Large differences in carbohydrate degradation and transport potential among lichen fungal symbionts

Abstract

Lichen symbioses are thought to be stabilized by the transfer of fixed carbon from a photosynthesizing symbiont to a fungus. In other fungal symbioses, carbohydrate subsidies correlate with reductions in plant cell wall-degrading enzymes, but whether this is true of lichen fungal symbionts (LFSs) is unknown. Here, we predict genes encoding carbohydrate-active enzymes (CAZymes) and sugar transporters in 46 genomes from the Lecanoromycetes, the largest extant clade of LFSs. All LFSs possess a robust CAZyme arsenal including enzymes acting on cellulose and hemicellulose, confirmed by experimental assays. However, the number of genes and predicted functions of CAZymes vary widely, with some fungal symbionts possessing arsenals on par with well-known saprotrophic fungi. These results suggest that stable fungal association with a phototroph does not in itself result in fungal CAZyme loss, and lends support to long-standing hypotheses that some lichens may augment fixed CO₂ with carbon from external sources.

Fig. 1. Distribution and ancestral states of CAZymes and selected sugar transporters across the evolution of Lecanoromycetes and related classes of Ascomycota projected onto a maximum likelihood phylogenomic tree based on 1310 loci.

Symbols beside tree tips refer to life history traits and phototrophic partners of LFS under study. Heatmaps with shades of red indicate the number of genes in different CAZyme classes or involved in degrading complex PCW components. Columns from left to right: AA Auxiliary Activities, CBM Carbohydrate binding module, CE Carbohydrate Esterases, GH Glycoside Hydrolases, GT Glycosyl Transferases, PL Polysaccharide Lyases. cell - Number of genes in 35 CAZyme families involved in cellulose and hemicellulose breakdown. pec - Number of genes in 11 CAZyme families involved in pectin breakdown. lign - Number of genes in three CAZyme families involved in lignin modification. Selection of CAZyme sets follows^, . PODs: numbers of heme haloperoxidase and DyP peroxidases potentially involved in lignin modification from Redoxibase (see text). Sugar transporters - selected PF00083 transporters. celldex - Number of cellodextrin transporters. malt - number of maltose transporters. total - Total number of other PF00083 transporters. Below the heatmap are ancestral sizes of CAZyme families involved in (hemi-)cellulose, pectin and lignin modification. Colored circles on tree branches indicate significantly expanded CAZyme families. The size of the circles indicates the number of individual CAFE runs (out of 20 total runs) in which a family was found to be significantly expanded. Expanded gene families recovered in less than five runs are omitted here. Exact numbers are given in Supplementary Fig. 12. Source data are provided as a Source Data file.

Fig. 2. Similarity of CAZyme sets involved in the breakdown of different complex plant-based polysaccharides based on phylogenetically corrected Principal Components Analysis.

Different colors indicate taxonomic groups; genomes from the subclasses Acarosporomycetidae and Umbilicariomycetidae use the same color codes as Lecanoromycetidae for simplicity. A Similarity of CAZyme families involved in cellulose and hemicellulose breakdown. B Similarity of CAZyme families involved in lignin breakdown, C Similarity of CAZyme families involved in pectin breakdown. Displayed below are representative members of the two subclasses Ostropomycetidae (with light green border) and Lecanoromycetidae (dark green border). D Graphis scripta, E Icmadophila ericetorum, F Mycoblastus sanguinarius, G Peltigera leucophlebia, H Xylographa carneopallida, I Agyrium rufum, J Evernia prunastri, K Cladonia macilenta. Image credits: Agyrium rufum: Paul Cannon (fungi.myspecies.info); Creative Commons: BY-NC 4.0. Peltigera leucophlebia: Jason Hollinger, uploaded by Amada44, CC-BY 2.0, https://commons.wikimedia.org/w/index.php?curid=24213606. Evernia prunastri: by Jason Hollinger, CC-BY 2.0, https://commons.wikimedia.org/w/index.php?curid=50595319. Cladonia macilenta: Bruce McCune & Sunia Yang - Lichen, CC-BY 4.0-NC, https://lichens.twinferntech.net/pnw/species/Cladonia_macilenta.shtml; other images by the authors. Source data are provided as a Source Data file.

Fig. 3. Gene trees of two CAZyme families involved in cellulose (GH5) and hemicellulose (GH43) breakdown. Each tree includes all experimentally characterized sequences combined with all sequences from the 83 genomes studied here.

CAZyme Subfamilies are labeled with numbers and gray rectangles. Three columns along tree tips display additional information of corresponding sequences when available. EC - Enzyme Code of experimentally characterized sequences downloaded from cazy.org. Sub Loc - Predicted subcellular location of Enzyme with DeepLoc. Tax - Taxonomic assignment of organisms from which the sequence comes from. For larger subfamilies, functions of characterized sequences based in Enzyme Code numbers are given as colored squares. Sequences used for heterologous expression experiments are marked with red arrows in GH5 subfamily 5. Source data are provided as a Source Data file.

Fig. 4. Enzymatic activity of two putative cellulases A and B from Xylographa bjoerkii and orthologs of these genes.

Activity of cellulases A and B at different pH conditions (a), at different temperatures (b), and with different substrates (c). Y axis represents mean absorbance (+/− standard error of mean) at 595 nm after adjusting for the dilution factor. Experiments were performed in technical and biological triplicates. d Number of orthologs of cellulase A and B in the 72 genomes that possess them. Different colors indicate taxonomic groups; genomes from the subclasses Acarosporomycetidae and Umbilicariomycetidae use the same color codes as Lecanoromycetidae for simplicity. Source data are provided as a Source Data file.