A comparative genomic analysis of lichen-forming fungi reveals new insights into fungal lifestyles

Abstract

Lichen-forming fungi are mutualistic symbionts of green algae or cyanobacteria. We report the comparative analysis of six genomes of lichen-forming fungi in classes Eurotiomycetes and Lecanoromycetes to identify genomic information related to their symbiotic lifestyle. The lichen-forming fungi exhibited genome reduction via the loss of dispensable genes encoding plant-cell-wall-degrading enzymes, sugar transporters, and transcription factors. The loss of these genes reflects the symbiotic biology of lichens, such as the absence of pectin in the algal cell wall and obtaining specific sugars from photosynthetic partners. The lichens also gained many lineage- and species-specific genes, including those encoding small secreted proteins. These genes are primarily induced during the early stage of lichen symbiosis, indicating their significant roles in the establishment of lichen symbiosis.Our findings provide comprehensive genomic information for six lichen-forming fungi and novel insights into lichen biology and the evolution of symbiosis.

Figure 1

Phylogenomic and syntenic relationships among lichen-forming fungi and their repeat contents. (A) The phylogenomic tree shows that lichen-forming fungi are derived from many ancestors. Red branches indicate lichen-forming fungal lineages, and colored squares indicate lifestyle of a fungal species. The scale of the phylogenomic tree is millions of years, as calculated using the mcmctree function in the Phylogenetic Analysis by Maximum Likelihood software package. Blue error bars at each node indicate 95% highest posterior density (HPD) for node age. (B) Synteny dot plots of lichen-forming fungi. Red (blue) dots indicate forward (reverse) matches. (C) Repetitive sequence contents of lichen-forming fungi identified using RepeatMasker software. DNA transposons, retroelements, and unclassified repeats are classes of interspersed repeats.

Figure 2

Gene family evolution in lichen-forming fungi and their relatives. Estimation of gene family expansion and contraction in lichen-forming fungi using the CAFE computational tool (P > 0.01). Red arrows indicate branch points where lichen-forming fungi diverged from non-lichenized ancestors. + and – indicate the numbers of expanded and contracted gene families, respectively. Only 16 species belonging to the Lecanoromyces, Eurotiomyces, and Dotidomyces closely related to lichen-forming fungi were shown, and the analysis results for all 56 species were placed in the Supplementary Figure S2. Species abbreviations: EpusR, Endocarpon pusillum R61883; EpusZ, E. pusillum Z07020; Gfla, Gyalolechia flavorubescens; Umue, Umbilicaria muehlenbergii; Cmet, Cladonia metacorallifera; Cmac, Cladonia macilenta. Abbreviations of other species names are provided in Supplementary Dataset S1.

Figure 3

Loss of plant cell wall-degrading enzymes (PCWDEs) in lichen-forming fungi for association with algal partners. (A) Distribution of selected carbohydrate-active enzyme (CAZyme) families related to cellulose, hemicellulose, and pectin degradation among lifestyles. Red, green, and orange boxes indicate lichen-forming fungi, plant pathogens, and mycorrhizal fungi, respectively. Asterisk indicates ectomycorrhizal fungi, a class of mycorrhizal fungi. The distribution of PCWDEs in lichen-forming fungi was compared with plant pathogens known to have a large number of PCWDEs for pathogenicity and mycorrhizal fungi known to be associated with symbiosis formations. Analysis of other lifestyles is in Supplementary Dataset S2. CAZyme family abbreviations: GH, glycoside hydrolase; AA, auxiliary activities; CE, carbohydrate esterase; PL, polysaccharide lyase. Gene gain and loss analysis of (B) cellulose, (C) hemicellulose, and (D) pectin-degrading CAZyme families through species tree–gene tree reconciliation. Blue (red) circles indicate the number of gene gains (losses). Bubble size indicates the number of genes gained or lost. Bar graph indicates the total number of genes encoding PCWDEs in each species.

Figure 4

Distribution of expanded and contracted gene families in lichen-forming fungi. (A) Comparative analysis of selected families among the cytochrome P450 (CYP) and transcription factor (TF) families and major facilitator superfamily (MFS), which expanded or contracted in lichen-forming fungi. Colored boxes indicate fungal species lifestyles. Asterisk indicates rapidly contracted gene families. (B) Expression patterns of diverse polyol transporters in G. flavorubescens.

Figure 5

Major contributors of TF gene family contraction in lichen-forming fungi. (A) Correlation between the number of TF genes and the total number of genes. Red and grey lines are regression and error lines, respectively. Colored dots indicate fungal species lifestyles. (B–D) The three major TF families known to affect the total TF size. Red boxes indicate the distribution of lichen-forming fungi. (B) Zn2 Cys6 Zn cluster; (C) zinc finger C2H2-type; and (D) homeodomain-like.

Figure 6

Symbiosis-induced genes in G. flavorubescens. Gene expression profiles of conserved and lichen-specific genes in G. flavorubescens. (A) Differentially up- (log₂ fold change > 1) and downregulated (log₂ fold change ≤  − 1) genes at each time stage. Gray bars indicate genes that were not significantly expressed. (B) Expression patterns of lichen-specific and conserved genes.

Figure 7

Lichen-specific small secreted proteins (SSPs) from G. flavorubescens induced in early lichen symbiosis. (A) Box plot of the number of secretomes and SSP distributions in lichen-forming fungi compared with other lifestyles. (B) Lichen-specific SSPs in C. macilenta and E. pusillum R61883. Ortholog SSPs of 56 fungal species were identified using blast and SSPs of C. macilenta and E. pusillum R61883 as references (E = 1 × 10⁻⁵). Abbreviations for fungal species are provided in Supplementary Dataset S1. (C) SSP expression in G. flavorubescens was classified as conserved (top) or lichen-specific (bottom).