Bacterial communities associated with the lichen symbiosis

Abstract

Lichens are commonly described as a mutualistic symbiosis between fungi and "algae" (Chlorophyta or Cyanobacteria); however, they also have internal bacterial communities. Recent research suggests that lichen-associated microbes are an integral component of lichen thalli and that the classical view of this symbiotic relationship should be expanded to include bacteria. However, we still have a limited understanding of the phylogenetic structure of these communities and their variability across lichen species. To address these knowledge gaps, we used bar-coded pyrosequencing to survey the bacterial communities associated with lichens. Bacterial sequences obtained from four lichen species at multiple locations on rock outcrops suggested that each lichen species harbored a distinct community and that all communities were dominated by Alphaproteobacteria. Across all samples, we recovered numerous bacterial phylotypes that were closely related to sequences isolated from lichens in prior investigations, including those from a lichen-associated Rhizobiales lineage (LAR1; putative N(2) fixers). LAR1-related phylotypes were relatively abundant and were found in all four lichen species, and many sequences closely related to other known N(2) fixers (e.g., Azospirillum, Bradyrhizobium, and Frankia) were recovered. Our findings confirm the presence of highly structured bacterial communities within lichens and provide additional evidence that these bacteria may serve distinct functional roles within lichen symbioses.

FIG. 1.

Species of foliose lichens containing green algal photobionts collected on rock outcrops at the sampling site. (A) Parmelia sulcata; (B) Rhizoplaca chrysoleuca (image courtesy of T. Wheeler); (C) Umbilicaria americana; (D) Umbilicaria phaea (image courtesy of F. Bungartz).

FIG. 2.

Relative abundances of various major bacterial lineages recovered from lichen species. Relative abundance was calculated as the percentage of sequences belonging to a particular lineage of all 16S rRNA gene sequences recovered from a given lichen species (4 samples per species).

FIG. 3.

Heat map showing the 15 most dominant phylotypes in each of four lichen species sampled for this study and their average relative abundances (as percentages of all sample 16S rRNA gene sequences recovered). Phylotypes were considered dominant if they were both highly abundant and occurred frequently in samples of a given lichen species. Color tones moving from red to yellowish white indicate the highest to lowest relative abundance values (by species). “NA” indicates that the phylotype was not included within the 15 most dominant phylotypes for that species.

FIG. 4.

ARB maximum parsimony constraint tree of Alphaproteobacteria sequences recovered from our lichen samples as well as closely related sequences obtained from GenBank (accession numbers are indicated, and those from previous lichen-bacterium studies show a citation). A large asterisk indicates that the lineage contains phylotypes closely related to sequences within genera containing known N₂-fixing bacteria. PT, phylotype (those PT numbers with asterisks are found among the 15 most dominant phylotypes per species) (Fig. 3). Red branches indicate the LAR1 lineage. Bar = 1% sequence dissimilarity. The tree is rooted with Acinetobacter baumannii (GenBank accession no. AY847284).

FIG. 5.

Nonmetric multidimensional scaling plots (Bray-Curtis distance matrices) depicting patterns of beta diversity for bacterial communities associated with lichen thalli. Points that are closer together on the ordination have communities that are more similar, and dot- ted lines were added postanalysis to highlight species clusters. PERMANOVA indicated that differences between bacterial communities mapped according to the lichen species of origin (A) for the sample were highly significant (P = 0.001), while differences mapped according to rock outcrop of origin (B) were not significant (P = 0.65).