The generation of thousands of fungal genomes is leading to a better understanding of genes and genomic organization within the kingdom. However, the epigenome, which includes DNA and chromatin modifications, remains poorly investigated in fungi. Large comparative studies in animals and plants have deepened our understanding of epigenomic variation, particularly of the modified base 5-methylcytosine (5mC), but taxonomic sampling of disparate groups is needed to develop unifying explanations for 5mC variation. Here, we utilize the largest phylogenetic resolution of 5mC methyltransferases (5mC MTases) and genome evolution to better understand levels and patterns of 5mC across fungi. We show that extant 5mC MTase genotypes are descendent from ancestral maintenance and de novo genotypes, whereas the 5mC MTases DIM-2 and RID are more recently derived, and that 5mC levels are correlated with 5mC MTase genotype and transposon content. Our survey also revealed that fungi lack canonical gene-body methylation, which distinguishes fungal epigenomes from certain insect and plant species. However, some fungal species possess independently derived clusters of contiguous 5mC encompassing many genes. In some cases, DNA repair pathways and the N6-methyladenine DNA modification negatively coevolved with 5mC pathways, which additionally contributed to interspecific epigenomic variation across fungi.
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Genome assemblies and gene annotations are available via the URL links listed in Supplementary Table 2. Gene Expression Omnibus and SRA accessions for RNA-Seq and WGBS data generated and used in this study are provided in the Methods.
Niederhuth, C. E. et al. Widespread natural variation of DNA methylation within angiosperms. Genome Biol. 17, 194 (2016).
Takuno, S., Ran, J.-H. & Gaut, B. S. Evolutionary patterns of genic DNA methylation vary across land plants. Nat. Plants 2, 15222 (2016).
Bewick, A. J., Vogel, K. J., Moore, A. J. & Schmitz, R. J. Evolution of DNA methylation across insects. Mol. Biol. Evol. 34, 654–665 (2017).
Glastad, K. G. et al. Variation in DNA methylation is not consistently reflected by sociality in Hymenoptera. Genome Biol. Evol. 9, 1687–1698 (2017).
Feng, S. et al. Conservation and divergence of methylation patterning in plants and animals. Proc. Natl Acad. Sci. USA 107, 8689–8694 (2010).
Zemach, A., McDaniel, I. E., Silva, P. & Zilberman, D. Genome-wide evolutionary analysis of eukaryotic DNA methylation. Science 328, 916–919 (2010).
Bewick, A. J. et al. The evolution of CHROMOMETHYLASES and gene body DNA methylation in plants. Genome. Biol. 18, 65 (2017).
Rošić, S., Amouroux, R. et al. Evolutionary analysis indicates that DNA alkylation damage is a byproduct of cytosine DNA methyltransferase activity. Nat. Genet. 50, 452–459 (2018).
Galagan, J. E. & Selker, E. U. RIP: the evolutionary cost of genome defense. Trends Genet. 20, 417–423 (2004).
Gladyshev, E. & Kleckner, N. DNA sequence homology induces cytosine-to-thymine mutation by a heterochromatin-related pathway in Neurospora. Nat. Genet. 49, 887–894 (2017).
Spatafora, J. W. et al. A phylum-level phylogenetic classification of zygomycete fungi based on genome-scale data. Mycologia 108, 1028–1046 (2016).
Stajich, J. E. Fungal genomes and insights into the evolution of the kingdom. Microbiol. Spectr. 5, FUNK-0055-2016 (2017).
Lewis, Z. A. et al. Relics of repeat-induced point mutation direct heterochromatin formation in Neurospora crassa. Genome Res. 19, 427–437 (2009).
Liu, S.-Y. et al. Bisulfite sequencing reveals that Aspergillus flavus holds a hollow in DNA methylation. PLoS ONE 7, e30349 (2012).
Huff, J. T. & Zilberman, D. Dnmt1-independent CG methylation contributes to nucleosome positioning in diverse eukaryotes. Cell 156, 1286–1297 (2014).
Jeon, J. et al. Genome-wide profiling of DNA methylation provides insights into epigenetic regulation of fungal development in a plant pathogenic fungus, Magnaporthe oryzae. Sci. Rep. 5, 8567 (2015).
Morselli, M. et al. In vivo targeting of de novo DNA methylation by histone modifications in yeast and mouse. eLife 4, e06205 (2015).
Wang, Y. L. et al. Genome-wide analysis of DNA methylation in the sexual stage of the insect pathogenic fungus Cordyceps militaris. Fungal Biol. 119, 1246–1254 (2015).
Honda, S. et al. Dual chromatin recognition by the histone deacetylase complex HCHC is required for proper DNA methylation in Neurospora crassa. Proc. Natl Acad. Sci. USA 113, E6135–E6144 (2016).
Li, W. et al. Differential DNA methylation may contribute to temporal and spatial regulation of gene expression and the development of mycelia and conidia in entomopathogenic fungus Metarhizium robertsii. Fungal Biol. 121, 293–303 (2017).
Selker, E. U. Premeiotic instability of repeated sequences in Neurospora crassa. Annu. Rev. Genet. 24, 579–613 (1990).
Singer, M. J., Marcotte, B. A. & Selker, E. U. DNA methylation associated with repeat-induced point mutation in Neurospora crassa. Mol. Cell. Biol. 15, 5586–5597 (1995).
Rhounim, L., Rossignol, J. L. & Faugeron, G. Epimutation of repeated genes in Ascobolus immersus. EMBO J. 11, 4451–4457 (1992).
Rossignol, J. L. & Faugeron, G. Gene inactivation triggered by recognition between DNA repeats. Experientia 50, 307–317 (1994).
Mondo, S. J. et al. Widespread adenine N6-methylation of active genes in fungi. Nat. Genet. 49, 964–968 (2017).
Cokus, S. J. et al. Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452, 215–219 (2008).
Urich, M. A., Nery, J. R., Lister, R., Schmitz, R. J. & Ecker, J. R. MethylC-Seq: base resolution whole genome bisulfite sequencing library preparation. Nat. Protoc. 10, 475–483 (2015).
Hofmeister, B. T. & Schmitz, R. J. Enhanced JBrowse plugins for epigenomics data visualization. BMC Bioinformatics 19, 159 (2018).
Catania, S. et al. Epigenetic maintenance of DNA methylation after evolutionary loss of the de novo methyltransferase. Preprint at https://www.biorxiv.org/content/early/2017/06/13/149385 (2017).
Goll, M. G. et al. Methylation of tRNAAsp by the DNA methyltransferase homolog Dnmt2. Science 311, 395–398 (2006).
Goll, M. G. & Bestor, T. H. Eukaryotic cytosine methyltransferases. Annu. Rev. Biochem. 74, 481–514 (2005).
Stroud, H. et al. Comprehensive analysis of silencing mutants reveals complex regulation of the Arabidopsis methylome. Cell 152, 352–364 (2013).
Bewick, A. J. & Schmitz, R. J. Gene body DNA methylation in plants. Curr. Opin. Plant Biol. 36, 103–110 (2017).
Takuno, S. & Gaut, B. S. Body-methylated genes in Arabidopsis thaliana are functionally important and evolve slowly. Mol. Biol. Evol. 1, 219–227 (2012).
Zhang, X. et al. Genome-wide high-resolution mapping and functional analysis of DNA methylation in Arabidopsis. Cell 126, 1189–1201 (2006).
Zilberman, D. et al. Genome-wide analysis of Arabidopsis thaliana DNA methylation uncovers an interdependence between methylation and transcription. Nat. Genet. 39, 61–69 (2007).
Bewick, A. J. et al. On the origin and evolutionary consequences of gene body DNA methylation. Proc. Natl Acad. Sci. USA 113, 9111–9116 (2016).
Bonasio, R. et al. Genome-wide and caste-specific DNA methylomes of the ants Camponotus floridanus and Harpegnathos saltator. Curr. Biol. 22, 1755–1764 (2012).
Glastad, K. M., Gokhale, K., Liebig, J. & Goodisman, M. A. D. The caste- and sex-specific DNA methylome of the termite Zootermopsis nevadensis. Sci. Rep. 6, 37110 (2016).
Law, J. A. & Jacobsen, S. E. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat. Rev. Genet. 11, 204–220 (2010).
Duncan, B. K. & Miller, J. H. Mutagenic deamination of cytosine residues in DNA. Nature 287, 560–561 (1980).
Sedgwick, B. Repairing DNA-methylation damage. Nat. Rev. Mol. Cell Biol. 5, 148–157 (2004).
Xiao, C. L. et al. N6-methyladenine DNA modification in the human genome. Mol. Cell 71, 306–318 (2018).
Carlile, M. J., Watkinson, S. C. & Gooday G. W. The Fungi 2nd edn (Academic Press, London, 2001).
Jones, P. et al. InterProScan 5: genome-scale protein function classification. Bioinformatics 30, 1236–1240 (2014).
Mirarab, S. et al. PASTA: ultra-large multiple sequence alignment for nucleotide and amino-acid sequences. J. Comput. Biol. 22, 377–386 (2015).
Bouckaert, R. et al. BEAST 2: a software platform for Bayesian evolutionary analysis. PLoS Comput. Biol. 10, e1003537 (2014).
Eddy, S. R. Accelerated profile HMM searches. PLoS Comput. Biol. 7, e1002195 (2011).
Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312–1313 (2014).
Paradis, E., Claude, J. & Strimmer, K. APE: analyses of phylogenetics and evolution in R language. Bioinformatics 20, 289–290 (2004).
Popescu, A.-A., Huber, K. T. & Paradis, E. ape 3.0: new tools for distance based phylogenetics and evolutionary analysis in R. Bioinformatics 28, 1536–1537 (2012).
Rouxel, T. et al. Effector diversification within compartments of the Leptosphaeria maculans genome affected by repeat-induced point mutations. Nat. Commun. 2, 202 (2011).
Revell, L. J. phytools: an R package for phylogenetic comparative biology (and other things). Methods Ecol. Evol. 3, 217–223 (2011).
O’Donnell, K., Cigelnik, E. & Benny, G. L. Phylogenetic relationships among the Harpellales and Kickxellales. Mycologia 90, 624–639 (1998).
O’Donnell, K., Lutzoni, F., Ward, T. J. & Benny, G. L. Evolutionary relationships among mucoralean fungi (Zygomycota): evidence for family polyphyly on a large scale. Mycologia 93, 286–296 (2000).
Jones, T. et al. The diploid genome sequence of Candida albicans. Proc. Natl Acad. Sci. USA 101, 7329–7334 (2003).
Van het Hoog, M. et al. Assembly of the Candida albicans genome into sixteen supercontigs aligned on the eight chromosomes. Genome Biol. 8, R52 (2007).
Espagne, E. et al. The genome sequence of the model ascomycete fungus Podospora anserina. Genome Biol. 9, R77 (2008).
Butler, G. et al. Evolution of pathogenicity and sexual reproduction in eight Candida genomes. Nature 459, 657–662 (2009).
Stajich, J. E. et al. Insights into evolution of multicellular fungi from the assembled chromosomes of the mushroom Coprinopsis cinerea (Coprinus cinereus). Proc. Natl Acad. Sci. USA 107, 11889–11894 (2010).
Amselem, J. et al. Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea. PLoS Genet. 7, e1002230 (2011).
Floudas, D. et al. The Paleozoic origin of enzymatic lignin decomposition reconstructed from 31 fungal genomes. Science 336, 1715–1719 (2012).
Olson, A. et al. Insight into trade-off between wood decay and parasitism from the genome of a fungal forest pathogen. New Phytol. 194, 1001–1013 (2012).
Staats, M. & van Kan, J. A. Genome update of Botrytis cinerea strains B05.10 and T4. Eukaryot. Cell 11, 1413–1414 (2012).
Chibucos, M. C., Crabtree, J., Nagaraj, S., Chaturvedi, S. & Chaturvedi, V. Draft genome sequences of human pathogenic fungus Geomyces pannorum sensu lato and bat white nose syndrome pathogen Geomyces (Pseudogymnoascus) destructans. Genome Announc. 1, e01045–13 (2013).
Muzzey, D., Schwartz, K., Weissman, J. S. & Sherlock, G. Assembly of a phased diploid Candida albicans genome facilitates allele-specific measurements and provides a simple model for repeat and indel structure. Genome Biol. 14, R97 (2013).
Toome, M. et al. Genome sequencing provides insight into the reproductive biology, nutritional mode and ploidy of the fern pathogen Mixia osmundae. New Phytol. 202, 554–564 (2013).
Walter, G. et al. DNA barcoding in Mucorales: an inventory of biodiversity. Persoonia 30, 11–47 (2013).
Wiemann, P. et al. Deciphering the cryptic genome: genome-wide analyses of the rice pathogen Fusarium fujikuroi reveal complex regulation of secondary metabolism and novel metabolites. PLoS Pathog. 9, e1003475 (2013).
Gostincar, C. et al. Genome sequencing of four Aureobasidium pullulans varieties: biotechnological potential, stress tolerance, and description of new species. BMC Genomics 15, 549 (2014).
Ohm, R. A. et al. Genomics of wood-degrading fungi. Fungal Genet. Biol. 72, 82–90 (2014).
Riley, R. et al. Extensive sampling of basidiomycete genomes demonstrates inadequacy of the white-rot/brown-rot paradigm for wood decay fungi. Proc. Natl Acad. Sci. USA 111, 9923–9928 (2014).
Tretter, E. D. et al. An eight-gene molecular phylogeny of the Kickxellomycotina, including the first phylogenetic placement of Asellariales. Mycologia 106, 912–935 (2014).
Chang, Y. et al. Phylogenomic analyses indicate that early fungi evolved digesting cell walls of algal ancestors of land plants. Genome Biol. Evol. 7, 1590–1601 (2015).
Chatterjee, S. et al. Draft genome of a commonly misdiagnosed multidrug resistant pathogen Candida auris. BMC Genomics 16, 686 (2015).
Perlin, M. H. et al. Sex and parasites: genomic and transcriptomic analysis of Microbotryum lychnidis-dioicae, the biotrophic and plant-castrating anther smut fungus. BMC Genomics 16, 461 (2015).
Drees, K. P. et al. Use of multiple sequencing technologies to produce a high-quality genome of the fungus Pseudogymnoascus destructans, the causative agent of bat white-nose syndrome. Genome Announc. 4, e00445–16 (2016).
Kijpornyongpan, T. et al. Broad genomic sampling reveals a smut pathogenic ancestry of the fungal clade Ustilaginomycotina. Mol. Biol. Evol. 35, 1840–1854 (2018).
Schultz, M. D. et al. Human body epigenome maps reveal noncanonical DNA methylation variation. Nature 523, 212–216 (2015).
Martin, M. & Marcel, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 17, 10–12 (2011).
Langmead, B., Trapnell, C. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).
Goffeau, A. et al. Life with 6000 genes. Science 274, 563–567 (1996).
Galagan, J. E. et al. The genome sequence of the filamentous fungus Neurospora crassa. Nature 422, 859–868 (2003).
Dean, R. A. et al. The genome sequence of the rice blast fungus Magnaporthe grisea. Nature 434, 980–986 (2005).
Martin, F. et al. The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis. Nature 452, 88–92 (2008).
Martinez, D. et al. Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion. Proc. Natl Acad. Sci. USA 106, 1954–1959 (2009).
Sharpton, T. J. et al. Comparative genomic analyses of the human fungal pathogens Coccidioides and their relatives. Genome Res. 19, 1722–1731 (2009).
Gao, Q. et al. Genome sequencing and comparative transcriptomics of the model entomopathogenic fungi Metarhizium anisopliae and M. acridum. PLoS Genet. 7, e1001264 (2011).
Zheng, P. et al. Genome sequence of the insect pathogenic fungus Cordyceps militaris, a valued traditional Chinese medicine. Genome Biol. 12, R116 (2011).
Arnaud, M. B. et al. The Aspergillus genome database (AspGD): recent developments in comprehensive multispecies curation, comparative genomics and community resources. Nucleic Acids Res. 40, D653–D659 (2012).
Hu, X. et al. Trajectory and genomic determinants of fungal-pathogen speciation and host adaptation. Proc. Natl Acad. Sci. USA 111, 16796–16801 (2014).
Janbon, G. et al. Analysis of the genome and transcriptome of Cryptococcus neoformans var. grubii reveals complex RNA expression and microevolution leading to virulence attenuation. PLoS Genet. 10, e1004261 (2014).
Corrochano, L. M. et al. Expansion of signal transduction pathways in fungi by extensive genome duplication. Curr. Biol. 26, 1577–1584 (2016).
Schultz, M. D., Schmitz, R. J. & Ecker, J. R. ‘Leveling’ the playing field for analyses of single-base resolution DNA methylomes. Trends Genet. 28, 583–585 (2012).
Alexa, A. & Rahnenfuhrer, J. topGO: enrichment analysis for Gene Ontology. R package version 2.32.0 (2016).
Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).
Pertea, M., Kim, D., Pertea, G. M., Leek, J. T. & Salzberg, S. L. Transcript-level expression analysis of RNA-Seq experiments with HISAT, StringTie and Ballgown. Nat. Protoc. 11, 1650–1667 (2016).
Grafen, A. The phylogenetic regression. Phil. Trans. R. Soc. Lond. B 326, 119–157 (1989).
Martins, E. P. & Hansen, T. F. Phylogenies and the comparative method: a general approach to incorporating phylogenetic information into the analysis of interspecific data. Am. Nat. 149, 646–667 (1997).
Pagel, M. Detecting correlated evolution on phylogenies: a general method for the comparative analysis of discrete characters. Proc. R. Soc. Lond. B 255, 37–45 (1994).
Flutre, T., Duprat, E., Feuillet, C. & Quesneville, H. Considering transposable element diversification in de novo annotation approaches. PLoS ONE 6, e16526 (2011).
Bao, W., Kojima, K. K. & Kohany, O. RepBase Update, a database of repetitive elements in eukaryotic genomes. Mob. DNA 6, 11 (2015).
Margolin, B. S. et al. A methylated Neurospora 5S rRNA pseudogene contains a transposable element inactivated by repeat-induced point mutation. Genet. 149, 1787–1797 (1998).
Selker, E. U. et al. The methylated component of the Neurospora crassa genome. Nature 422, 893–897 (2003).
Liaw, A. & Wiener, M. Classification and regression by randomForest. R News 2, 18–22 (2002).
We thank E. Demers for DNA from Candida albicans, Clavispora lusitaniae and Candida auris, T. Giraud for DNA from M. lychnidis-dioicae A1, A. Idnurm for DNA from S. roseus, and N. Ponts for DNA from A. bisporus, B. cinerea, Fusarium fujikuroi, L. maculans ‘brassicae’ and P. anserina. We also thank M. Perlin for DNA from M. lychnidis-dioicae. We thank N. Rohr and T. Ethridge for WGBS library preparation for all species sequenced in this study except C. cinerea, H. irregulare and W. cocos. We thank D. Carter-House and J. Ortanez for DNA preparation of Zygomycetes Coemansia spiralis, Hesseltinella vesiculosa, Kirkomyces cordense, Lobosporangium transversale, Parasitella parasitica, P. blakesleeanus, R. spectabilis, S. fusiger and Syncephalis fuscata. We thank N. Morffy and Z. Lewis for useful feedback during manuscript preparation. We thank the following collaborators for the use of unpublished genic data: C. Aime, A. Andrianopoulos, D. Armaleo, G. Bills, G. Bonito, S. Branco, T. Bruns, K. Bushley, Y. Chang, I.-G. Choi, A. Churchill, L. Corrochano, C. Cuomo, A. Desirò, P. Dyer, J. Franciso, R. Gazis, J. Gladden, S. Goodwin, A. Gryganskyi, D. Hibbett, D. Johnson, A. Kohler, B. Lindahl, F. Lutzoni, J. Magnuson, J. Maria Barrasa, F. Martin, M. Milgroom, L. Nagy, W. Nierman, M. Nowrousian, D. Nuss, K. O’Donnell, R. Ohm, C. Pires, B. Schwessinger, S. Singer, B. Slippers, J. Spatafora, J. Taylor, A. Tsang, S. Unruh, K. Wolfe and L. Zettler. We also thank the Georgia Advanced Computing Resource Center and Georgia Genomics and Bioinformatics Core at the University of Georgia for sequencing and computational resources, respectively. This work was supported by the Office of the Vice President for Research at the University of Georgia (to R.J.S.) and US National Science Foundation grant DEB 1441715 (to J.E.S.). R.J.S. is a Pew Scholar in the Biomedical Sciences, supported by The Pew Charitable Trusts. Computational analysis on the University of California, Riverside High-Performance Computing Center cluster were supported by grants from the National Science Foundation (DBI-1429826) and National Institutes of Health (S10-OD016290). The work conducted by the US Department of Energy Joint Genome Institute—a DOE Office of Science User Facility—is supported by the Office of Science of the US Department of Energy under contract number DE-AC02-05CH11231.
The authors declare no competing interests.
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Supplementary Figs. 1–16
5mC DNA MTase and the tRNA methyltransferase DNMT2 annotations for 528 fungal species investigated in this study.
WGBS and mapping statistics for 40 fungal species investigated in this study.
Number of species per phylum for each observed 5mC MTase genotype.
5mC DNA MTase and the tRNA methyltransferase DNMT2 annotations for a subset of Animalia, Chlorophyta, Fungi, and Prokaryota. Protein models correspond to those used in Supplementary Fig. 2.
Number of CG-, CH-, and CN-enriched genes across fungal species investigated.
ALKBH annotations for Chordata, Fungi, and Nematoda investigated in this study. Protein models correspond to those used in Supplementary Fig. 15.
Results from Pagel’s test for correlated evolution.
Results from phylogenetic generalized least squares (PGLS).
Annotated proteins from fungal species containing the N-6 DNA Methylase domain (PF02384) as identified by Interproscan v5.23-62.0.
METTL annotations for fungal species investigated in this study. Protein models correspond to those used in Supplementary Fig. 16.
Annotated proteins for fungal species containing the domain the methyltransferase small domain (N6AMT1 proteins) as identified by Interproscan v5.23-62.0.
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Bewick, A.J., Hofmeister, B.T., Powers, R.A. et al. Diversity of cytosine methylation across the fungal tree of life. Nat Ecol Evol 3, 479–490 (2019). https://doi.org/10.1038/s41559-019-0810-9
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