Network Analysis Reveals Ecological Links between N-Fixing Bacteria and Wood-Decaying Fungi

<div><p>Nitrogen availability in dead wood is highly restricted and associations with N-fixing bacteria are thought to enable wood-decaying fungi to meet their nitrogen requirements for vegetative and generative growth. We assessed the diversity of <i>nifH</i> (dinitrogenase reductase) genes in dead wood of the common temperate tree species <i>Fagus sylvatica</i> and <i>Picea abies</i> from differently managed forest plots in Germany using molecular tools. By incorporating these genes into a large compilation of published <i>nifH</i> sequences and subsequent phylogenetic analyses of deduced proteins we verified the presence of diverse pools corresponding to functional <i>nifH</i>, almost all of which are new to science. The distribution of <i>nifH</i> genes strongly correlated with tree species and decay class, but not with forest management, while higher fungal fructification was correlated with decreasing nitrogen content of the dead wood and positively correlated with <i>nifH</i> diversity, especially during the intermediate stage of wood decay. Network analyses based on non-random species co-occurrence patterns revealed interactions among fungi and N-fixing bacteria in the dead wood and strongly indicate the occurrence of at least commensal relationships between these taxa.</p></div

Network organized around 23 <i>nifH</i> MOTUs and 27 fungal species (abbreviations according to the legend).

<p>Fungi (sporocarps) and <i>nifH</i> MOTUs serve as connected nodes, solid lines display co-occurrence patterns (Z-Score < -1.96) and dotted lines avoidance patterns. Edge widths display significance levels from thinnest  =  0.049 to thickest  =  0.0017. Differently shaped and colored nodes/ hubs display taxonomic differences on phylum level and their ecological role in wood decay. Subnetworks are grouped by tree species, and colored background circles indicate affiliations of included taxa to substrate dead wood species (green  =  <i>Fagus</i> affiliated, red  =  <i>Picea</i> affiliated, blue  =  unaffiliated “Generalists”).</p

Rank abundance chart displaying the distribution of the 12 most abundant <i>nifH</i> MOTUs derived from the dead wood dataset within the compiled <i>nifH</i> dataset comprising 26,383 sequences.

<p>Only the 200 largest MOTUs are shown due to space limitations. Colored bars indicate dead wood tree species (green, <i>Fagus sylvatica</i>; red, <i>Picea abies</i>). The inserted table lists the best BLASTn hit reference sequences in NCBI Genbank for the same 12 most abundant wood-derived MOTUs from our study.</p

Distribution of the 7,730 <i>nifH</i> MOTUs according to the environments where they have been detected, and whether described as originating from an isolate in GenBank.

<p>168 of the 176 MOTUs derived from this dead wood study have been exclusively identified in wood samples. The integrated heatmap displays proportions of rare sequence types (singletons).</p

Results of perMANOVA analysis of Bray-Curtis dissimilarities in <i>nifH</i> MOTU community structure in relation to tree species, decay class (based on remaining mass after decay) and management type and their interactions, Df = degrees of freedom; SS = sum of squares; MS = mean sum of squares; Pseudo-F = F value by permutation, boldface indicates statistical significance at p<0.05, p-values based on 999 permutations (lowest p-value possible is 0.001).

<p>Results of perMANOVA analysis of Bray-Curtis dissimilarities in <i>nifH</i> MOTU community structure in relation to tree species, decay class (based on remaining mass after decay) and management type and their interactions, Df  =  degrees of freedom; SS  =  sum of squares; MS  =  mean sum of squares; Pseudo-F  =  F value by permutation, boldface indicates statistical significance at p<0.05, p-values based on 999 permutations (lowest p-value possible is 0.001).</p

ANOVA table of effects of the indicated factors on fungal fructification ability.

<p>Complete model summary representing <i>R², F, P</i> statistics. Abbreviations of the depicted ANOVA table Df  =  degrees of freedom, SS  =  sum of squares, MS  =  mean sum of squares. The summary model is as follows: <i>R²</i>, <i>F</i>, and <i>p</i> were 0.5208,</p><p>8.476 and <0.001 (significant), respectively. Boldface indicates statistical significance.</p

Second part of three (Figs. 3, 4 and 5): Phylogenetic tree of <i>nifH</i> protein sequences.

<p>50% majority rule consensus tree of 13,500 PhyloBayes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088141#pone.0088141-Lartillot1" target="_blank">[52]</a> post burn-in trees, unrooted. Black values at internodes  =  Bayesian Posterior Probability (if >0.5). Pink values  =  MEGA5 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088141#pone.0088141-Tamura1" target="_blank">[56]</a> Maximum Parsimony (MP) bootstrap support (if >50). Green values  =  GARLI <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088141#pone.0088141-Zwickl1" target="_blank">[54]</a> Maximum Likelihood bootstrap support (if >50). Terminal triangles represent monophyletic clades with MOTUs solely of one tree species origin, collapsed but keeping the internal distance (substitutions per site, see scale bar), in light pink  =  50–79 MP bootstrap support, dark pink  =  80–100 MP bootstrap support. Green color indicates MOTUs solely from <i>Fagus</i> origin, red color <i>Picea</i> origin and dark blue color mixed origin (with bars showing ratio of [green] vs. [red]). Terminal labels with sequences from this study: MOTU ID (SMOTU  =  singleton MOTU), total number of sequences, FASY  =  from <i>Fagus</i>, PIAB  =  from <i>Picea</i>, followed by number of sequences in the same order, then forest management type(s) (AC.Conif  =  managed spruce forests, AC.Decid  =  managed beech forests, Extensiv  =  extensively managed beech forests) and number of sequences in same order. Terminal labels with sequences from other sources: near BLAST hit, summary of ecological data of sequences in that MOTU. The width of visible terminal branches represents the number of sequences (size correct up to 10 sequences). To the right, amino acid sequence logos and Kyte-Doolittle hydophobicity alignments for labeled nodes on the tree. The small tree shape (based on screenshot from Archaeopteryx v.0.972 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088141#pone.0088141-Han1" target="_blank">[66]</a>) shows the position within the complete phylogenetic tree.</p

Third part of three (Figs. 3, 4 and 5): Phylogenetic tree of <i>nifH</i> protein sequences.

<p>50% majority rule consensus tree of 13,500 PhyloBayes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088141#pone.0088141-Lartillot1" target="_blank">[52]</a> post burn-in trees, unrooted. Black values at internodes  =  Bayesian Posterior Probability (if >0.5). Pink values  =  MEGA5 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088141#pone.0088141-Tamura1" target="_blank">[56]</a> Maximum Parsimony (MP) bootstrap support (if >50). Green values  =  GARLI <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088141#pone.0088141-Zwickl1" target="_blank">[54]</a> Maximum Likelihood bootstrap support (if >50). Terminal triangles represent monophyletic clades with MOTUs solely of one tree species origin, collapsed but keeping the internal distance (substitutions per site, see scale bar), in light pink  =  50–79 MP bootstrap support, dark pink  =  80–100 MP bootstrap support. Green color indicates MOTUs solely from <i>Fagus</i> origin, red color <i>Picea</i> origin and dark blue color mixed origin (with bars showing ratio of [green] vs. [red]). Terminal labels with sequences from this study: MOTU ID (SMOTU  =  singleton MOTU), total number of sequences, FASY  =  from <i>Fagus</i>, PIAB  =  from <i>Picea</i>, followed by number of sequences in the same order, then forest management type(s) (AC.Conif  =  managed spruce forests, AC.Decid  =  managed beech forests, Extensiv  =  extensively managed beech forests) and number of sequences in same order. Terminal labels with sequences from other sources: near BLAST hit, summary of ecological data of sequences in that MOTU. The width of visible terminal branches represents the number of sequences (size correct up to 10 sequences). To the right, amino acid sequence logos and Kyte-Doolittle hydophobicity alignments for labeled nodes on the tree. The small tree shape (based on screenshot from Archaeopteryx v.0.972 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088141#pone.0088141-Han1" target="_blank">[66]</a>) shows the position within the complete phylogenetic tree.</p

Interrelations between sporocarp richness and remaining mass after decay in %, <i>nifH</i> MOTU richness and log-transformed nitrogen content per density unit (N (g/cm³))(A, B, C).

<p>The figure displays interrelations separately per dead wood species.</p

Dissimogram for the bird communities showing the Jaccard dissimilarity between each possible site pair <i>versus</i> Euclidian distance between the forests sites.

<p>(<b>a–c</b>) all sites in 2011, (<b>d–f</b>) sites with low land use intensity, and (<b>g–i</b>) sites with high land use intensity. The <i>x</i>-axis is the distance between each pair of sites. The <i>grey line</i> is the fitted line estimated based on non-linear least squares. Note the different scales in each <i>x</i>-axis.</p