Large-Scale Genome Sequencing of Mycorrhizal Fungi Provides Insights Into the Early Evolution of Symbiotic Traits

Lawrence Berkeley National Laboratory Recent Work

Title Large-scale genome sequencing of mycorrhizal fungi provides insights into the early evolution of symbiotic traits.

Permalink https://escholarship.org/uc/item/1pc0z4nx

Journal Nature communications, 11(1)

ISSN 2041-1723

Authors Miyauchi, Shingo Kiss, Enikő Kuo, Alan et al.

Publication Date 2020-10-12

DOI 10.1038/s41467-020-18795-w

Peer reviewed

eScholarship.org Powered by the California Digital Library University of California ARTICLE

https://doi.org/10.1038/s41467-020-18795-w OPEN Large-scale genome sequencing of mycorrhizal fungi provides insights into the early evolution of symbiotic traits

Shingo Miyauchi et al.#

Mycorrhizal fungi are mutualists that play crucial roles in nutrient acquisition in terrestrial ecosystems. Mycorrhizal symbioses arose repeatedly across multiple lineages of Mucor- 1234567890():,; omycotina, Ascomycota, and Basidiomycota. Considerable variation exists in the capacity of mycorrhizal fungi to acquire carbon from soil organic matter. Here, we present a combined analysis of 135 fungal genomes from 73 saprotrophic, endophytic and pathogenic species, and 62 mycorrhizal species, including 29 new mycorrhizal genomes. This study samples ecologically dominant fungal guilds for which there were previously no symbiotic genomes available, including ectomycorrhizal Russulales, Thelephorales and Cantharellales. Our analyses show that transitions from saprotrophy to symbiosis involve (1) widespread losses of degrading enzymes acting on lignin and cellulose, (2) co-option of genes present in saprotrophic ancestors to fulfill new symbiotic functions, (3) diversification of novel, lineage- specific symbiosis-induced genes, (4) proliferation of transposable elements and (5) divergent genetic innovations underlying the convergent origins of the ectomycorrhizal guild.

#A list of authors and their afﬁliations appears at the end of the paper.

NATURE COMMUNICATIONS | (2020) 11:5125 | https://doi.org/10.1038/s41467-020-18795-w | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18795-w

ycorrhizal fungi are central to the evolution, biology, additional symbiosis-related functional traits, such as nitrogen Mand physiology of land plants because they promote and phosphate acquisition. Finally, we investigate the age dis- plant growth by facilitating the acquisition of scarce tribution of symbiosis-upregulated genes across a phylogeneti- and essential nutrients, such as phosphorus and nitrogen1–3. cally representative set of ectomycorrhizal fungi, using a They are also major drivers of carbon sequestration and they phylostratigraphic approach to resolve lineage-specific and con- have a well-documented impact on the composition of microbial served elements of symbiotic transcriptomes. We show that and plant communities1,2. The most ubiquitous classes of transitions from saprotrophy to ectomycorrhizal symbiosis mycorrhizal symbioses are ectomycorrhiza, arbuscular mycor- involve widespread losses of degrading enzymes acting on lig- rhiza, orchid mycorrhiza, and ericoid mycorrhiza4,5. Each class is nocellulose, co-option of genes present in saprotrophic ancestors classified based on host plant and characteristic symbiotic to fulfill novel symbiotic functions, diversification of lineage- structures. Although mycorrhizal fungi are highly diverse in specific symbiosis-induced genes, proliferation of transposable terms of their evolutionary history, the independent evolution of elements (TEs), and divergent genetic innovations underlying the similar symbiotic morphological structures, and physiological convergent origins of the ectomycorrhizal guild. traits in divergent fungal taxa provides a striking example of convergent evolution3–5. Although unique and common traits in mycorrhizal symbioses have recently been reviewed2, molecular Results mechanisms underlying these convergent phenotypes remain Main features of mycorrhizal genomes. We compared 62 draft largely undetermined1,3–8. genomes from mycorrhizal fungi, including 29 newly released gen- Prior comparisons of genomes from ectomycorrhizal, orchid omes, and predicted 9344–31,291 protein-coding genes per species and ericoid mycorrhizal fungi, wood decayers and soil decom- (see “Methods”, Supplementary Information and Supplementary posers have elucidated the mechanisms of several transitions from Data1).Thissetincludesnewgenomesfromtheearlydiverging saprotrophy to mutualism in Dikarya9–16. These analyses have fungal clades in the Russulales, Thelephorales, Phallomycetidae, and shown that multiple lineages of ectomycorrhizal fungi have lost Cantharellales (Basidiomycota), and Helotiales and Pezizales (Asco- most genes encoding lignocellulose‐degrading enzymes present in mycota). We combined these mycorrhizal fungal genomes with 73 their saprotrophic ancestors, explaining the reduced capacity of fungal genomes from wood decayers, soil/litter saprotrophs, and root ectomycorrhizal fungi to acquire C complexed in soil organic endophytes (Fig. 1 and Supplementary Data 2). There was little matter (SOM) and plant cell walls17 and, as a consequence, their variation in the completeness of the gene repertoires, based on increasing dependence on the host plant sugars. The diversity of Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis trophic states in extant ectomycorrhizal fungi may be a con- (coefficient of variation, c.v. = 7.98), despite variation in assembly sequence of their multiple origins from saprotrophic ancestors contiguity (Fig. 1). Genome size varied greatly within each phylum, with varied decay capabilities, including white and brown rot with genomes of mycorrhizal fungi being larger than those of wood decayers, and soil and litter decomposers3–5. However, the saprotrophic species (Figs. 1 and 2, and Supplementary Data 2; extent to which ectomycorrhizal fungi make use of their secreted P < 0.05, generalized least squares with the Brownian motion model plant cell wall degrading enzymes (PCWDEs) and microbial cell (GLS)). Glomeromycotina had exceptionally large genomes (Figs. 1 wall degrading enzymes (MCWDE) to decay or decompose SOM and 2, and Supplementary Data 2), with Gigaspora rosea having the is not well understood17–24. largest genome (567 Mb) among the 135 fungi compared27. Despite their ecological prominence, much remains to be learned about the evolution and functional diversification of mycorrhizal symbionts and the crucial acquisitions that allow TEs in mycorrhizal genomes. The main driver of genome colonization of and nutrient exchange with plants3,25. Here, we inflation appeared to be repeat content (Fig. 3 and Supplementary present a combined analysis of 135 fungal genomes from Data 3; P < 0.05 GLS), such as long terminal repeat retro- 73 saprotrophic, endophytic and pathogenic fungal species, and transposons (LTRs), which ranged from 0.01 to 46.4% of the 62 mycorrhizal fungal species, including 29 new mycorrhizal assembly (Fig. 3 and Supplementary Data 3). The distribution of genomes. This study approximately doubles the number of TE categories varies between ectomycorrhizal fungal taxa even published genomes of mycorrhizal fungi, and it samples major within the same fungal orders or genera (Fig. 3), indicating that groups, for which there were previously no symbiotic genomes invasions by different TE families took place independently in available, including Russulales, Thelephorales, Phallomycetidae, different clades. However, the total TE coverage in genomes of and Cantharellales. These groups are important, because they (1) ectomycorrhizal Ascomycota and Glomeromycota was sig- are often ecologically dominant (Russulales and Thelephorales), nificantly higher than in Basidiomycota (P < 0.05, GLS; Supple- (2) represent early diverging clades for which no ectomycorrhizal mentary Data 3). In Ascomycota, the most abundant TE families genomes were previously available (Phallomycetidae and Can- are LTRs, such as Gypsy and Copia, and non-LTR I, whereas in tharellales), and (3) include groups that arose before (Canthar- Basidiomycota, Gypsy and Copia LTRs, Tad1, helitrons, and ellales) or after the origin of ligninolytic peroxidases (class II Zizuptons are abundant (Fig. 3). Lifestyle has a higher impact POD) implicated in white rot26. than phylogeny on TE coverage with a significantly higher TE This dataset presents an opportunity to carry out a broader content in ectomycorrhizal symbionts compared to other life- analysis of the evolution of saprotrophic capabilities than that we styles for several TE categories (14–35% of total variances, P value previously attempted in our large-scale comparative analysis of <0.05; PERMANOVA) (Supplementary Fig. 1a, b, and Supple- Agaricomycetidae12. In the present study, we hypothesize that the mentary Data 3 and 4). In Agaricales, a large proportion of LTR evolutionary mechanisms in action in Agaricomycetidae can be insertions occurred during the past 5 million years (Mya) in traced back to the early diverging clades of ectomycorrhizal fungi. Amanita rubescens, Laccaria bicolor, Laccaria amethystina, and To assess gains of ectomycorrhizal lifestyle traits, we discuss the Cortinarius glaucopus, while the major bursts are more ancient in fundamental adaptations that underlie convergent evolution of Tricholoma matsutake (Supplementary Fig. 1c). These LTRs have ectomycorrhizal fungi, including the loss of some metabolic been proliferating over the past 5–6 Mya for Tuber melanos- functions, such as PCWDEs, and the acquisition of small secreted porum, Tuber aestivum, and Choiromyces venosus, while LTR effector-like proteins that may facilitate the accommodation of accumulation in Tuber magnatum occurred between 6 and 14 symbiotic fungi within their host plants. We also compare Mya (ref. 16).

2 NATURE COMMUNICATIONS | (2020) 11:5125 | https://doi.org/10.1038/s41467-020-18795-w | www.nature.com/naturecommunications AUECOMMUNICATIONS NATURE 1. Fig. of data number Source transposable (K): survey. of Scaffolds BUSCO (%) proteins, on secreted coverage based predicted (%): % content of in number TE completeness (K): Mb, genome Secreted in (%): genes, size BUSCO predicted genome length, (Mb): of N50 Genome number (Mb): values. (K): L50 median Genes scaffolds, show assemblies, lines in dotted analyzed. (TE) genomes elements The fungal panel. 135 bottom the see of coded, features General 1 Fig. https://doi.org/10.1038/s41467-020-18795-w | COMMUNICATIONS NATURE

Mucoromycota Ascomycota Basidiomycota Glomerales PezizomycetesDothideomycetes Leotiomycetes CantharellalesRussulales Boletales Agaricales Sistotremastrum niveocremeum Rhizophagus irregularis Phanerochaete chrysosporium Wickerhamomyces anomalus Acephala macrosclerotiorum Monacrosporium haptotylum Melanogaster broomeianus Rhizophagus irregularis Paxillus ammoniavirescens Rhizophagus irregularis Rhizophagus irregularis Rhizophagus irregularis Rhizophagus irregularis Saccharomyces cerevisiae Hysterangium stoloniferum Botryobasidium botryosum Punctularia strigosozonata Rhizophagus cerebriforme Phialocephala scopiformis Hebeloma cylindrosporum Dacryopinax primogenitus Moniliophthora perniciosa Heterobasidion annosum Schizophyllum commune Gautieria morchelliformis Hypholoma sublateritium Aureobasidium pullulans Gymnopus androsaceus Rhizopogon vesiculosus Arthroderma benhamiae Rhizophagus diaphanus Cenococcum geophilum Rhizophagus irregularis Sclerotinia sclerotiorum Arthrobotrys oligospora Lepidopterella palustris Meliniomyces variabilis Sphaerobolus stellatus Candida tanzawaensis Amorphotheca resinae Morchella importuna. Sarcoscypha coccinea Pisolithus microcarpus Cantharellus anzutake Choiromyces venosus Tricholoma matsutake Ascocoryne sarcoides Cortinarius glaucopus Tremella mesenterica Tuber melanosporum Coprinellus micaceus Thelephora ganbajun Laccaria amethystina Scleroderma citrinum Auricularia subglabra Cladosporium fulvum Piriformospora indica Neolentinus lepideus Rhizopogon vinicolor Meliniomyces bicolor Rhizoscyphus ericae Thelephora terrestris Ascobolus immersus Fibulorhizoctonia Tulasnella calospora Omphalotus olearius Pyronema confluens Trichophaea hybrida Coniophora puteana Morchella importuna Taphrina deformans Oidiodendron maius Gyromitra esculenta Magnaporthe grisea Aspergillus clavatus Coprinopsis cinerea Russula ochroleuca Caloscypha fulgens Lentinellus vulpinus Pleurotus ostreatus Sebacina vermifera Ceratobasidium Trichoderma reesei Plicaturopsis crispa Piloderma croceum Amanita rubescens Pisolithus tinctorius Kalaharituber pfeilii Alternaria alternata Neurospora crassa Hydnum rufescens Xerocomus badius Phlebia brevispora Serpula lacrymans Rhizoctonia solani Glonium stellatum Amanita muscaria Wilcoxina mikolae Glarea lozoyensis Stagonospora Xylaria hypoxylon Paxillus adelphus Aspergillus flavus Crucibulum laeve Paxillus involutus Tuber magnatum Lactarius quietus Marasmius fiardii Chalara longipes Jaapia argillacea Terfezia boudieri Gigaspora rosea Russula emetica Pluteus cervinus Mycena galopus Terfezia claveryi Armillaria gallica Boletus edulis Ramaria rubella Suillus brevipes Laccaria bicolor Tuber aestivum Botrytis cinerea Gyrodon lividus Postia placenta Amanita thiersii Xylona heveae Tirmania nivea Sistotrema Boletus edulis Wallemia sebi Bisporella Usnea florida Suillus luteus Tuber borchii Ectomycorrhiza 22)1:15 tp:/o.r/013/44700175w|www.nature.com/naturecommunications | https://doi.org/10.1038/s41467-020-18795-w | 11:5125 (2020) | sp. sp. sp. P sp. sp. DAOM C C2 B3 A5 A4 A1 Saprotroph 0 400 200 0 Genome (Mb) Ericoid mycorrhiza Pathogen 020406080 TE content(%) eoeadasml etrs h ifrn ietls(.. coyoria r color are ectomycorrhiza) (e.g., lifestyles different The features. assembly and Genome 10203040500 Parasite Genes (K) Orchid mycorrhiza 0123 Secreted (K) Endophyte 0102030 Scaffolds (K) Arbuscular mycorrhiza Wood decayer 01234 L50 (Mb) Yeast 58 59 95100 90 85 80 75 BUSCO (%) ARTICLE 3 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18795-w

Genomes (Mbp) Repeat element coverage (%)

Ectomycorrhiza

Saprotroph

Pathogen

Ericoid mycorrhiza

Orchid mycorrhiza

Arbuscular mycorrhiza

Endophyte

Wood decayer

0 50 100 150 0 20 40 60 80

Fig. 2 The distribution of genome size (in Mb) and repeat element coverage (%) for each lifestyles. The boxes represent median, upper, and lower quartiles with the whiskers showing minimal and maximal values, and outliers in circle. The small dots show single observations. The number of species per lifestyle is as follows: ectomycorrhizal fungi (n = 45), arbuscular mycorrhizal fungi (n = 10), ericoid mycorrhizal fungi (n = 4), orchid mycorrhizal fungi (n = 3), endophytes (n = 5), pathogens (n = 14), soil/litter saprotrophs (n = 32), and wood decayers (n = 17). Source data Fig. 1.

Phylogeny, orthologous, and paralogous genes. Reconstructed Pfam pattern with soil saprotrophic ascomycete species, such as phylogenetic relationships and estimated divergence dates are Chalara longipes and the dark septate endophyte (DSE) Phialo- generally consistent with the dates recovered by previous studies. cephala scopiformis. Ectomycorrhizal fungi in Basidiomycota are For example, we estimated the age of the most recent common grouped with soil saprotrophs and wood decayers, and displayed ancestor (MRCA) of the Agaricomycetidae to be 132 Mya no specific pattern in their primary and secondary metabolism (Fig. 4a), while it was estimated to be 149 or 125 Mya in phylo- gene repertoires that may explain their symbiosis-related ability. genomic analyses by Floudas et al.26 and Kohler et al.12, but Similarly, hierarchical clustering of the presence and abundance 191–176 Mya in a multigene megaphylogeny by Varga et al.28. of the different membrane transporters and transcriptional reg- We estimated the ages of the MRCAs of Agaricomycetes, Agar- ulators revealed no specific pattern(s) for ectomycorrhizal fungi icales, Polyporales, Russulales, and Boletales to be 280, 116, 104, (Supplementary Fig. 2b, c). 88, and 82 Mya (Fig. 4a), respectively. We estimated the MRCA of 26 Pezizomycotina at 326 Mya (Fig. 4b), while Floudas et al. Predicted secretomes of saprotrophs and symbiotrophs.As obtained a mean age of 344 Mya. secreted proteins play a key role in SOM decomposition and We inferred gene families from the predicted proteomes using symbiosis development, we compared predicted secretomes, gene family clustering. A total of 68,923 and 129,258 gene families including carbohydrate-active enzymes (CAZymes), lipases, pro- fi were identi ed in Ascomycota and Basidiomycota, respectively, teases, and other secreted proteins, such as effector-like small and were used to infer orthology and paralogy (Supplementary secreted proteins (SSPs; Supplementary Data 6). The number of Data 5). The species in our datasets contained substantial novelty genes encoding secreted proteins represents 4.6–5.7% of the total fi in gene content. Species-speci c genes ranged from 994 in T. protein repertoire for Basidiomycota and Ascomycota, respectively melanosporum to 39,410 in Mycena galopus, for a total of 251,392 (Supplementary Fig. 3). The proportions of secreted proteins fi and 606,378 taxon-speci c genes in Ascomycota and Basidiomy- within the different protein categories were consistent in both fi cota, respectively.The large number of species-speci c genes in M. phyla (Supplementary Fig. 3, and Supplementary Data 6 and 7). galopus is the result of a series of striking expansion of gene About half of the secreted proteins were SSPs (Supplementary families. Fig. 3a and Supplementary Data 7c). Only secreted CAZymes and SSPs showed significant differences in relative abundance among Functional gene categories encoded by mycorrhizal genomes. lifestyles (P < 0.01; generalized Campbell and Skillings procedure; Hierarchical clustering of the presence and abundance of different Supplementary Fig. 3 and Supplementary Data 8), with orchid and Pfam protein domains identified genome-wide patterns of func- ericoid mycorrhizal symbionts possessing the largest CAZyme tional domain content among some of these fungi (Supplemen- repertoires. On the other hand, the average number of secreted tary Fig. 2a). Arbuscular mycorrhizal fungi clustered together proteases and lipases is similar in saprotrophs and symbiotrophs. with Pfam categories showing a substantial differential abundance No expansion of gene families coding for secreted proteases, in genes encoding proteins with NUDIX, tetratricopeptide repeat, secreted phosphatases, and phytases were found in ectomycor- BTB/POZ, Sel1 repeat, ubiquitin, and high-mobility group box rhizal fungi (Supplementary Fig. 3c). domains, corroborating our previous study27. This comparison also emphasizes some of the unique aspects of the genomes from Losses of PCWDEs. In Basidiomycota, ectomycorrhizal species the ectomycorrhizal ascomycete Acephala macrosclerotiorum contain significantly fewer secreted CAZymes acting on cellu- (Helotiaceae) and ericoid mycorrhizal fungi (e.g., Oidiodendron lose, hemicellulose, pectins, lignin, suberins, and tannins (P < maius, Meliniomyces species), such as the expansion of genes 0.01, the generalized Campbell and Skillings procedure; Fig. 5, encoding proteins with FAD and AMP-binding domains, alde- Supplementary Fig. 3c and Supplementary Data 6) than all other hyde dehydrogenases and sugar transporters. They share their ecological guilds, i.e., lifestyles. They are also reduced in secreted

4 NATURE COMMUNICATIONS | (2020) 11:5125 | https://doi.org/10.1038/s41467-020-18795-w | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18795-w ARTICLE

LTR Non-LTREnSpm/CACTA DNA Ma Ginger2/TDD Penelope Non−LTRSINE3/5SHarbinger riner/Tc1 CryptonFpiggyBac Unkno Helitron ZisuptonK Academ P DIRSGypsyCopia R Dada olobokIS3EU MuDR olintonMe L T TEX CRE hAT DNA Total TR ad1 L1 rlin wn Coverage (%) I P 0204060

Laccaria amethystina 0.2 8.7 1.5 0.5 0 0.2 0.2 0.4 0.3 23.7 Laccaria bicolor 0.6 10.11.60.4 0 0.3 0.60.4 0.2 1.2 0.6 0.1 24.8 Coprinopsis cinerea 0 3.4 0.6 0 1.3 Coprinellus micaceus 0.12.422.10.100.1 0 0.20.2 0.1 0.2 7.4 Hypholoma sublateritium 0.2 0.50.2 0 0.3 4.2 Hebeloma cylindrosporum 0.4 0.1 2.5 Cortinarius glaucopus 0.5 7 1.81 0.1 0.60.4 0.1 0.2 0.1 0.1 28.5 Crucibulum laeve 3.10.6 0.1 0.2 0.3 9.3 Tricholoma matsutake 0.1 34.45.7 3.300.30 0 00.3 0.2 0.90 0.1 0.60 0 33.8 Amanita thiersii 16.95.3 2.5 9.1 Amanita rubescens 0.15.6 4.3 0.6 0 0.20.9 1.1 0.50.1 0.1 0.1 33.5 Amanita muscaria 0 2.6 0.8 0.4 0 0 12.3 Pluteus cervinus 0.2 0 4.3 Gymnopus androsaceus 0.4 3.3 3.5 0.10.1 0 00 0.3 0.1 0.2 23.8 Omphalotus olearius 0.2 0.8 Marasmius fiardii 0.19.5 1.5 3.5 11.8 Moniliophthora perniciosa 1.6 1.4 Mycena galopus 0.2 7.3 1.100.40 0.4 0 0.40.60.2 0.1 0.3 0.3 0 0.2 0.3 0 0 033.6 Armillaria gallica 0.1 7.50.2 00 0 0.3 0 17.4 Pleurotus ostreatus 3.50.5 0 0.1 0.2 0.3 0.2 3.9 Schizophyllum commune 1.4 1 00.22.9 Paxillus ammoniavirescens 003.8 2.2 0.1 0.1 0.2 0.3 0 11.1 Paxillus adelphus 1.77.2 9.7 0 0 1.10.220 2.34.7 0.4 0 0 0 33.6 Paxillus involutus 0.23.6 1.7 0 0.2 0.2 0.20.2 0.6 0.1 19.7 Gyrodon lividus 00.123.44.3 0.1 0.10.1 0.9 0 1.2 19.5 Xerocomus badius 2.3 2.50 0.1 11.2 Boletus edulis 0.2 9.4 4.7 0.10.1 0.20.2 0.7 0.1 0.3 0.8 0.2 25.7 Boletus edulis P 0.1 8.15.6 0.10.20 0 0.2 0.60.1 0.2 1.60.1 25.7 Pisolithus microcarpus 8.1 3.81.2 1.2 0.6 0.71 0.4 0.1 23.5 Pisolithus tinctorius 0.420.4 2.1 0.80 0.91.1 0.9 0.6 0.4 0.1 24.9 Scleroderma citrinum 0.1 12.3 4.50.1 0.4 0.3 0.2 0 0.2 0.5 0.1 23.9 Suillus luteus 0 2.7 1.40.20.1 0.4 0.1 0.1 2.1 0.1 0.6 17.2 Suillus brevipes 01.9 1.20.20.1 0 0.1 0.1 0 0.1 0.1 0.2 0 17.2 Coniophora puteana 0.20.8 0 3 Serpula lacrymans 12.9 3.9 0.20.4 0.4 0.4 00 09.6 Rhizopogon vesiculosus 3 0.3 0.200 0.10.2 0 0 21 Rhizopogon vinicolor 1.20.2 0.1 0.1 0.1 014.8 Melanogaster broomeianus 0.2 6 1.9 00.60.1 0.20.1 0 27.5 Fibulorhizoctonia sp. 02.40.7 0.8000 0 0 00 27.6 Piloderma croceum 00.72.4 0.20 0 0 0 0.1 0.2 0 13.2 Plicaturopsis crispa 0.1 0.5 00 4.3 Phanerochaete chrysosporium 0.13.90.8 0 0 0.1 0.1 0.1 2 Basidiomycota Phlebia brevispora 2.2 1.6 0003.60.1 0.1 0.1 Postia placenta 12.10.3 0 0 4.8 Neolentinus lepideus 003.1 2.5 0.10 0.1 6.6 Jaapia argillacea 3.6 0.1 0.1 0 0.1 0.1 6.1 Punctularia strigosozonata 0.1 1.7 0.4 3 Sistotremastrum niveocremeum 0.10.1 0 3.4 Heterobasidion annosum 8.70.7 0.1 0.40.2 0 9.9 Lactarius quietus 0.235 4.2 1.501.900 0.10.1 4.4 0.1 1.7 1.30.1 0.2 0.1 43.8 Lentinellus vulpinus 3 0.8 0 0 3.5 Russula emetica 0.29.21.2 10.300.2 6.8 0.6 0.3 0.1 25.4 Russula ochroleuca 201.30 0.1 0 00.1 0 21.9 Russulales Thelephora terrestris 06.8 1.6 0.10 0 0 0.1 0.5 23.8 Thelephora ganbajun 0.30.4 0.2 0 0 27 Ramaria rubella 0.2 27.52.90.1 0.900.2 1.3 0.6 0.1 0.3 0 0.4 36.6 Gautieria morchelliformis 0.3 28.7 11.4 0.1 4.6 0 0.20.5 0.6 4.2 1.60.1 0.9 0 0.1 0 42.6 Hysterangium stoloniferum 0.3 34.18.9 0.6 0.6 0.1 1.35.4 0 0 0.1 0 0.6 46.7 Sphaerobolus stellatus 0.1 6.80.20.300.40.4 0.2 0 0.3 0 0 0.1 19.8 Auricularia subglabra 1.3 0.1 0.1 0 0 5.5 Piriformospora indica 0.3 0.2 4.5 Sebacina vermifera 0.3 10 Hydnum rufescens 3.815.82.1 0.4 30.1 0.40.2 1 3.2 0.2 44 Cantharellus anzutake 0028.5 2.20.8 0.7 0.10.2 0.4 0.70.4 0.1 0 0 0 44.1 Sistotrema sp. 0.1 0.1 0 3.6 Botryobasidium botryosum 001.4 0.4 3.7 0.10.1 0 0 7.9 Tulasnella calospora 1.8 0.9 00.1 0 09.4 Ceratobasidium sp. 3.5 0.50.400 0.1 0 0.1 13.9 Rhizoctonia solani 8.1 0.2 0 0 0.1 0 0 0.1 11.2

CantharellalesTremella mesenterica 12.8 2.20.8 Boletales Agaricales 0.1 0.2 0.1 00.40.1 6.8 Dacryopinax primogenitus 20.10.4 0.1 0 2.9 Wallemia sebi 0.4 0.4 Chalara longipes 0.4 0.3 0 1 Meliniomyces variabilis 1.90.8 0.1 02 Meliniomyces bicolor 10.25.3 0.9 0.8 0.2 1 1 0.1 9.8 Rhizoscyphus ericae 2.51.4 1.2 0.80.2 0.6 0.1 1 0.1 0.30.2 7.5 Acephala macrosclerotiorum 00.52.4 0.2 0.2 3.7 Phialocephala scopiformis 0.5 0.3 0.3 1 Ascocoryne sarcoides 0.2 0.3 0.6 Glarea lozoyensis 2.20.2 0.2 0.9 Amorphotheca resinae 3.70.9 5.2 0.1 04.7 Oidiodendron maius 1.3 1.4 0.9 0 00.50.3 0 0 2.3 Bisporella sp. 1.8 0.80.9 0.3 0.3 1 000.4 5 Leotiomycetes Sclerotinia sclerotiorum 1.40.6 1.3 0.1 1.2 0.5 3.5 Botrytis cinerea 0.5 0.1 0 0.1 0.7 Magnaporthe grisea 5.2 1.6 1.9 1.7 0.2 0.4 0.6 Neurospora crassa 2.4 0.2 1.2 0.1 0.1 4.8 Trichoderma reesei 0 00.3 Xylaria hypoxylon 1.5 Cenococcum geophilum 35.812.84.7 7 0.7 0.1 1.4 1.4 1.4 0.5 0.1 0.1 1.3 1 35.3 Glonium stellatum 1.4 Lepidopterella palustris 4.9 0.9 0200.10 7.6 Stagonospora sp. 0.1 0 0.2 Alternaria alternata 0 0.2 0.2 Aureobasidium pullulans 0.2 0.4 0.2 0 0.8 Cladosporium fulvum 19.9 8.3 15.1 0.3 0 1.7 0.2 7.9 Aspergillus clavatus 3.7 0.50.4 0.1 2.1 Aspergillus flavus 0 0 0.1 Dothideomycetes Arthroderma benhamiae 0 Usnea florida 1.6 0.30.4 0.4 0.4 0 9.6 0.5 0.3 0.2 Ascomycota Xylona heveae Tuber magnatum 0 46.4 1.3 1.3 0 0.80.5 0.5 3.6 0.3 0 13.2 Tuber aestivum 03.1019.6 1.6 11.700.6 0.1 1.3 0 0 0 24.5 Tuber melanosporum 0.338.9 1.7 7.20.1 00 1.8 0.4 0.13.7 0 0.1 0.1 0.1 9.7 Tuber borchii 0.512.30.6 8.3 0.10 7.10.3 0.7 1.7 0 0.1 30.2 Choiromyces venosus 00.10.218.5 3.1 4.5 05.93.7 1.90 0.1 0.20 29.7 Morchella importuna.C 2.1 0.41.6 0.1 5.2 Morchella importuna 2.5 0.3 1.90.1 0.2 5.2 Gyromitra esculenta 2.2 00.13.80.2 0 0.1 7.7 Caloscypha fulgens 18.40.68.6 0.1 0.1 0.1 0.1 19.4 Wilcoxina mikolae 28.7 1.5 0 6.6 0.4 0 34.1 Trichophaea hybrida 35.76.1 6.6 03.30.20.2 0.2 0 0 0.6 0 0.1 43 Pyronema confluens 8.20 2.2 0.1 0 7.5 Sarcoscypha coccinea 0.4 1.4 0.1 0.2 3.6 Kalaharituber pfeilii 5.81.8 8.81.2 2.1 0.1 0.4 2.2 0.1 0.1 0.4 0.1 31.3 Terfezia boudieri 9.21.41.5 0.2 0.60.2 0.60.1 0.1 0.5 0.1 21.2 Terfezia claveryi 23.4 3 0.1 1.9 0.1 0.1 0.3 1.2 0.1 1.3 0.10.10.10.1 0.4 0.6 0 22.9 Tirmania nivea 17.51.1 0 3.4 0.3 0 0.10.6 0.2 1.5 0.2 0.4 0.2 0.4 19.4 Ascobolus immersus 0.10.50.8 0.2 0 0 0 1.8 Monacrosporium haptotylum 1.6 0.1 0.3 Arthrobotrys oligospora 1.3 0.1 Saccharomyces cerevisiae 0.1 0.2 0.6 0.6 Wickerhamomyces anomalus 0 1.5 Candida tanzawaensis 0.1 0.5 Taphrina deformans 0.9 0 0.2 0.6 2.7 Gigaspora rosea 030.74.50.1 5.8 0.1 0 0.8 0 2.90.6 0.1 0.4 0.4 0.1 0 0.2 0.5 0.5 53.2 Rhizophagus cerebriforme 1.11.1 0 0 0.20.2 0.8 0.4 0 0.30 0 0 0.3 22.4 Rhizophagus diaphanus 0.2 0 0.5 000.10.40.30 0.1 0 0 0 0.4 20.6 Rhizophagus irregularis 0.700.402.8 0 0.20.60.4 0.1 0.1 0 0 0.6 26.8 Rhizophagus irregularis DAOM 0.200.2 0 0.10.10.1 0 0.1 0 0.1 13.2 Rhizophagus irregularis A1 0.20.3 00.1 0 0.10.10.4 0 0 0.1 0.1 0 20.8 Rhizophagus irregularis A4 0.20.7 0.1 0 0.1 0.10.20.1 0 0.1 0 0 0 0 24.9 Rhizophagus irregularis A5 0.300.10.100 0.3 0.20 0.1 0.1 24.3 Rhizophagus irregularis B3 0.1 0.3000.30.10.4 0.1 0 0.2 0.1 0 22.3 GlomeralesRhizophagus irregularis Pezizomycetes C2 000.20.50.2 000.4 0.1 000 0.122.1 Mucoromycota

Ectomycorrhiza Ericoid mycorrhiza Orchid mycorrhiza Arbuscular mycorrhiza

Saprotroph Pathogen Parasite Endophyte Wood decayer Yeast

Fig. 3 Distribution and coverage (%) of transposable elements families in genome assemblies. The bubble size is proportional to the coverage of each of TE family (% indicated inside bubbles). The bars show the total coverage per genome. See also Supplementary Data 2–4. Source data are provided as a Source data Fig. 1.

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a Cellulose All Hemicellulose Lignin Pod Pectin 76 Laccaria amethystina 83 47 28 1 8 147 Laccaria bicolor 89 48 29 1 12 141 Coprinopsis cinerea 164 115 37 1 12 201 Coprinellus micaceus 216 142 54 320

Hypholoma sublateritium 256 144 182 106 54 14 22 Hebeloma cylindrosporum 84 49 24 311 250 146 Cortinarius glaucopus 98 49 41 12 8 Crucibulum laeve 238 157 54 14 27 266 Tricholoma matsutake 97 51 39 0 7 125 Amanita thiersii 142 95 27 020 175 Amanita muscaria 63 32 29 0 2 369 Pluteus cervinus 177 113 45 16 19 Ectomycorrhiza Gymnopus androsaceus 164 253 146 77 10 30 Omphalotus olearius 119 73 29 417 Saprotroph 2266 375 172 Marasmius fiardii 207 134 46 1 27 Pathogen 255 Moniliophthora perniciosa 101 50 36 1 15 Armillaria gallica 303 237 133 54 850 Orchid mycorrhiza Mycena galopus 454 253 138 19 63 Pleurotus ostreatus 194 216 141 47 9 28 Wood decayer Schizophyllum commune 151 114 15 0 22 Paxillus involutus 777 76 4426 6 0 Endophyte 79 Paxillus adelphus 70 40 25 0 5 Gyrodon lividus 379 86 79 48 22 09 Xerocomus badius 85 82 48 28 0 6 90 Boletus edulis 87 46 34 0 7 Pisolithus microcarpus 52 53 34 17 0 2 91 Scleroderma citrinum 63 33 23 0 7 112 86 Suillus luteus 86 50 29 0 7 Suillus brevipes 96 58 31 0 7 Coniophora puteana 122 139 104 17 0 18 Serpula lacrymans 380 184 98 71 14 0 13 Fibulorhizoctonia sp. 328 205 82 0 41 122 156 Piloderma croceum 108 52 38 1 18 Plicaturopsis crispa 125 82 21 7 22 Phanerochaete chrysosporium 151 140 100 39 16 12 137 Phlebia brevispora 159 92 49 17 18 144 Postia placenta 90 58 19 1 13 351 Thelephora ganbajun 44 34 6 0 4 184 Lentinellus vulpinus 91 114 76 31 11 7 128 Lactarius quietus 76 30 43 13 237 Heterobasidion annosum 141 84 37 8 20 367 Neolentinus lepideus 120 110 75 19 0 16 176 Jaapia argillacea 147 116 18 1 13 Punctularia strigosozonata 181 105 45 11 31 336 Sistotremastrum niveocremeum 166 94 68 15 4 Ramaria rubella 177 101 58 13 18 158 320 223 Gautieria morchelliformis 154 53 91 36 10 Sphaerobolus stellatus 532 208 297 63 27 Auricularia subglabra 259 162 60 18 37 Piriformospora indica 156 121 14 0 21 328 128 Sebacina vermifera 177 139 18 0 20 Hydnum rufescens 64 36 23 0 5 55 Cantharellus anzutake 80 47 06 269 120 27 164 Sistotrema sp. 208 144 23 0 41 Botryobasidium botryosum 154 121 20 0 13 1198 166 Tulasnella calospora 246 188 9 0 49 Ceratobasidium sp. 321 175 70 0 76 235 115 Rhizoctonia solani 341 164 59 0 118 108 Dacryopinax primogenitus 80 54 14 0 12 92 Tremella mesenterica 24 19 4 0 1 Wallemia sebi 28 26 2 00 Choiromyces venosus 88 64 7 0 17 Carb. Permian Triassic Jurassic Cretaceous Cenozoic

881 491 416 325 300 200 100 0 Mya

Cellulose b 380 AllHemicellulose Lignin Pod Pectin 403 Chalara longipes 324 243 432 38 404 Meliniomyces variabilis 328 248 42 1 38 485 Meliniomyces bicolor 238 165 371 36 Rhizoscyphus ericae 174 127 23 0 24 483 Acephala macrosclerotiorum 282 198 430 41 509 357 Phialocephala scopiformis 287 201 522 34 Ascocoryne sarcoides 630 145 119 100 16 Glarea lozoyensis 192 140 27 5 25 Amorphotheca resinae 74 57 12 0 5 Ectomycorrhiza 564 326 Oidiodendron maius 332 254 34 0 44 Saprotroph Bisporella sp. 263 197 313 35 595 452 Sclerotinia sclerotiorum 145 100 160 29 173 Pathogen Botrytis cinerea 156 101 22 0 33 Magnaporthe grisea 175 145 18 2 12 Parasite 200 Neurospora crassa 111 91 12 0 8 245 Trichoderma reesei 109 90 130 6 Ericoid mycorrhiza 302 Xylaria hypoxylon 181 137 222 22 627 Endophyte Cenococcum geophilum 92 66 171 9 210 Glonium stellatum 196 139 35 6 22 Yeast 213 Lepidopterella palustris 83 56 15 2 12 364 Stagonospora sp. 239 188 314 20 291 431 Alternaria alternata 228 164 264 38 Aureobasidium pullulans 190 141 193 30 244 Cladosporium fulvum 174 98 482 28 493 Aspergillus clavatus 107 93 40 10 190 183 Aspergillus flavus 186 128 140 44 220 Arthroderma benhamiae 26 20 60 0 Usnea florida 65 43 18 0 4 228 357 Xylona heveae 34 28 60 0 Tuber magnatum 52 40 5 0 7 60 Tuber aestivum 52 38 60 8 63 Tuber melanosporum 49 39 550 64 Tuber borchii 48 38 5 0 5 112 Choiromyces venosus 105 80 70 18 178 Morchella importuna.C 159 122 5 0 32 160 Morchella importuna 159 5 0 33 173 121 187 Gyromitra esculenta 161 125 5 0 31 252 Caloscypha fulgens 90 66 5 0 19 222 Wilcoxina mikolae 65 48 80 9 68 56 39 90 8 91 Trichophaea hybrida 153 Pyronema confluens 77 63 5 1 9 243 Sarcoscypha coccinea 143 106 80 29 Kalaharituber pfeilii 78 68 60 4 37 82 Terfezia boudieri 43 35 5 1 3 130 Ascobolus immersus 114 102 6 0 6 Monacrosporium haptotylum 179 125 9 0 45 183 19 Arthrobotrys oligospora 142 108 60 28 Wickerhamomyces anomalus 19 18 10 0 16 Saccharomyces cerevisiae 20 13 12 010 Candida tanzawaensis 15 14 00 1 Taphrina deformans 16 14 2 00

Ediacaran Cambrian Ord. Sil. Devonian Carb. Permian Triassic Jurassic Cretaceous Cenozoic

600 500 400 300 200 100 0 Mya auxiliary activity (AA) enzymes (including class II PODs, lac- Cantharellus anzutake, implying that ectomycorrhizal basidio- cases, lytic polysaccharide monooxygenases (LPMO)), carbo- mycetes are unable to use sucrose directly from the plant. Except hydrate esterases (CE), polysaccharide lyases (PL), and for Acephala macrosclerotiorum,thenumberofPCWDEsin associated cellulose-binding modules (e.g., CBM1; Supplemen- ectomycorrhizal Ascomycota is lower than those of most of tary Figs. 3–5, and Supplementary Data 7 and 8). Notably, there other groups, excluding yeasts and arbuscular mycorrhizal fungi is no invertase GH32 gene in their genome, except for (Figs. 4 and 5, and Supplementary Figs. 3–5). According to

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Fig. 4 Evolution of gene families encoding PCWDEs in Basidiomycota and Ascomycota. a Basidiomycota. b Ascomycota. Fungal taxa are displayed according to their phylogeny (left panel). Bubbles with numbers at the tree nodes represent the total number of genes coding for total PCWDEs (intracellular and secreted) for ancestral nodes determined by COMPARE. The bubble size is proportional to the number of PCWDE genes. Heat maps contain the number of genes coding for substrate-speciﬁc PCWDEs for the extant species. Relative abundance of genes is represented by a color scale, from the minimum (blue) to maximum (red) number of copies per species. Ectomycorrhizal fungi are framed with a black line. See also Supplementary Data 8 and Supplementary Fig. 5. The tree is a chronogram estimated with r8s on the basis of a maximum likelihood phylogeny inferred with RAxML. The geological timescale (in million year) is indicated at the bottom. Source data are provided as a Source data Fig. 2.

RedoxiBase (http://peroxibase.toulouse.inra.fr), none of the class ectomycorrhizal Pezizales, such as the newly sequenced Wilcox- II PODs of ectomycorrhizal species are ligninolytic (i.e., lig- ina mikolae and Trichophaea hybrida (Pyronemataceae), also ninolytic POD or LiP), except those of Gautieria morchelliformis have few PCWDEs. The desert truffles Kalaharituber pfeilii and (see below; Supplementary Fig. 4 and Supplementary Data 6p). Terfezia boudieri (Pezizaceae) both have ectomycorrhiza-like Below, we discuss the PCWDEs of ectomycorrhizal fungi, restricted suites of PCWDEs, but they differ in the numbers of emphasizing newly sampled lineages with previously unknown genes encoding cellulose-acting LPMOs (ten and one copies, decomposition capacity. respectively) and they encode one copy of GH32 invertase. Like Cantharellales arose before the origin of ligninolytic class II other ectomycorrhizal species, A. macrosclerotiorum contained no POD26,28–30. Both saprotrophs (Botryobasidium botryosum, class II PODs, but had a large set of PCWDEs (282 genes, Sistotrema sp.) and orchid symbionts (Tulasnella calospora, including 17 cellulose-acting LPMOs, and genes encoding GH6, Ceratobasidium sp.) in Cantharellales possess large sets of GH7, GH32, GH45, PL1, and PL3; Figs. 4 and 5, Supplementary enzymes acting on cellulose, hemicellulose, and pectins, including Figs. 3–5 and Supplementary Data 8). Closely related taxa to A. LPMOs (AA9; Figs. 4 and 5, and Supplementary Figs. 3–5). We macrosclerotiorum mainly contain DSEs, such as P. scopiformis, sequenced the first genomes of ectomycorrhizal Cantharellales, saprotrophs, and pathogens, which are characterized by a larger Hydnum rufescens, and C. anzutake, and found that they have set of PCWDEs compared to ectomycorrhizal fungi. To assess highly reduced repertoires of secreted PCWDEs. In this regard, whether this large repertoire of PCWDEs is expressed during the ectomycorrhizal symbionts of Cantharellales are more similar symbiosis development, we carried out transcript profiling of to ectomycorrhizal Agaricales and Boletales than to orchid Pinus sylvestris–A. macrosclerotiorum ectomycorrhizas using symbionts of Cantharellales. These results provide another RNA-Seq. The fungal symbiont develops a dense Hartig net independent example of convergent loss of PCWDEs in within the root cortex, but no mantle sheath (Supplementary ectomycorrhizal lineages, and highlight the different decomposi- Fig. 6a). Transcript profiling of ectomycorrhizas showed that tion capacities of two guilds of plant symbionts. most of the PCWDEs are not induced in symbiotic tissues In contrast to Cantharellales, Phallomycetidae diverged shortly (Supplementary Fig. 6b and Supplementary Data 16), suggesting a after the evolution of ligninolytic class II POD. The one tight transcriptional control of the PCWDE gene expression. previously published genome from this group, the saprotrophic “cannonball fungus”, Sphaerobolus stellatus, has an astonishingly large repertoire of PCWDEs, including 63 class II PODs. The two MCWDEs are retained in ectomycorrhizal fungi. We explored genomes of ectomycorrhizal Phallomycetidae reported here, G. the genetic capabilities of the sequenced fungi to decompose morchelliformis (Gomphales) and Hysterangium stoloniferum microbial (i.e., bacterial and fungal) cell walls by comparing 31 PCWDE to MCWDE repertoires (Figs. 4 and 5, Supplementary (Hysterangiales) , have diverse PCWDEs acting on cellulose, – hemicellulose, pectins, and lignin, with 31 and five genes Figs. 3 5 and Supplementary Data 7). The proportion of encoding ligninolytic class II PODs, respectively (Figs. 4 and 5, MCWDE (acting on chitin, glucans, mannans, and peptidogly- Supplementary Figs. 3–5 and Supplementary Data 8). Although cans) in ectomycorrhizal fungi is similar to that in saprotrophs. many Ramaria species (Gomphales) form ectomycorrhiza, the Thus, ectomycorrhizal fungi have generally retained MCWDE newly sequenced Ramaria rubella (subgenus Lentoramaria)is genes (e.g., chitinases, ß-1,3-glucanases) although they have lost likely a litter decomposer, which is consistent with its possession most PCWDEs (e.g., endo- and exocellulases). Ectomycorrhizal of 13 class II PODs, 6 cellulose-acting LPMOs, and GH6 and fungi may use secreted MCWDE to scavenge nitrogen compounds trapped in SOM (e.g., chitin) by selectively using these GH7 cellobiohydrolases. Thus, a robust suite of PCWDEs appears 32 to be a characteristic of both saprotrophs and ectomycorrhizal hydrolytic enzymes in addition to oxidative mechanisms . Some species in Phallomycetidae. of these chitin-, glucan-, and mannan-active enzymes are likely Ectomycorrhizal Russulales and Thelephorales, for which we involved in fungal cell wall remodeling during complex devel- fi opmental processes, such as ectomycorrhiza and sporocarp report the rst genomes, both have highly reduced complements 10,11,33 of PCWDEs. The two Thelephorales species (Thelephora terrestris formation . and T. ganbajun) resemble Boletales in having a highly reduced suite of the major enzymes acting on crystalline cellulose and Mycorrhizal development is driven by gene co-option. We used lignin (three cellulose-acting LPMOs, no class II POD, and no a phylostratigraphic approach to characterize the evolutionary GH6 or GH7). Ectomycorrhizal Russulales (Lactarius quietus, origins of ectomycorrhizal lineages on the basis of gene functions Russula emetica, and Russula ochroleuca) also have small in extant organisms. We examined the age distribution of genes repertoires of PCWDEs (no CBM1, one or two cellulose-acting induced at different stages of ectomycorrhiza establishment, so- LPMOs, no GH6 or GH7, no CE1, and no LiP), but they retain called symbiosis-induced genes, by defining phylogenetic ages one atypical Mn POD gene (Supplementary Data 6p). In contrast, (phylostrata), that correspond to internal nodes of the tree along saprotrophic Russulales (e.g., Lentinellus vulpinus) have a typical the lineage leading from the root to the symbiotic species for white rot array of PCWDEs. which transcriptomic data are available (Fig. 6, Supplementary In Ascomycota, species in Tuberaceae followed the general Fig. 7 and Supplementary Data 9). In Ascomycota and Basidio- symbiotroph trend, except C. venosus, which has a large mycota, an average of 74% and 67% of the ectomycorrhiza- complement of PCWDEs (Figs. 4a and 5, see ref. 16). Other induced genes predated the evolution of ectomycorrhizal

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CBM1 AA1_1AA1_2AA1_3 GH45 CBM1 AA1_1AA1_2AA1_3 GH45 AA9 AA1 AA2 GH6 GH7 CE1 AA9 AA1 AA2 GH6 GH7 CE1

Chalara longipes 12 24 4 6 8 1 2 9 2 7 Laccaria amethystina 1 2 5 1 Laccaria bicolor 1 4 1 9 1 1 Meliniomyces variabilis 11 18 2 7 5 1 2 8 4 4 Coprinopsis cinerea 30 28 17 1 5 6 1 3 Meliniomyces bicolor 10 3 2 4 10 1 1 3 2 4 Coprinellus micaceus 44 38 17 3 2 7 1 Rhizoscyphus ericae 11 6 3 3 4 1 1 2 1 Hypholoma sublateritium 26 12 9 14 1 4 1 2 Acephala macrosclerotiorum 17 12 3 7 8 1 3 2 3 Hebeloma cylindrosporum 2 1 2 3 1 Phialocephala scopiformis 19 19 2 7 10 1 3 6 2 4 Cortinarius glaucopus 3 2 5 1 9 2 Ascocoryne sarcoides 28 5 1 3 2 5 3 4 Crucibulum laeve 39 31 15 13 4 8 3 2 Glarea lozoyensis 12 22 2 1 5 4 3 5 1 9 Tricholoma matsutake 1 2 10 2 1 Amorphotheca resinae 2 1 1 1 3 Amanita thiersii 8 12 10 1 1 2 1 Amanita rubescens 1 8 1 Oidiodendron maius 30 8 1 10 2 5 2 4 Amanita muscaria 2 12 Bisporella sp. 20 24 2 7 3 2 2 1 8 Pluteus cervinus 24 23 9 12 2 4 2 2 16 8 4 1 2 2 Sclerotinia sclerotiorum Gymnopus androsaceus 29 5 1 23 9 8 2 2 Botrytis cinerea 14 7 4 1 3 1 Omphalotus olearius 12 9 3 4 1 2 3 2 Magnaporthe grisea 17 18 3 1 5 1 2 3 1 8 Marasmius fiardii 10 17 16 2 3 2 1 Neurospora crassa 19 13 1 7 2 4 1 5 Moniliophthora perniciosa 2 11 3 1 2 Trichoderma reesei 13 2 3 1 1 1 2 Mycena galopus 44 11 3 24 2 17 1 11 2 4 Armillaria gallica 13 17 1 21 8 2 2 4 1 Xylaria hypoxylon 16 18 2 6 1 3 4 1 4 Pleurotus ostreatus 32 25 11 1 9 2 16 2 2 Cenococcum geophilum 3 5 5 1 1 1 2 1 Schizophyllum commune 5 19 2 1 2 1 7 13 16 1 1 13 5 2 3 3 5 Glonium stellatum Paxillus ammoniavirescens 3 10 Lepidopterella palustris 2 2 1 5 1 Paxillus adelphus 3 13 Stagonospora sp. 15 30 2 4 3 5 4 3 11 Paxillus involutus 3 12 1 Alternaria alternata 12 25 1 1 4 4 3 3 3 4 Gyrodon lividus 1 4 6 Aureobasidium pullulans 4 4 1 1 4 2 1 3 2 Xerocomus badius 3 9 1 Boletus edulis 2 4 1 Cladosporium fulvum 2 1 8 2 2 2 Dothideomycetes Boletus edulis P 2 6 2 Aspergillus clavatus 13 7 2 2 2 3 5 Pisolithus microcarpus 3 8 Aspergillus flavus 4 7 5 1 1 3 3 Pisolithus tinctorius 4 7 Arthroderma benhamiae 1 Scleroderma citrinum 2 8 Boletales Agaricales Ascomycota Usnea florida 2 1 4 1 Suillus luteus 2 16 1 Xylona heveae 1 2 2 Suillus brevipes 2 14 1 Tuber magnatum 1 1 2 2 Coniophora puteana 2 10 3 1 2 1 Serpula lacrymans 7 5 4 1 1 Tuber aestivum 2 2 1 4 1 Rhizopogon vesiculosus 3 11 Tuber melanosporum 1 3 1 2 1 Rhizopogon vinicolor 2 14 Tuber borchii 1 3 1 2 Melanogaster broomeianus 1 9 13 Choiromyces venosus 4 8 1 2 2 1 Fibulorhizoctonia sp. 31 14 26 1 1 20 7 7

Morchella importuna.C 16 22 1 3 2 1 2 Basidiomycota Piloderma croceum 1 5 9 2 1 1 2 Morchella importuna 16 19 2 3 2 1 2 Plicaturopsis crispa 8 8 5 7 2 1 4 Gyromitra esculenta 18 24 2 2 2 1 Phanerochaete chrysosporium 34 12 1 14 1 8 2 1 Phlebia brevispora 24 9 6 1 11 1 4 3 1 Caloscypha fulgens 6 12 1 2 Postia placenta 2 2 1 Wilcoxina mikolae 2 2 2 1 2 Neolentinus lepideus 4 3 1 2 Trichophaea hybrida 1 3 1 3 Jaapia argillacea 23 14 1 1 3 4 1 2 Pyronema confluens 6 10 1 1 1 2 1 Punctularia strigosozonata 20 11 12 11 1 4 1 2 Pezizomycetes Leotiomycetes Sarcoscypha coccinea 14 18 1 2 1 2 2 1 Sistotremastrum niveocremeum 48 21 1 9 14 4 3 2 2 Kalaharituber pfeilii 8 7 1 1 1 Heterobasidion annosum 17 9 2 11 7 1 1 2 1 Terfezia boudieri 3 2 1 Lactarius quietus 2 14 1 9 Lentinellus vulpinus 20 9 1 6 11 1 3 1 2 Terfezia claveryi 2 2 1 1 Russula emetica 1 9 2 4 Tirmania nivea 5 5 1 1 Russulales Russula ochroleuca 2 10 2 7 Ascobolus immersus 38 29 1 2 4 5 8 Thelephora terrestris 1 3 1 Monacrosporium haptotylum 98 21 2 1 2 4 3 5 Thelephora ganbajun 3 1 Arthrobotrys oligospora 100 22 2 5 4 6 Ramaria rubella 16 6 5 9 13 2 3 2 4 Saccharomyces cerevisiae 1 Gautieria morchelliformis 3 1 10 25 1 1 2 Wickerhamomyces anomalus Hysterangium stoloniferum 1 1 13 5 1 45 34 2 14 41 4 8 2 5 Candida tanzawaensis 1 Sphaerobolus stellatus Auricularia subglabra 44 16 2 3 15 2 6 3 4 Taphrina deformans Piriformospora indica 60 22 1 2 1 2 9 Gigaspora rosea 16 Sebacina vermifera 47 24 5 5 4 1 6 Rhizophagus cerebriforme 5 1 Hydnum rufescens 1 2 4 1 1 Rhizophagus diaphanus 3 Cantharellus anzutake 1 7 7 1 1 3 Rhizophagus irregularis 5 Sistotrema sp. 49 18 1 2 4 3 2 Rhizophagus irregularis DAOM 3 Botryobasidium botryosum 27 31 3 1 2 7 2 Rhizophagus irregularis A1 4 Tulasnella calospora 76 28 1 5 17 2 2 Ceratobasidium sp. 50 27 10 6 6 12 4 3 Rhizophagus irregularis A4 6

Cantharellales Rhizoctonia solani 34 31 11 7 3 8 5 Glomerales Mucoromycota Rhizophagus irregularis A5 8 Tremella mesenterica 2 4 1 Rhizophagus irregularis B3 Dacryopinax primogenitus 1 Rhizophagus irregularis C2 7 Wallemia sebi 8

Ectomycorrhiza Orchid mycorrhiza Ericoid mycorrhiza Arbuscular mycorrhiza

Saprotroph Pathogen Parasite EndophyteWood decayer Yeast

Fig. 5 Distribution of key secreted PCWDEs in analyzed fungi. Bubbles with numbers contain the number of genes coding for a series of secreted PCWDEs needed for cellulose and lignin degradation. Taxa are color coded according to their lifestyle (see bottom panel). See also Supplementary Data6a, j. Left panel, Ascomycota and Mucoromycota; right panel: Basidiomycota. Source data are provided as a Source data Fig. 3. symbiosis, respectively (Fig. 6). Approximately 6% and 18% of in symbiosis-induced genes (Fisher’s exact test, P < 0.005; these genes were already present in the MRCAs of the Ascomy- Supplementary Data 9b), suggesting that there was no dis- cota and that of the Basidiomycota (e.g., for L. bicolor, hydro- tinguished period during ectomycorrhiza evolution caracterized phobins, GH131, GH28, and CBM1_GH5), respectively (Fig. 6 by an excess number of gene birth events. These observations and Supplementary Fig. 7). No speciﬁc phylostrata was enriched imply that symbiosis-induced genes have mostly been co-opted

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a Genes mapping to specific phylostrata

MRCA of Before emergence At the emergence Total number Ascomycota/ of mycorrhizal of mycorrhizal of induced Basidiomycota symbiosis symbiosis genes Chalara longipes Meliniomyces variabilis Meliniomyces bicolor PS11 Rhizoscyphus ericae PS10 Aceph. macrosclerotiorum 501 (22%) 1666 (73.5%) 602 (26.5%) 2268 (100%) PS9 Phialocephala scopiformis Ascocoryne sarcoides PS8 Glarea lozoyensis Amorphotheca resinae Ectomycorrhiza PS7 Oidiodendron maius Bisporella sp. Emergence of mycorrhizal symbiosis Sclerotinia sclerotiorum PS6 Botrytis cinerea Magnaporthe grisea Neurospora crassa Trichoderma reesei Xylaria hypoxylon Cenococcum geophilum 78 (16%) 358 (73.5%) 129 (26.5%) 487 (100%) PS5 Glonium stellatum ECM-induced in 1 species Lepidopterella palustris Stagonospora sp. ECM-induced in 2 species Alternaria alternata Aureobasidium pullulans ECM-induced in 3 species Cladosporium fulvum Aspergillus clavatus Aspergillus flavus Arthroderma benhamiae PS4 Usnea florida Xylona heveae Tuber magnatum 86 (17%) 383 (74%) 73(14%) 514 (100%) Tuber aestivum Tuber melanosporum Tuber borchii Choiromyces venosus 18% 74% 22% Morchella importuna.C Morchella importuna PS3 Gyromitra esculenta Caloscypha fulgens Wilcoxina mikolae Trichophaea hybrida Pyronema confluens PS2 Sarcoscypha coccinea Kalaharituber pfeilii Terfezia boudieri PS1 Ascobolus immersus Monacrosporium haptotylum Arthrobotrys oligospora Wickerhamomyces anomalus Saccharomyces cerevisiae Candida tanzawaensis Taphrina deformans

70.0

b Laccaria amethystina Laccaria bicolor 74 (8%) 666 (69%) 99 (10%) 964 (100%) PS18 Coprinopsis cinerea Coprinellus micaceus PS17 Hypholoma sublateritium Hebeloma cylindrosporum 29 (6%) 368 (75%) 123 (25%) 491 (100%) PS16 Cortinarius glaucopus Crucibulum laeve PS15 Tricholoma matsutake 6 (11%) 37 (68.5%) 17 (31.5%) 54 (100%) Amanita thiersii Amanita muscaria 26 (5%) 314 (61%) 200 (39%) 514 (100%) PS14 Pluteus cervinus Ectomycorrhiza Gymnopus androsaceus Omphalotus olearius PS13 Marasmius fiardii Emergence of mycorrhizal symbiosis Moniliophthora perniciosa Armillaria gallica Mycena galopus Pleurotus ostreatus Schizophyllum commune Paxillus involutus 17 (7%) 206 (85.5%) 2 (0.8%) 241 (100%) Paxillus adelphus PS12 Gyrodon lividus ECM-induced in 1 species Xerocomus badius Boletus edulis ECM-induced in 2 species Pisolithus microcarpus 0 (0%) 18 (51%) 0 (0%) 35 (100%) Scleroderma citrinum Suillus luteus ECM-induced in 3 species Suillus brevipes Coniophora puteana ECM-induced in 4 species PS11 Serpula lacrymans Fibulorhizoctonia sp. ECM-induced in 5 species Piloderma croceum 13 (4%) 227 (63%) 131 (37%) 358 (100%) Plicaturopsis crispa ECM-induced in 6 species Phan. chrysosporium Phlebia brevispora Postia placenta 6% 67% 20% PS10 Thelephora ganbajun Lentinellus vulpinus Lactarius quietus Heterobasidion annosum PS9 Neolentinus lepideus Jaapia argillacea PS8 Punctularia strigosozonata Sistotr. niveocremeum Ramaria rubella PS7 Gautieria morchelliformis Sphaerobolus stellatus Auricularia subglabra PS6 Piriformospora indica Sebacina vermifera Hydnum rufescens PS5 Cantharellus anzutake Sistotrema sp. PS4 Botryobasidium botryosum PS3 Tulasnella calospora Ceratobasidium sp. PS2 Rhizoctonia solani PS1 Dacryopinax primogenitus Tremella mesenterica Wallemia sebi Choiromyces venosus 50.0

Fig. 6 Phylostratigraphy for ectomycorrhiza-specific upregulated genes. Pie charts on the time-calibrated trees represent the number of gene clusters containing ectomycorrhiza-specific upregulated genes from the species compared. Numbers and percentage of genes mapping to phylostrata are shown on the right of the trees. Ectomycorrhizal species used for the comparison are in green boxes. Upregulated genes were selected according to FDR adjusted P value < 0.05. a Ascomycota, the fold change (FC) used for defining ectomycorrhiza-specific upregulated genes was FC ≥ 5. b Basidiomycota, the FC used for defining ectomycorrhiza-specific upregulated genes was FC ≥ 5. See Supplementary Data 9 for the identified phylostrata containing ectomycorrhiza- upregulated genes and enrichment statistics. Source data are provided as a Source data Fig. 4.

NATURE COMMUNICATIONS | (2020) 11:5125 | https://doi.org/10.1038/s41467-020-18795-w | www.nature.com/naturecommunications 9 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18795-w for ectomycorrhiza development during evolution from sapro- MiSSPs, CAZymes, proteins with unknown KOG function and trophic ancestors. However, the phylostratigraphic analysis also proteins of signaling and metabolic pathways. Altogether, 31.2% suggested that an average of 9–22 % (in Ascomycota; Fig. 6a) and of these 1028 symbiosis-induced genes are mostly species specific 19–20 % (in Basidiomycota; Fig. 6b) of ectomycorrhiza-induced (clusters V–VII). Most of these genes encode MiSSPs, proteins genes are restricted to specific mycorrhizal lineages. Given our with unknown KOG functions and proteins of signaling path- current taxon sampling, it is difficult to distinguish genes that ways. The phylostratigraphic analysis of secreted proteins and coincidentally evolved with mycorrhiza formation from species- MiSSPs corroborated these results (Supplementary Fig. 7). specific orphan genes, except in the case of three clades that comprise more than a single ectomycorrhizal species, Boletales, the genus Laccaria and Tuberaceae, which comprise more than a Discussion single ectomycorrhizal species. In these clades, the proportion of After the origin of Pinaceae (ca. 200 Mya), ectomycorrhizal symbiosis-induced genes that map to the origin of ectomycor- symbiosis arose repeatedly, perhaps 80 times or more, across rhiza showed a large variation, from 0 to 0.8% in the Boletales multiple lineages of Mucoromycotina (Endogonales), Ascomy- (two species), to 10% in Laccaria, and 14% in the Tuberaceae cota, and especially Basidiomycota2–4,8,34,35. The ancestors of (Fig. 6). ectomycorrhizal fungi are genetically and ecologically diverse, To test our phylostratigraphic results, we assessed the evolu- making this a superb example of convergent evolution3,7,9,12.To tionary conservation of 5917 ectomycorrhiza-induced transcripts assess the general shared properties of the lifestyle evolution and identified in ten different ectomycorrhizal interactions among the functional biology of ectomycorrhizal symbioses, we conducted a 135 studied fungal genomes (Fig. 7a). We found that 15.2 % of the comparative analysis of 62 mycorrhizal and 73 non-mycorrhizal 5917 symbiosis-induced genes are shared by all species in our fungal species. Our dataset, the most comprehensive so far, dataset (clusters IV and V). Most encode proteins involved in core includes several major fungal clades that have not been sampled metabolic or signaling functions. Genes from cluster III are previously for ectomycorrhizal genomes, i.e., Cantharellales, conserved within Basidiomycota only, while genes from cluster VII Phallomycetidae, Thelephorales, and Russulales. For the sake of are shared by Ascomycota only. Most of them have no known comparison, it also includes genomes of arbuscular mycorrhizal function (i.e., no KOG domain). Altogether, 31.2% of these fungi, orchid mycorrhizal fungi, and ericoid mycorrhizal fungi symbiosis-induced genes are species specific(clusterVI).Mostof providing a unique opportunity to highlight differences and these genes encode proteins with unknown KOG functions or similarities between the major types of mycorrhizal symbioses. mycorrhiza-induced small secreted proteins (MiSSPs). The Our analyses of early diverging clades of ectomycorrhizal fungi proportion of species-specific, ectomycorrhiza-induced genes is (e.g., Cantharellales) support the general view that transitions therefore substantial, as reported earlier10,12,16 and suggested by from saprotrophy to ectomycorrhizal symbiosis involve (1) our phylostratigraphic analysis. widespread losses of PCWDEs acting on lignin, cellulose, hemi- Next, we examined whether the same or different gene families cellulose, pectins, suberins, and tannins, (2) co-option of meta- were co-opted in independent ectomycorrhizal lineages, i.e., bolic and signaling genes present in saprotrophic ancestors to whether co-option was convergent or divergent during fungal fulfill new symbiotic functions, (3) diversification of novel, evolution. We quantified convergence by the extent of overlap lineage-specific symbiosis-induced orphan genes, and (4) massive among conserved ectomycorrhiza-induced gene families within proliferation of TEs. In addition, they corroborated and extended independent lineages. Conserved ectomycorrhiza-induced genes our previous analyses of the genomes of arbuscular mycorrhizal showed little or no overlap among the analyzed species (Fig. 6 fungi27 and ericoid mycorrhizal fungi15. Despite the general trend and Supplementary Data 9). For example, there were only eight toward losses of PCWDEs in ectomycorrhizal lineages in both clusters of ectomycorrhiza-induced genes common to at least four Ascomycota and Basidiomycota, there is considerable diversity in Basidiomycota symbionts (Supplementary Data 9). Similarly, only the apparent decay capacities of ectomycorrhizal fungi. For 12 gene clusters were shared by at least three Ascomycota example, in Agaricales (Agaricomycetidae), C. glaucopus pos- symbionts (Supplementary Data 9). This low overlap between co- sesses 12 recently duplicated copies of atypical class II POD genes, opted genes suggests that independently evolved ectomycorrhizal which may confer some ability to obtain nutrients from soil lineages recruited different ancestral gene families for symbiosis, phenolic compounds, as previously suggested36. However, in addition to a likewise unique set of novel genes, that evolved secreted PODs can play a variety of additional roles, such as after the origins of symbiosis12. biosynthesis of cell wall melanins, detoxifying the immediate hyphae environment, or/and converting plant polymers into more oxidized, recalcitrant components of SOM. Similarly, the Symbiosis-induced secreted proteins in symbionts. The genome of T. matsutake (also Agaricales) encodes two GH7 expression of genes encoding secreted and symbiosis-induced cellobiohydrolases, in agreement with its known facultative secreted proteins from ectomycorrhizal fungi was measured by saprotrophic ability37. Ectomycorrhizal fungi that evolved from RNA-Seq profiling in ten ectomycorrhizal associations (Supple- brown rot fungi in the Boletales (also Agaricomycetidae), such as mentary Methods). By using a BLASTP-based analysis, we Paxillus involutus, appear to have adapted the oxidative decom- assessed the evolutionary conservation of the expressed position system from their saprotrophic ancestors to liberate N 6669 secreted proteins (Fig. 7b) and 1028 symbiosis-induced entrapped in decaying SOM38–41. Their secreted proteases, secreted proteins (Fig. 7c) among 135 fungal species (Supple- LPMOs, and laccases may act in concert to decay available SOM mentary Datas 12, 13 and 14). A substantial proportion of the compounds. Alternative hypotheses for the role of the remaining secreted proteins are species-specific SSPs (cluster III, Fig. 7b). In PCWDEs in ectomycorrhizal fungi include the remodeling of root addition, we found that 38.1% of symbiosis-induced secreted cell walls during host colonization42. proteins are shared by all species of fungi (clusters I and III). Among the newly sampled fungal groups, ectomycorrhizal Most code for core metabolic or signaling functions and Phallomycetidae and Cantharellales present highly variable suites CAZymes. Genes from cluster II (9.3%) are conserved within of PCWDEs. Most striking is the ectomycorrhizal G. morchelli- Basidiomycota only. Genes from cluster IV (21.4%) are conserved formis (Gomphales, Phallomycetidae), which has 25 class II mainly in Ascomycota, showed a lower similarity in Basidiomy- manganese PODs (MnP) that may be involved in the decay of cota and are poorly conserved in Glomeromycota. They encode SOM and/or detoxification of soil polyphenolic compounds, as

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well as numerous enzymes active on cellulose and hemicellulose. ligninolytic enzymes. In contrast to their saprotrophic cousin The class II PODs of G. morchelliformis are members of a gene Sistotrema sp., C. anzutake and H. rufescens are ectomycorrhizal family that underwent expansion after the divergence of Aur- species of Cantharellales that lack not only class II LiP, but also iculariales and other Agaricomycetes. In contrast, Cantharellales many glycoside hydrolases acting on cellulose, hemicellulose and evolved prior to the diversiﬁcation of class II LiP30, and none pectins, LPMOs, and CBM1 motifs that are needed for plant cell of its species—saprotrophic or mycorrhizal—possesses these wall and SOM decomposition. In this regard, they resemble some

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Fig. 7 Phylogenetic conservation of ectomycorrhiza-induced genes. We analyzed genes coding for ectomycorrhiza-induced proteins, secreted proteins and ectomycorrhiza-induced secreted proteins among 135 fungi. Heat maps show BLASTP sequence similarity of 5917 ectomycorrhiza-induced proteins (a), 6669 genes coding for secreted proteins (b), and 1028 ectomycorrhiza-induced secreted proteins (c) from ten ectomycorrhizal fungi, with available symbiotic transcriptomes among 135 genomes of Basidiomycota and Ascomycota. The heat maps depict a double-hierarchical clustering of protein sequences encoded by symbiosis-upregulated genes, genes coding for secreted proteins and genes coding for symbiosis-upregulated secreted proteins (rows, fold change ≥ 5 in symbiotic tissues compared to free-living mycelium, false discovery rate-corrected P ≤ 0.05); right panel, functional categories (KOG) are given for each cluster of sequences in % as bargrams. Clusters I–VIII correspond to group of sequences sharing the same level of protein sequence similarity based on BlastP. The distribution of transcripts belonging to Ascomycota and Basidiomycota in each cluster is shown as pie charts. Data were visualized and clustered using R (package HeatPlus97). The hierarchical clustering was done by using a Euclidian distance and Ward clustering method. Color scale on the left (white to red) shows the % of sequence identity according to BLASTP. The symbiosis-induced genes were retrieved from the ectomycorrhizal transcriptomes of ten species: A. macrosclerotiorum EW76-UTF0540, A. muscaria Koide, C. geophilum 1.58, H. cylindrosporum h7, L. bicolor S238N, P. involutus ATCC 200175, P. croceum F1598, P. microcarpus 441, T. matsutake 945, and T. magnatum (see Supplementary Data 10, 13 and 14). GEO accession codes are provided in the “Methods/Phylostratigraphy” section. Source data are provided as a Source data Fig. 5.

of the most derived ectomycorrhizal Agaricomycetidae with Further insight into the differences in gene composition highly reduced saprotrophic capabilities, such as species of between saprotrophic and symbiotic fungi was obtained by Boletales. The ectomycorrhizal Russulales and Thelephorales that focusing on genes involved in adaptations to the host plant we sampled also conform to this model of a greatly reduced habitat in which symbionts thrive. Novel genes, such as effector- saprotrophic apparatus in prominent symbiotic fungi playing like MiSSPs, are likely required for symbiosis development (e.g., major roles in forest ecosystems. dampening of plant defense reactions43,44)andmetabolism45, Ectomycorrhizal fungi are not all depauperate in PCWDEs. and have evolved in every ectomycorrhizal lineage. Transcript Species such as A. macrosclerotium in the Leotiomycetes, may profiling of several ectomycorrhizal interactions has shown that represent transitional steps from pure saprotrophy toward pure 7–38% of genes that are upregulated during symbiosis are ectomycorrhizal symbiosis. Such taxa could be capable of facul- species-specific genes, i.e., they are restricted to a single tative saprotrophy, i.e., SOM decomposition. As suggested for mycorrhizal species. Among all symbiosis-upregulated genes, ericoid mycorrhizal fungi, which exhibit a large PCWDE reper- 8–28% encode candidate secreted effector-like MiSSPs. Their toire15, a dual saprotrophic/mutualistic habit may provide greater functional analysis is hampered by the the lack of high- ecological flexibility and fitness under specific environmental throughput genetic engineering techniques for ectomycorrhizal conditions. Moreover, in such lineages, evolutionary reversal to fungi, but some of these MiSSPs have been functionally char- saprotrophy might be possible (although that has not been acterized, including LbMiSSP7, LbMiSSP8, LbMiSSP7.6, and demonstrated). We showed that the PCWDEs of A. macro- PaMiSSP10 (refs. 44,46–48). The evolution of innovations, such as sclerotium are downregulated at the transcriptional level during the control of the host plant immunity, development, and the symbiotic interaction to avoid triggering plant defense reac- metabolism by these lineage-specific symbiotic effectors was tions and/or digesting host root tissues. This suggests that loss of likely a necessary step to colonized the new niches represented genes encoding PCWDEs is a consequence of, but not a by tree roots. Some of the effector-like SSPs, but also structural requirement for, the evolution of ectomycorrhizal mutualisms. SSPs like hydrophobins, may have evolved from SSPs used by What remains to be determined is why ectomycorrhizal fungi do the saprotrophic ancestors to communicate or compete in the not retain their ancestral dual lifestyle, and how the ectomycor- soilandwoodenvironments,46,49. rhizal associations become so intimate that saprotrophic cap- Novel and recently evolved genes, including MiSSPs, are abilities are irretrievably lost. The lack of invertase and sucrose undoubtedly responsible for the particular attributes of individual transporter genes in most ectomycorrhizal fungi means that they mycorrhizal lineages. Indeed, arrays of ectomycorrhiza-induced are unable to use apoplastic sucrose released by their host plant. genes are unique to each specific clade with between 14 and 39% They fully rely on their partner for their glucose supply, a of symbiosis-upregulated genes being species-specific genes with mechanism reinforcing their dependence on the plant partner. no known function12 (present study). At the same time, our Our results, and those of prior studies3,14, suggest that the phylostratigraphic analysis showed that a large proportion of predominant mechanism for the transition from saprotrophy to genes that are upregulated in ectomycorrhizas arose long before ectomycorrhizal symbiosis appears to involve restricted secretion the evolution of the mutualistic associations. In other words, of hydrolytic enzymes acting on plant cell walls (whether via numerous genes used by saprotrophic ancestors were co-opted regulatory shifts or gene loss), which enables the symbiont to be for the symbiotic lifestyle. Important ecological traits, such as N accommodated in roots. An example of an ultimate adaptation to and P acquisition traits (e.g., organic N- and P-degrading secreted symbiosis is illustrated by the arbuscular mycorrhizal fungi that enzymes) were already present in free-living saprotrophic are obligate symbionts. They display the lowest repertoire of ancestors of ectomycorrhizal symbionts. Despite the well- PCWDEs of the current set of sequenced mycorrhizal fungi. documented ability of ectomycorrhizal fungi to hydrolyze Unfortunately, no genomic data exist yet to support the origin of organic phosphate compounds and scavenge nitrogen through arbuscular mycorrhizal fungi from saprotrophic CAZyme-rich the degradation of litter proteins accumulating in soil litter, we Mucoromycotina. In contrast, ericoid mycorrhizal fungi and found that ectomycorrhizal genomes generally contain a similar orchid mycorrhizal fungi have the higher set of PCWDEs sup- number or less copies of genes coding for secreted N- and P- porting their dual saprotrophic/symbiotic lifestyles3,15. We thus targeting hydrolases than saprotrophs, pathogens, or ericoid propose that the PCWDE repertoire is reflective of the age of the mycorrhizal fungi. symbiosis along the saprotrophy to symbiosis continuum with the One of the most striking genomic features of mycorrhizal fungi arbuscular mycorrhizal symbiosis emerging in the early Devonian is their high content in TEs. A large proportion of the genomes of (393–419 Mya), the ectomycorrhizal symbiosis during the Jurassic mycorrhizal fungi can be ascribed to past transposition, providing (ca. 200 Mya) and mycorrhizal associations with Ericaceae species a major contribution to their genome landscape. It has been to the Cretaceous (ca. 117 Mya)4. proposed that the lack of sexual recombination (i.e., asexual

12 NATURE COMMUNICATIONS | (2020) 11:5125 | https://doi.org/10.1038/s41467-020-18795-w | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18795-w ARTICLE reproduction) can favor the uncontrolled proliferation of TEs47. were inferred using FastTree 2.1.10 (refs. 69,70) under the Whelan and Goldman It may be true for arbuscular mycorrhizal fungi with no known (WAG) model of protein evolution with gamma-distributed rate heterogeneity. We sexual reproduction, but this hypothesis cannot stand for the excluded gene trees with deep paralogs to identify single-copy gene. Gene trees of not strictly single-copy gene families were checked if the duplications are deep in ectomycorrhizal fungi investigated in the present study as they the tree, or species specific. In those cases where the duplications were species are known to produce ascocarps or basidiocarps. In biotrophic specific, the gene was marked as suitable for phylogenetic inference and the protein pathogens, such as Blumeria graminis f. sp. hordei and B. gra- that was closest to the root (in terms of patristic distance) for each species was minis f. sp. tritici, it has been proposed that burst(s) of TEs offer a chosen for inclusion in the phylogenetic analyses. Single-copy genes were realigned using PRANK v. 150803 (ref. 67) with the template for rapid evolution of virulence genes by duplications, default settings and one round of alignment improvement. Poorly aligned regions 48 small-scale rearrangements, or deletions . It is tempting to were removed using trim-Al v1.2 (ref. 68) with the -strict setting. The alignments speculate that the observed TE proliferation(s) play a key role in were concatenated into a supermatrix using a custom Perl script, excluding single promoting the rapid evolution of symbiosis-related factors, such alignments that were <50 amino acids long. We included genes in the supermatrix only if they were present in >40 and >50 Ascomycota and Basidiomycota species, as MiSSPs. respectively. ML phylogenies were inferred using RAxML 8.2.4 (ref. 71) with 100 Collectively, our results suggest that each independently bootstrap replicates and a partitioned model, where each gene was treated as a evolved ectomycorrhizal lineage uses species-specific novel genes, separate partition. The PROTGAMMAWAG model was used for each partition. as well as members of ancestral gene families to develop sym- The resulting phylogenies were used in the molecular dating analyses. In addition, we constructed an organismal phylogeny restricted to the 62 biotic interactions, indicating that divergent genetic innovations mycorrhizal fungi. Orthologous genes among the fungi were identified using underlie the convergent origins of the ectomycorrhizal guild. FastOrtho with the parameters set to 50% identity, 50% coverage, and inflation 3.0 Thus, the functional heterogeneity in symbiosis-related gene (ref. 72). Protein sequences were downloaded from MycoCosm (mycocosm.jgi.doe. networks in ectomycorrhizal fungi is not only found among gov). Clusters with single-copy genes were identified and aligned with MAFFT 73 species-specific genes as evidenced for MiSSPs3,14,43–45,50,51, but 7.221 (ref. ), ambiguous regions (containing gaps and poorly aligned) were eliminated, and single-gene alignments were concatenated with Gblocks 0.91b also in conserved genes (e.g., hydrophobins) that predate the (ref. 74). A phylogenetic tree was constructed with RAxML 7.7.2 (ref. 75), the gains of mycorrhizal symbiosis. standard algorithm, the PROTGAMMAWAG model of sequence evolution, and The large number of independently evolved ectomycorrhizal 1000 bootstrap replicates. lineages raises questions as to why the emergence of this guild is so common in nature. Furthermore, the absence of known reversals to Molecular dating. In order to time calibrate the phylogenies, we used the pena- the saprotrophic lifestyle suggests that genetic traits underlying the lized likelihood algorithm as implemented in r8s (ref. 76) with the POWELL transitions to ectomycorrhizal lifestyles represent fixed evolutionary optimization. To identify the appropriate smoothing parameters, we performed a transitions. Future studies should focus on conditions that may cross-validation analysis for each dataset. For the Basidiomycota phylogeny, the 77 predispose some groups to evolve ectomycorrhizal symbioses, or fossil Archaeomarasmius legetti from the mid-Cretaceous (90-94 Ma) was used to calibrate the node containing the suborder Marasmiineae, and the fossil of a other mechanisms that increase the likelihood of convergent evo- 78 – suilloid ectomycorrhiza from the middle Eocene (50 Mya) was used to calibrate lution52 55. The present study roughly doubles the number of the node containing the suborders Boletineae, Paxillineae, Sclerodermatineae, and published ectomycorrhizal genomes. Even greater sampling of key Suillineae. For the Ascomycota phylogeny, the fossil Paleopyrenomycites devonicus lineages, particularly early divergent clades, is needed to elucidate from the early Devonian (~400 Ma)79 was used to calibrate the split between Pezizomycotina and Saccharomycotina. The Basidiomycota supermatrix consisted the macro- and microevolutionary mechanisms that were respon- of 940,059 sites from 1042 genes. All nodes were recovered with a bootstrap sible for the diversification of ectomycorrhizal fungi. support of 96% or higher. The Ascomycota supermatrix consisted of 1,474,883 sites from 1432 genes. All nodes were recovered with bootstrap support of 100%. Methods Strains and fungal material used for genome sequencing. Fungal strains used Analysis of PCWDE evolution. A comprehensive list of CAZyme annotations can for genome sequencing are described in Supplementary Data 1. Assignment of be found in Supplementary Data 11 and at the Mycorrhizal Fungi page at the JGI individual taxa to functional guilds was based on common categorizations in the MycoCosm database by following this link: https://mycocosm.jgi.doe.gov/ literature34,56,57. DNA was extracted with a modified cetyltrimethylammonium = 12 mycocosm/annotations/browser/cazy/summary;-6jBQE?p Mycorrhizal_fungi. bromide protocol . We investigated the evolution of gene families encoding CAZymes with a particular interest in PCWDEs, which include enzymes active on cellulose (various Genome sequencing, assembly, and annotation. All sequencing, assembly, and GH families, AA3_1, LPMO, and CBM1), lignin (AA1_1, AA1_3, AA2_class II annotation was performed at JGI. Genome sequencing was done with either Illu- POD, AA5_1, DyP, HTP, and OXO), and pectin (CE8, PL, and GH families; see fi mina or Pacific Biosciences (PacBio) technology, or both (“hybrid”). Genomes were also Supplementary Data 8). We rst performed all-vs-all Blast using mpiBlast 1.6.0 80 assembled from Illumina reads using ALLPATHS-LG58, followed by patching (ref. ) for 51 Ascomycota and 61 Basidiomycota proteomes, and clustered fi using PacBio with PBJelly59 in the case of hybrid assemblies. All other genomes proteins into gene families using the Hi x 1.0.5 clustering method, with default fi were assembled from PacBio reads using Falcon60 or Celera Assembler61. Mito- parameters resulting in 243,784 and 408,904 clusters, respectively. Identi cation of chondrial genomes were assembled separately. All transcriptome sequencing was PCWDE families was performed based on InterPro (IPR) domains in the done with Illumina only, and subsequently assembled into putative transcripts similarity-based clustering (see above). IPR data from the JGI website were used to using either Rnnotator11 or Trinity62. Each genome was annotated using the JGI annotate proteins with IPR domains for all species. We considered clusters as Annotation Pipeline63,64, aided by the transcriptome when available. Detailed PCWDE families if at least 50% of the proteins had the appropriate IPR domain. fi + methods are described in the Supplementary Methods. Further classi cation of PCWDE families was supported by BLASTP 2.6.0 search80, and validation by the CAZy annotation pipeline81. Multiple sequence alignments were inferred using PRANK 140,603 with default settings, and trim-Al Organismal phylogeny. We assembled two datasets, one of Ascomycota that 1.4.rev15 was used for removing ambiguously aligned regions (with the argument included 51 genomes and one of Basidiomycota that included 62 genomes. We -gt 0.1). ML gene trees of the identified PCWDE clusters were inferred using opted for analyzing Ascomycota and Basidiomycota genomes separately, and only FastTree 2.1.10 under the WAG model of protein evolution with gamma- fungal genomes to improve computational efficiency and because the origins of the distributed rate heterogeneity. TreeFix 1.1.10 (ref. 82) was used for gene tree/species ectomycorrhizal symbioses are shallow evolutionary events, so we reasoned that tree reconciliation (-m PROTGAMMAWAG, –alpha 0.001, –niter 300) with the additional resolution cannot be added by including non-fungal outgroup taxa. The genome-based ML species trees. Gene clusters containing less than four proteins computational step of similarity-based clustering, multiple sequence alignments, were excluded from the analysis. For the reconstruction of gene duplication and and tree inference are particularly sensitive to the phylogenetic breadth that the loss history of PCWDEs, we used the COMPARE pipeline54. Orthogroups were species cover. Analyzing Ascomycota and Basidiomycota separately improved the identified, and duplications and losses were inferred for each enzyme group across accuracy of our predictions, by not having to analyze as deep nodes of the tree as species trees based on Dollo parsimony. The ancestral gene copy numbers for every the Dikarya (MRCA of Ascomycota and Basidiomycota). We performed all-vs-all internal node were calculated by summing the mapped gains and losses over the Blast with mpiBlast 1.6.0 (ref. 65) and clustered proteins into gene families by Hifix species tree. 1.0.5 (ref. 66), using default parameters. Clusters having 0.5n–2n proteins Class I and class II PODs were also curated by the RedoxiBase database (http:// (n = number of species) were aligned by PRANK140603 (ref. 67) with default peroxibase.toulouse.inra.fr) to distinguish between LiP, versatile PODs, MnP, or settings, and trimmed with trim-Al 1.4.rev15 (ref. 68) to remove ambiguously atypical Class II PODs. The latter PODs have been annotated as basidiomycete aligned regions (with the argument -gt 0.1). Maximum likelihood (ML) gene trees sub-class B (CIIBB) or sub-class C (CIIBC)83.

NATURE COMMUNICATIONS | (2020) 11:5125 | https://doi.org/10.1038/s41467-020-18795-w | www.nature.com/naturecommunications 13 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18795-w

Phylostratigraphy. Phylogenetic ages of ectomycorrhiza-induced genes were Code availability assigned based on the most phylogenetically distant orthologous gene in the PRINGO R scripts are deposited at GitHub: https://github.com/ShingoMiyauchi/ containing gene family, using a phylostratigraphic approach84. Ectomycorrhiza- PRINGO. The program COMPARE is also available at Github: https://github.com/ induced transcripts were retrieved from RNA-seq-based transcriptome profil- laszlognagy/COMPARE. ings: Amanita muscaria, GSE63867; Cenococcum geophilum, GSE83909; Hebe- loma cylindrosporum, GSE63868; P. involutus, GSE63924; Piloderma croceum, GSE63925; T. magnatum, GSE116692; A. macrosclerotiorum, SRP130276 and Received: 22 January 2020; Accepted: 16 September 2020; SRP130279-82; L. bicolor, SRP164436-38, SRP164526, SRP164559, and SRP164564; Pisolithus microcarpus, SRP122806, SRP122812, SRP122818, SRP122826, SRP122829, and SRP122850; and T. matsutake, SRP103258). Details of the RNA-seq experiments are described on Gene Expression Omnibus (GEO), NCBI. Note that this pooled set of ectomycorrhiza-induced genes represent a heterogeneous set of symbiosis-induced transcripts from ectomy- References corrhizal roots from different fungal-tree associations sampled at different 1. Van der, Heijden, M. G. A., Martin, F. M., Selosse, M.-A. & Sanders, I. R. stages of development, from early to late stage of symbiosis establishment. We Mycorrhizal ecology and evolution: the past, the present, and the future. New selected those having an expression fold change (FC) > 5 and FC > 2 (Supple- Phytol. 205, 1406–1423 (2015). mentary Data 9). The phylogenetic trees of the Asco- and Basidiomycota were 2. Genre, A., Lanfranco, L., Perotto, S. & Bonfante, P. Unique and common traits divided into phylostrata, based on the phylogenetic distance in terms of internal in mycorrhizal symbioses. Nat. Rev. Microbiol. https://doi.org/10.1038/ nodes from the species being examined. Each phylostratum corresponded to an s41579-020-0402-3 (2020). ancestral species; genes were assigned to phylostrata based on the species dis- 3. Martin, F., Kohler, A., Murat, C., Veneault-Fourrey, C. & Hibbett, D. S. tribution of orthogroups the studied genesbelongto.Usingthismethod,the Unearthing the roots of ectomycorrhizal symbioses. Nat. Rev. Microbiol. 14, ages of upregulated genes were defined and mapped to species trees. We 760–773 (2016). thoroughly selected a subset of the sequenced genomes of species belonging to ‐ either the Ascomycota or the Basidiomycota. Running COMPARE on the 4. Strullu Derrien, C., Selosse, M.-A., Kenrick, P. & Martin, F. M. 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completeness with single-copy orthologs. Bioinformatics 31, 3210–3212 Author contributions (2015). F.M.M. conceived and coordinates the Mycorrhizal Genomics Initiative. F.M.M., L.G.N., 86. Konietschke, F. Simultane Konﬁdenzintervalle fuer nichtparametrische D.H., and I.V.G. designed the project. I.V.G. coordinated genome sequencing and relative Kontrasteffekte. PhD thesis, University of Goettingen (2009). annotation at JGI A.Kuo, B.A., K.W.B., A.Carver, A.C.l., C.C., A.L., K.L., M.N., R.A.O., 87. Pinheiro, J., Bates, D., DebRoy, S., Sarkar, D. & R Core Team. nlme: Linear R.D.H., R.R., J.P., and M.Y. performed genome and transcriptome sequencing, assembly and Nonlinear Mixed Effects Models. R package version 3.1–137 https:// and annotation at J.G.I. E.D., V.L., and B.H. performed CAZyme annotations. S.M., F.M., CRAN.R-project.org/package=nlme (2018). F.M.M., and M.B. analyzed CAZyme distribution. E.K., A.Kohler, L.G.N., and F.M.M. 88. Auguie, B. egg: https://cran.r-project.org/web/packages/egg/index.html (2017). performed analyses of PCWDE evolution, phylostratigraphic studies and analyses of 89. Yu, G., Smith, D. K., Zhu, H., Guan, Y. & Lam, T. T.-Y. ggtree: an R package convergence. C.D. annotated peroxidases. S.M. performed comparative genome analyses for visualization and annotation of phylogenetic trees with their covariates and with the help of E.M. and C.M., and developed scripts for the visual display of data. other associated data. Methods Ecol. Evol. 8,28–36 (2017). M.S.G. and D.H. constructed dated trees. M.F.P., M.P., S.P., and L.M.V. performed 90. Wickham, H. ggplot2: Elegant Graphics for Data Analysis (Springer, New mycorrhization experiments for RNA-Seq. A.Kohler and E.M. analyzed transcriptome York, 2009). data. Z.K. and G.S. performed clustering analyses using HiFix and Silix. A.Y., B.S., C.M., 91. Kassambara, A. ggpubr R package: ggplot2-based publication ready plots.R D.C., F.M., L.F., G.B., J.B.S., J.M., J.W.S., J.X., L.T., L.Z., M.B., M.G., M.P., M.S., M.Y., N.C., package version 0.2.5.999 https://rpkgs.datanovia.com/ggpubr/ (2020). P.B., P.W., P.W.C., R.V., S.P, T.B. and Y.S. provided biological material, DNA and RNA 92. Paradis, E. & Schliep, K. ape 5.0: an environment for modern phylogenetics for sequencing or genomes. F.M.M., and S.M. wrote the manuscript with the help of D.H. and evolutionary analyses in R. Bioinformatics 35, 526–528 (2018). and L.G.N. 93. Mensy, F. Detecting the effect of biological categories on genome composition. https://github.com/fantin-mesny/Effect-Of-Biological-Categories-On- Competing interests Genomes-Composition (2020). 94. Oksanen, J. et al. Vegan: community ecology package. R package version 2.5–6 The authors declare no competing interests. https://CRAN.R-project.org/package=vegan (2019). 95. Hervé, M. RVAideMemoire: testing and plotting procedures for biostatistics. R Additional information package version 0.9-75 https://CRAN.R-project.org/ Supplementary information is available for this paper at https://doi.org/10.1038/s41467- package=RVAideMemoire (2020). 020-18795-w. 96. Castanera, R. et al. Transposable elements versus the fungal genome: Impact on whole-genome architecture and transcriptional proﬁles. PLoS Genet. 12, Correspondence and requests for materials should be addressed to F.M.M. e1006108 (2016). 97. Ploner, A. Heatplus: Heatmaps with row and/or column covariates and Peer review information Nature Communications thanks the anonymous reviewers for colored clusters. R package version 2.34.0, https://github.com/alexploner/ their contribution to the peer review of this work. Peer reviewer reports are available. Heatplus (2020). Reprints and permission information is available at http://www.nature.com/reprints

’ Acknowledgements Publisher s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. The project was funded by the U.S. Department of Energy (DOE) Joint Genome Insti- tute, a DOE Office of Science User Facility, and supported by the Office of Science of the U.S. DOE under Contract No. DE-AC02-05CH11231 within the framework of the Mycorrhizal Genomics Initiative (CSP # 305), Metatranscriptomics of Forest Soil Eco- Open Access This article is licensed under a Creative Commons systems project (CSP # 570), and the 1000 Fungal Genome projects (CSP # 662 and Attribution 4.0 International License, which permits use, sharing, 1974). This research was also supported by the Laboratory of Excellence ARBRE (ANR- adaptation, distribution and reproduction in any medium or format, as long as you give 11-LABX-0002-01), the Region Lorraine, the European Regional Development Fund, and appropriate credit to the original author(s) and the source, provide a link to the Creative the Plant–Microbe Interfaces Scientific Focus Area in the Genomic Science Program, the Commons license, and indicate if changes were made. The images or other third party Office of Biological and Environmental Research in the US DOE Office of Science. L.G.N. material in this article are included in the article’s Creative Commons license, unless is funded by the Momentum Program of the Hungarian Academy of Sciences (Grant No. indicated otherwise in a credit line to the material. If material is not included in the LP2019/13-2019) and the National Research, Development and Innovation office article’s Creative Commons license and your intended use is not permitted by statutory (Contract No: GINOP-2.3.2-15-2016-00052). G.S. received funding from the European regulation or exceeds the permitted use, you will need to obtain permission directly from Research Council under the European Union’s Horizon 2020 research and innovation the copyright holder. To view a copy of this license, visit http://creativecommons.org/ programme under grant agreement no. 714774 and the grant GINOP-2.3.2.-15-2016- licenses/by/4.0/. 00057. We also thank the many colleagues whom provided fungal strains and material, including Jan Colpaert, Pierre-Emmanuel Courty, Joanna F. Dames, Patricia Jargeat, Björn Lindahl, Asuncion Morte, Jose-Eduardo Marque-Galvez, Andy Taylor, and © The Author(s) 2020 Yukari Kuga.

Shingo Miyauchi 1,25, Enikő Kiss2,25, Alan Kuo3,25, Elodie Drula4, Annegret Kohler1, Marisol Sánchez-García5, Emmanuelle Morin1, Bill Andreopoulos3, Kerrie W. Barry3, Gregory Bonito6, Marc Buée1, Akiko Carver3, Cindy Chen3, Nicolas Cichocki1, Alicia Clum3, David Culley7, Pedro W. Crous8, Laure Fauchery 1, Mariangela Girlanda9, Richard D. Hayes 3, Zsóﬁa Kéri2, Kurt LaButti 3, Anna Lipzen3, Vincent Lombard4, Jon Magnuson 7, François Maillard1, Claude Murat 1, Matt Nolan3, Robin A. Ohm3, Jasmyn Pangilinan3, Maíra de Freitas Pereira1, Silvia Perotto 9, Martina Peter10, Stephanie Pﬁster10, Robert Riley3, Yaron Sitrit11, J. Benjamin Stielow8, Gergely Szöllősi 2, Lucia Žifčáková12, Martina Štursová12, Joseph W. Spatafora13, Leho Tedersoo14, Lu-Min Vaario15, Akiyoshi Yamada16, Mi Yan3, Pengfei Wang17, Jianping Xu 18, Tom Bruns19,

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Petr Baldrian 12, Rytas Vilgalys 20, Christophe Dunand21, Bernard Henrissat 4,22,23, ✉ Igor V. Grigoriev 3,19,26, David Hibbett5,26, László G. Nagy2,26 & Francis M. Martin1,24,26

1Université de Lorraine, Institut national de recherche pour l’agriculture, l’alimentation et l’ environnement, UMR Interactions Arbres/ Microorganismes, Centre INRAE Grand Est-Nancy, 54280 Champenoux, France. 2Synthetic and Systems Biology Unit, Biological Research Centre, 6726 Szeged, Hungary. 3US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. 4INRAE, USC1408 Architecture et Fonction des Macromoleculeś Biologiques, 13009 Marseille, France. 5Biology Department, Clark University, Lasry Center for Bioscience, 950 Main Street, Worcester, MA 01610, USA. 6Plant Soil and Microbial Sciences, Michigan State University, East Lansing, MI 48824, USA. 7Chemical & Biological Processes Development Group, Paciﬁc Northwest National Laboratory, Richland, WA, USA. 8Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584 CT Utrecht, Netherlands. 9Department of Life Sciences and Systems Biology, University of Torino, Viale Mattioli 25, 10125 Torino, Italy. 10Swiss Federal Institute for Forest, Snow and Landscape Research WSL, Zuercherstrasse 111, 8903 Birmensdorf, Switzerland. 11The Jacob Blaustein Institutes for Desert Research, Bergman Campus, Ben-Gurion University of The Negev, Beer-Sheva, Israel. 12Laboratory of Environmental Microbiology, Institute of Microbiology of the Czech Academy of Sciences, Videnska 1083, 14220 Praha 4, Czech Republic. 13Department Botany & Plant Pathology, Oregon State University, Corvallis, OR, USA. 14Natural History Museum, University of Tartu, 14a Ravila, 50411 Tartu, Estonia. 15Department of Forest Sciences, University of Helsinki, Helsinki, Finland. 16Institute of Mountain Science, Faculty of Agriculture, Shinshu University, Minami-minowa, Kami-ina, Nagano 399-4598, Japan. 17Department of Key Laboratory, The 2nd Afﬁliated Hospital of Kunming Medical University, 374 Dian Mian Road, Kunming 650101 Yunnan, China. 18Department of Biology, McMaster University, 1280 Main St. West, Hamilton, ON L8S 4K1, Canada. 19Department of Plant and Microbial Biology, University of California – Berkeley, Berkeley, CA, USA. 20Department of Biology, Duke University, Durham, NC 27708, USA. 21Laboratoire de Recherche en Sciences Végétales, Université de Toulouse, CNRS, UPS, Toulouse, France. 22Architecture et Fonction des Macromoleculeś Biologiques (AFMB), CNRS, Aix-Marseille Univ., 13009 Marseille, France. 23Department of Biological Sciences, King Abdulaziz University, Jeddah, Saudi Arabia. 24Beijing Advanced Innovation Centre for Tree Breeding by Molecular Design (BAIC-TBMD), Institute of Microbiology, Beijing Forestry University, Tsinghua East Road Haidian District, Beijing, China. 25These authors contributed equally: Shingo Miyauchi, Enikő Kiss, Alan Kuo. 26These authors jointly supervised this ✉ work: Igor V. Grigoriev, David Hibbett, László G. Nagy, Francis M. Martin. email: [email protected]

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