense bacterial diversity was transferred via the sporophyte to the gametophyte, which can explain the high specificity of Sphagnum-associated bacteria over long distances. In contrast to higher plants, which acquire their bacteria mainly from the environment, mosses as the phylogenetically oldest land plants maintain their bacterial diversity within the whole lifecycle.

Figure 1

Dendrogram based on amplified 16S rRNA gene fragments of bacterial communities associated with S. magellanicum (M) and S. fallax (F; FS=sporophyte) from different sites in Austria (R, Rotmoos; W, Wasenmoos; P, Pürgschachen Moor) obtained by using eubacterial primers and separated by single-strand conformation polymorphism (SSCP). The patterns obtained were grouped by unweighted pair group method with average linkages.

Figure 2

Bacterial community composition revealed by 16S rRNA gene clone libraries. Absolute and relative abundances of the taxonomic groups are shown for the S. magellanicum- (a) and S. fallax-associated (b) bacterial communities.

Figure 3

Taxonomic classification of Alphaproteobacteria associated with S. fallax and S. magellanicum. Pyrosequencing reads are classified at family (a) and genus level (b) with a confidence threshold of 50%. Unclassified reads are not shown. Groups not reaching 1% of relative abundance are included in ‘Other'. Multi-coloured charts at the legend are shown for each genus and sample correspondingly.

Figure 4

Localisation of bacteria in moss gametophytes. Fluorescent in situ hybridisation (FISH) of S. fallax leaves showed colonisation of the outer surface (a and b) and hyaline cells (c and d). Violet: cell walls of Sphagnum cells; green: chlorophyll-containing Sphagnum chlorocytes; yellow: Alphaproteobacteria; red: other bacteria. Images acquired by confocal laser scanning microscopy (CLSM; panels a and c) and processed by 3D computer reconstruction using Imaris7.0 (b and d). Scale bar=10 μm.

Figure 5

Localisation of bacteria in hyalocytes of Sphagnum. Internal space of hyalocytes of S. fallax (a and b) and S. magellanicum (c and d) hybridised with Alphaproteobacteria- and Planctomycetes-specific probes. Yellow: Alphaproteobacteria (a and c) or Planctomycetes (b and d) indicated by arrows; red: other bacteria; green: algae. Scale bar=10 μm (a and b) or 5 μm (c and d).

Figure 6

Canonical correspondence analysis biplot of operational taxonomic units identified by SSCP community fingerprints. Independent ecological gradients are given as unweighted average of indicator values by Ellenberg for vascular plants and bryophytes. Single fingerprints of S. fallax- and S. magellanicum-associated communities are depicted with black squares and gray circles, respectively. Names of the single SSCP patterns correspond to the names of the sample. Dashed ovals were drawn around samples of the same Sphagnum species. Species–environment correlations of the first and the second axes are 0.942 and 0.902, respectively. Sum of all Eigenvalues=0.563; significance for the first axis: P-value=0.002, F-ratio=2.795 tested by Monte-Carlo permutation test (1000 permutations).