Similar diversity of alphaproteobacteria and nitrogenase gene amplicons on two related sphagnum mosses

Abstract

Sphagnum mosses represent a main vegetation component in ombrotrophic wetlands. They harbor a specific and diverse microbial community with essential functions for the host. To understand the extend of host specificity and impact of environment, Sphagnum fallax and Sphagnum angustifolium, two phylogenetically closely related species, which show distinct habitat preference with respect to the nutrient level, were analyzed by a multifaceted approach. Microbial fingerprints obtained by PCR-single-strand conformation polymorphism of 16S rRNA and nitrogenase-encoding (nifH) genes were highly similar for both Sphagnum species. Similarity was confirmed for colonization patterns obtained by fluorescence in situ hybridization (FISH) coupled with confocal laser scanning microscopy (CLSM): Alphaproteobacteria were the main colonizers inside the hyaline cells of Sphagnum leaves. A deeper survey of Alphaproteobacteria by 16S rRNA gene amplicon sequencing reveals a high diversity with Acidocella, Acidisphaera, Rhodopila, and Phenylobacterium as major genera for both mosses. Nitrogen fixation is an important function of Sphagnum-associated bacteria, which is fulfilled by microbial communities of Sphagna in a similar way. NifH libraries of Sphagnum-associated microbial communities were characterized by high diversity and abundance of Alphaproteobacteria but contained also diverse amplicons of other taxa, e.g., Cyanobacteria and Deltaproteobacteria. Statistically significant differences between the microbial communities of both Sphagnum species could not be discovered in any of the experimental approach. Our results show that the same close relationship, which exists between the physical, morphological, and chemical characteristics of Sphagnum mosses and the ecology and function of bog ecosystems, also connects moss plantlets with their associated bacterial communities.

Keywords: Alphaproteobacteria; FISH–CLSM; SSCP fingerprints; Sphagnum angustifolium; Sphagnum fallax; amplicon library; nitrogenase.

Figure 1

High-performance liquid chromatography -UV secondary metabolite profiles of S. angustifolium and S. fallax at 229 nm (UV spectra (200–400 nm; 1, phenol; 2, 5, 6, indole derivative; 3, 4, phenols; 7, 9, sphagnic acid; 8, phenol; 10, naringenin type flavonoid; 11, 12, apigenin flavonoids.

Figure 2

Unweighted pair group method with average linkages dendrograms of bacterial communities associated with S. angustifolium (SA, squares) and S. fallax (SF, circles). The dendrograms were generated from the PCR-SSCP profiles of Bacteria (A), Alphaproteobacteria (B), and bacterial nifH genes (C) using unweighted pair group method with average linkages (UPGMA). Numbers in round brackets indicate replicates. Double-headed vertical arrows indicate the similarity for the groupings.

Figure 3

Detrended correspondence analysis (DCA, indirect unimodal gradient analysis) of operational taxonomic units (OTUs) identified by SSCP community fingerprints. Eigenvalues of first and second axis are 0.244 and 0.141, respectively; sum of all eigenvalues 1.715. Black stars and diamonds show the location of the 16 samples, colored circles the location of the 111 OTUs in the biplot. The colors indicate its preference to Sphagnum angustifolium (ang) or S. fallax, respectively.

Figure 4

Localization of bacteria in moss gametophytes. Fluorescent in situ hybridization of S. angustifolium (A–C) and S. fallax (D–F) leaves showed colonization of hyaline cells by Alphaproteobacteria. Images acquired by confocal laser scanning microscopy (CLSM) (A,B,D,E): violet – cell walls of Sphagnum cells; green – chlorophyll-containing Sphagnum chlorocytes; yellow – Alphaproteobacteria; red – other bacteria. 3D computer reconstructions of CLSM images using Imaris7.0 (C,F): blue – moss tissue; red: Alphaproteobacteria; green: other bacteria. Scale bar = 20 μm.

Figure 5

Rarefaction curves for amplicon libraries of Sphagnum samples. Saturation curves are presented for samples of S. angustifolium (SA) and S. fallax (SF). Alphaproteobacteria (A) and nifH datasets (B) were clustered with similarity cut-offs defined.

Figure 6

Taxonomic classification of Alphaproteobacteria associated with S. angustifolium and S. fallax. Pyrosequencing reads are classified at family (A) and genus level (B) with a confidence threshold of 80%. Groups below 1% of relative abundance are included in Other. Multi-colored charts at the legend are shown for each sample correspondingly.

Figure 7

Phylogenetic composition of the nitrogenase gene (nifH) amplicon libraries of S. angustifolium (SA, circles) and S. fallax (SF, squares). Neighbor-joining phylogenetic tree was constructed with both one representative sequence per NifH cluster (92% similarity) and the nearest reference sequences (accession numbers in brackets). A partial sequence of the light-independent photochlorophyllide reductase subunit L (BchL) from Chlorobaculum tepidum (Acc. Nr. AAG12203) was used as out-group. Reliability of the tree topology was evaluated by 100 bootstrap resamplings (bootstrap values not shown). Abundance and phylogenetic affiliation of the clusters are indicated at the legend. Scale bar = 0.1 substitutions per site.