Large-scale genome sequencing of mycorrhizal fungi provides insights into the early evolution of symbiotic traits - DORA 4RI
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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.#
1234567890():,;
Mycorrhizal fungi are mutualists that play crucial roles in nutrient acquisition in terrestrial
ecosystems. Mycorrhizal symbioses arose repeatedly across multiple lineages of Mucor-
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 ana-
lyses 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 sapro-
trophic ancestors to fulfill new symbiotic functions, (3) diversification of novel, lineage-
specific symbiosis-induced genes, (4) proliferation of transposable elements and (5) diver-
gent 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
M
ycorrhizal fungi are central to the evolution, biology, additional symbiosis-related functional traits, such as nitrogen
and 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 Data 1). This set includes new genomes from the early diverging
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
NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18795-w ARTICLE
Genome (Mb) TE content (%) Genes (K) Secreted (K) Scaffolds (K) L50 (Mb) BUSCO (%)
0 200 400 0 20 40 60 80 0 10 20 30 40 50 0 1 2 3 0 10 20 30 0 1 2 3 4 75 80 85 90 95 100
Laccaria amethystina
Laccaria bicolor
Coprinopsis cinerea
Coprinellus micaceus
Hypholoma sublateritium
Hebeloma cylindrosporum
Agaricales Cortinarius glaucopus
Crucibulum laeve
Tricholoma matsutake
Amanita thiersii
Amanita rubescens
Amanita muscaria
Pluteus cervinus
Gymnopus androsaceus
Omphalotus olearius
Marasmius fiardii
Moniliophthora perniciosa
Mycena galopus
Armillaria gallica
Pleurotus ostreatus
Schizophyllum commune
Paxillus ammoniavirescens
Paxillus adelphus
Paxillus involutus
Gyrodon lividus
Xerocomus badius
Boletus edulis
Boletales
Boletus edulis P
Pisolithus microcarpus
Pisolithus tinctorius
Scleroderma citrinum
Suillus luteus
Suillus brevipes
Basidiomycota
Coniophora puteana
Serpula lacrymans
Rhizopogon vesiculosus
Rhizopogon vinicolor
Melanogaster broomeianus
Fibulorhizoctonia sp.
Piloderma croceum
Plicaturopsis crispa
Phanerochaete chrysosporium
Phlebia brevispora
Postia placenta
Neolentinus lepideus
Jaapia argillacea
Russulales
Punctularia strigosozonata
Sistotremastrum niveocremeum
Heterobasidion annosum
Lactarius quietus
Lentinellus vulpinus
Russula emetica
Russula ochroleuca
Thelephora terrestris
Thelephora ganbajun
Ramaria rubella
Gautieria morchelliformis
Hysterangium stoloniferum
Sphaerobolus stellatus
Auricularia subglabra
Piriformospora indica
Cantharellales
Sebacina vermifera
Hydnum rufescens
Cantharellus anzutake
Sistotrema sp.
Botryobasidium botryosum
Tulasnella calospora
Ceratobasidium sp.
Rhizoctonia solani
Tremella mesenterica
Dacryopinax primogenitus
Wallemia sebi
Chalara longipes
Meliniomyces variabilis
Leotiomycetes
Meliniomyces bicolor
Rhizoscyphus ericae
Acephala macrosclerotiorum
Phialocephala scopiformis
Ascocoryne sarcoides
Glarea lozoyensis
Amorphotheca resinae
Oidiodendron maius
Bisporella sp.
Sclerotinia sclerotiorum
Botrytis cinerea
Magnaporthe grisea
Neurospora crassa
Trichoderma reesei
Dothideomycetes
Xylaria hypoxylon
Cenococcum geophilum
Glonium stellatum
Lepidopterella palustris
Stagonospora sp.
Alternaria alternata
Ascomycota
Aureobasidium pullulans
Cladosporium fulvum
Aspergillus clavatus
Aspergillus flavus
Arthroderma benhamiae
Usnea florida
Xylona heveae
Tuber magnatum
Tuber aestivum
Tuber melanosporum
Pezizomycetes
Tuber borchii
Choiromyces venosus
Morchella importuna.C
Morchella importuna
Gyromitra esculenta
Caloscypha fulgens
Wilcoxina mikolae
Trichophaea hybrida
Pyronema confluens
Sarcoscypha coccinea
Kalaharituber pfeilii
Terfezia boudieri
Terfezia claveryi
Tirmania nivea
Ascobolus immersus
Monacrosporium haptotylum
Arthrobotrys oligospora
Saccharomyces cerevisiae
Wickerhamomyces anomalus
Candida tanzawaensis
Taphrina deformans
Gigaspora rosea
Mucoromycota
Rhizophagus cerebriforme
Glomerales
Rhizophagus diaphanus
Rhizophagus irregularis
Rhizophagus irregularis DAOM
Rhizophagus irregularis A1
Rhizophagus irregularis A4
Rhizophagus irregularis A5
Rhizophagus irregularis B3
Rhizophagus irregularis C2
Ectomycorrhiza Ericoid mycorrhiza Orchid mycorrhiza Arbuscular mycorrhiza
Saprotroph Pathogen Parasite Endophyte Wood decayer Yeast
Fig. 1 General features of the 135 fungal genomes analyzed. Genome and assembly features. The different lifestyles (e.g., ectomycorrhiza) are color
coded, see bottom panel. The dotted lines show median values. Genome (Mb): genome size in Mb, TE content (%): coverage (%) of transposable
elements (TE) in assemblies, Genes (K): number of predicted genes, Secreted (K): number of predicted secreted proteins, Scaffolds (K): number of
scaffolds, L50 (Mb): N50 length, BUSCO (%): genome completeness in % based on BUSCO survey. Source data Fig. 1.
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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
Pezizomycotina at 326 Mya (Fig. 4b), while Floudas et al.26 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-
were identified 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
in gene content. Species-specific 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
and 606,378 taxon-specific genes in Ascomycota and Basidiomy- within the different protein categories were consistent in both
cota, respectively.The large number of species-specific 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/naturecommunicationsNATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18795-w ARTICLE
LTR Non-LTR En DNA
M Sp G
SI Ha ar m in
Pe No in Z / K A ge C pi P U
G n
DI y Co L Ta elo RT n −L
N E3 C in r b H el r/ D isu CAC olo IS3 cad Mu D r2/T rypt ggy olin M nkn
e
p
RS s pi TR d p EX L TR h i t T a p
/5 R ge A ro c d to T o E em D N b DD onF Ba to erli ow Total
Coverage (%) y a 1 e 1 I S E r T n 1 a n A k U R A P c n n n 0 20 40 60
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 10.1 1.6 0.4 0 0.3 0.6 0.4 0.2 1.2 0.6 0.1 24.8
Coprinopsis cinerea 0 3.4 0.6 0 1.3
Coprinellus micaceus 0.1 2.4 2 2.1 0.1 0 0 0.2 0.2 0.1 0.1 0.2 7.4
Hypholoma sublateritium 0.2 0.5 0.2 0 0.3 4.2
Hebeloma cylindrosporum 0.4 0.1 2.5
Agaricales
Cortinarius glaucopus 0.5 7 1.8 1 0.1 0.6 0.4 0.1 0.2 0.1 0.1 28.5
Crucibulum laeve 3.1 0.6 0.1 0.2 0.3 9.3
Tricholoma matsutake 0.1 34.4 5.7 3.3 0 0 0 0 0.3 0.2 0.9 0.3 0 0.1 0.6 0 0 33.8
Amanita thiersii 16.9 5.3 2.5 9.1
Amanita rubescens 0.1 5.6 4.3 0.6 0 0.2 0.9 1.1 0.5 0.1 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 0.1 0.1 0 0 0 0.3 0.1 0.2 23.8
Omphalotus olearius 0.2 0.8
Marasmius fiardii 0.1 9.5 1.5 3.5 11.8
Moniliophthora perniciosa 1.6 1.4
Mycena galopus 0.2 7.3 1.1 0 0.4 0 0.4 0.6 0.2 0.1 0.3 0.4 0.3 0 0.2 0.3 0 0 0 0 33.6
Armillaria gallica 0.1 7.5 0.2 0 0 0 0.3 0 17.4
Pleurotus ostreatus 3.5 0.5 0 0.1 0.2 0.3 0.2 3.9
Schizophyllum commune 1.4 1 0 0.2 2.9
Paxillus ammoniavirescens 0 3.8 2.2 0 0.1 0.1 0.2 0.3 0 11.1
Paxillus adelphus 1.7 7.2 9.7 0 0 1.1 0.2 2 2.3 4.7 0.4 0 0 0 0 33.6
Paxillus involutus 0.2 3.6 1.7 0 0.2 0.2 0.2 0.2 0.6 0.1 19.7
Gyrodon lividus 0 23.4 4.3 0.1 0.1 0.1 0.1 0.9 0 1.2 19.5
Xerocomus badius 2.3 2.5 0 0.1 11.2
Boletus edulis
Boletales
0.2 9.4 4.7 0.1 0.1 0.2 0.2 0.7 0.1 0.3 0.8 0.2 25.7
Boletus edulis P 0.1 8.1 5.6 0.1 0.2 0 0 0.2 0.6 0.1 0.2 1.6 0.1 25.7
Pisolithus microcarpus 8.1 3.8 1.2 1.2 0.6 0.7 1 0.4 0.1 23.5
Pisolithus tinctorius 0.4 20.4 2.1 0.8 0 0.9 1.1 0.9 0.6 0.4 0.1 24.9
Scleroderma citrinum 0.1 12.3 4.5 0.1 0.4 0.3 0.2 0 0.2 0.5 0.1 23.9
Basidiomycota
Suillus luteus 0 2.7 1.4 0.2 0.1 0.4 0.1 0.1 2.1 0.1 0.6 17.2
Suillus brevipes 0 1.9 1.2 0.2 0.1 0 0.1 0.1 0 0.1 0.1 0.2 0 17.2
Coniophora puteana 0.2 0.8 0 3
Serpula lacrymans 12.9 3.9 0.2 0.4 0.4 0.4 0 0 0 9.6
Rhizopogon vesiculosus 3 0.3 0.2 0 0.1 0 0.2 0 0 21
Rhizopogon vinicolor 1.2 0.2 0.1 0.1 0.1 0 14.8
Melanogaster broomeianus 0.2 6 1.9 0 0.1 0.2 0.6 0.1 0 27.5
Fibulorhizoctonia sp. 0 2.4 0.7 0.8 0 0 0 0 0 0 0 27.6
Piloderma croceum 0 2.4 0.2 0 0 0 0 0.1 0.7 0.2 0 13.2
Plicaturopsis crispa 0.1 0.5 0 0 4.3
Phanerochaete chrysosporium 0.1 3.9 0.8 0 0 0.1 0.1 0.1 2
Phlebia brevispora 2.2 1.6 0 0.1 0 0.1 0.1 0 3.6
Postia placenta 12.1 0.3 0 0 4.8
Neolentinus lepideus 0 3.1 2.5 0 0.1 0 0.1 6.6
Jaapia argillacea 3.6 0.1 0.1 0 0.1 0.1 6.1
Russulales
Punctularia strigosozonata 0.1 1.7 0.4 3
Sistotremastrum niveocremeum 0.1 0.1 0 3.4
Heterobasidion annosum 8.7 0.7 0.1 0.4 0.2 0 9.9
Lactarius quietus 0.2 35 4.2 1.5 0 0 0.1 0.1 4.4 0.1 1.7 1.3 1.9 0.1 0.2 0 0.1 43.8
Lentinellus vulpinus 3 0.8 0 0 3.5
Russula emetica 0.2 9.2 1.2 1 0.2 0.3 6.8 0.6 0.3 0 0.1 25.4
Russula ochroleuca 20 1.3 0 0.1 0 0 0.1 0 21.9
Thelephora terrestris 0 6.8 1.6 0.1 0 0 0 0.1 0.5 23.8
Thelephora ganbajun 0.3 0.4 0.2 0 0 27
Ramaria rubella 0.2 27.5 2.9 0.1 0.9 0 1.3 0.6 0.1 0.3 0 0.2 0.4 36.6
Gautieria morchelliformis 0.3 28.7 11.4 0.1 4.6 0 0.2 0.5 0.6 4.2 1.6 0.1 0.9 0 0.1 0 42.6
Hysterangium stoloniferum 0.3 34.1 8.9 0.6 0.6 0.1 1.3 5.4 0 0 0.1 0 0.6 46.7
Sphaerobolus stellatus 0.1 6.8 0.2 0.3 0 0.4 0.2 0 0.3 0.4 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
Cantharellales
Sebacina vermifera 0.3 10
Hydnum rufescens 3.8 15.8 2.1 0.4 3 0.4 0.2 1 3.2 0.2 0.1 44
Cantharellus anzutake 0 28.5 2.2 0.8 0 0.7 0.1 0.2 0.4 0.7 0.4 0.1 0 0 0 44.1
Sistotrema sp. 0.1 0.1 0 3.6
Botryobasidium botryosum 0 1.4 0.4 0 3.7 0.1 0.1 0 0 7.9
Tulasnella calospora 1.8 0.9 0 0.1 0 0 9.4
Ceratobasidium sp. 3.5 0.5 0.4 0 0.1 0 0 0.1 13.9
Rhizoctonia solani 8.1 0.2 0 0 0.1 0 0 0.1 11.2
Tremella mesenterica 12.8 2.2 0.8 0.1 0.2 0.1 0 0.1 0.4 6.8
Dacryopinax primogenitus 2 0.4 0.1 0.1 0 2.9
Wallemia sebi 0.4 0.4
Chalara longipes 0.4 0.3 0 1
Meliniomyces variabilis 1.9 0.8 0.1 0 2
Leotiomycetes
Meliniomyces bicolor 10.2 5.3 0.9 0.8 0.2 1 1 0.1 9.8
Rhizoscyphus ericae 2.5 1.4 1.2 0.8 0.2 0.6 0.1 1 0.1 0.3 0.2 7.5
Acephala macrosclerotiorum 0 2.4 0.2 0.5 0.2 3.7
Phialocephala scopiformis 0.5 0.3 0.3 1
Ascocoryne sarcoides 0.2 0.3 0.6
Glarea lozoyensis 2.2 0.2 0.2 0.9
Amorphotheca resinae 3.7 0.9 5.2 0.1 0 4.7
Oidiodendron maius 1.3 1.4 0.9 0 0 0.5 0.3 0 0 2.3
Bisporella sp. 1.8 0.8 0.9 0.3 0.3 1 0 0.4 0 5
Sclerotinia sclerotiorum 1.4 0.6 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
Dothideomycetes
Trichoderma reesei 0 0 0.3
Xylaria hypoxylon 1.5
Cenococcum geophilum 35.8 12.8 4.7 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 0 0 0 2 0.1 7.6
Ascomycota
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 0.5 0.4 0.1 2.1
Aspergillus flavus 0 0 0.1
Arthroderma benhamiae 0
Usnea florida 1.6 0.3 0.4 0.4 0.4 0 9.6
Xylona heveae 0.5 0.3 0.2
Tuber magnatum 0 46.4 1.3 1.3 0 0.8 0.5 0.5 3.6 0.3 0 13.2
Tuber aestivum 0 19.6 1.6 11.7 0 3.1 0.1 0.6 1.3 0 0 0 0 24.5
Tuber melanosporum 0.3 38.9 1.7 7.2 0.1 0 0 1.8 0.4 0.1 3.7 0 0.1 0.1 0.1 9.7
Pezizomycetes
Tuber borchii 0.5 12.3 0.6 8.3 0.1 0 7.1 0.3 0.7 1.7 0 0.1 30.2
Choiromyces venosus 0 18.5 3.1 4.5 0 3.7 0.1 1.9 5.9 0 0.1 0.2 0 0.2 29.7
Morchella importuna.C 2.1 0.4 1.6 0.1 5.2
Morchella importuna 2.5 0.3 1.9 0.1 0.2 5.2
Gyromitra esculenta 2.2 0 3.8 0.2 0.1 0 0.1 7.7
Caloscypha fulgens 18.4 0.6 8.6 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 35.7 6.1 6.6 0 3.3 0.2 0.2 0.2 0 0 0.6 0 0.1 43
Pyronema confluens 8.2 0 2.2 0.1 0 7.5
Sarcoscypha coccinea 0.4 1.4 0.1 0.2 3.6
Kalaharituber pfeilii 5.8 1.8 8.8 1.2 2.1 0.1 0.4 2.2 0.1 0.1 0.4 0.1 31.3
Terfezia boudieri 9.2 1.4 1.5 0.2 0.6 0.2 0.6 0.1 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 0.1 0.1 0.1 0.1 0.4 0.6 0 22.9
Tirmania nivea 17.5 1.1 0 3.4 0.3 0 0.1 0.6 0.2 1.5 0.2 0.4 0.2 0.4 19.4
Ascobolus immersus 0.1 0.5 0.8 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 0 4.5 0.1 5.8 0.1 0 0.8 0 3 0.7 2.9 0.6 0.1 0.4 0.4 0.1 0 0.2 0.5 0.5 53.2
Mucoromycota
Rhizophagus cerebriforme 1.1 1.1 0 0 0.2 0.2 0.8 0.4 0 0.3 0 0 0 0.3 22.4
Glomerales
Rhizophagus diaphanus 0.2 0 0.5 0 0 0.1 0.4 0.3 0 0.1 0 0 0 0.4 20.6
Rhizophagus irregularis 0.7 0 2.8 0 0.4 0.2 0.6 0.4 0 0.1 0.1 0 0 0.6 26.8
Rhizophagus irregularis DAOM 0.2 0 0.2 0 0.1 0.1 0.1 0 0.1 0 0.1 13.2
Rhizophagus irregularis A1 0.2 0.3 0 0 0.1 0.1 0.4 0 0 0.1 0.1 0.1 0 20.8
Rhizophagus irregularis A4 0.2 0.7 0.1 0 0.1 0.1 0.2 0.1 0 0.1 0 0 0 0 24.9
Rhizophagus irregularis A5 0.3 0 0.3 0.1 0.1 0.2 0 0 0.1 0.1 0 24.3
Rhizophagus irregularis B3 0.1 0.3 0 0 0.3 0.1 0.4 0.1 0 0.2 0.1 0 22.3
Rhizophagus irregularis C2 0 0.2 0 0 0.2 0.5 0.4 0.1 0 0 0 0 0.1 22.1
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.
NATURE COMMUNICATIONS | (2020)11:5125 | https://doi.org/10.1038/s41467-020-18795-w | www.nature.com/naturecommunications 5ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18795-w
a All
Cellulose
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 3 20
256 144
Hypholoma sublateritium 182 106 54 14 22
146 Hebeloma cylindrosporum 84 49 24 3 11
250 98 49 41 8
Cortinarius glaucopus 12
Crucibulum laeve 238 157 54 14 27
266
Tricholoma matsutake 97 51 39 0 7
125
Amanita thiersii 142 95 27 0 20
175 Amanita muscaria 63 32 29 0 2
369 Pluteus cervinus 177 113 45 16 19
Ectomycorrhiza Gymnopus androsaceus 253 146 77 10 30
164
Saprotroph Omphalotus olearius 119 73 29 4 17
226
6
375 172 Marasmius fiardii 207 134 46 1 27
Pathogen 255 Moniliophthora perniciosa 101 50 36 1 15
303
Armillaria gallica 237 133 54 8 50
Orchid mycorrhiza Mycena galopus 454 253 138 19 63
Pleurotus ostreatus 216 141 47 9 28
194
Wood decayer Schizophyllum commune 151 114 15 0 22
77
7
Paxillus involutus 76 44 26 0 6
Endophyte Paxillus adelphus 70 40 25 0 5
79
379 Gyrodon lividus 79 48 22 0 9
86
Xerocomus badius 82 48 28 0 6
85
90 Boletus edulis 87 46 34 0 7
Pisolithus microcarpus 53 34 17 0 2
91 52
Scleroderma citrinum 63 33 23 0 7
86 Suillus luteus 86 50 29 0 7
112
Suillus brevipes 96 58 31 0 7
Coniophora puteana 139 104 17 0 18
122
Serpula lacrymans 98 71 14 0 13
380 184
122
Fibulorhizoctonia sp. 328 205 82 0 41
156 Piloderma croceum 108 52 38 1 18
Plicaturopsis crispa 125 82 21 7 22
140
Phanerochaete chrysosporium 151 100 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 114 76 31 11 7
91
128
Lactarius quietus 76 30 43 1 3
237 Heterobasidion annosum 141 84 37 8 20
367 Neolentinus lepideus 110 75 19 0 16
120
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
328
Piriformospora indica 156 121 14 0 21
128 Sebacina vermifera 177 139 18 0 20
Hydnum rufescens 64 36 23 0 5
55
269 120 Cantharellus anzutake 80 47 27 0 6
164 Sistotrema sp. 208 144 23 0 41
Botryobasidium botryosum 154 121 20 0 13
1
198
166 Tulasnella calospora 246 188 9 0 49
Ceratobasidium sp. 321 175 70 0 76
115 235
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 0 0
Choiromyces venosus 88 64 7 0 17
Carb. Permian Triassic Jurassic Cretaceous Cenozoic
881 491 416 325 300 200 100 0 Mya
Cellulose
b 403
380
Chalara longipes 324
All Hemicellulose
243
Lignin
43
Pod
2
Pectin
38
404
Meliniomyces variabilis 328 248 42 1 38
485 Meliniomyces bicolor 238 165 37 1 36
Rhizoscyphus ericae 174 127 23 0 24
483
Acephala macrosclerotiorum 282 198 43 0 41
509 357
Phialocephala scopiformis 287 201 52 2 34
630
Ascocoryne sarcoides 145 119 10 0 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 31 3 35
595 452
Sclerotinia sclerotiorum 145 100 16 0 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
Ericoid mycorrhiza Trichoderma reesei 109 90 13 0 6
302
Xylaria hypoxylon 181 137 22 2 22
Endophyte 627
Cenococcum geophilum 92 66 17 1 9
210
Glonium stellatum 196 139 35 6 22
Yeast 213
Lepidopterella palustris 83 56 15 2 12
364 Stagonospora sp. 239 188 31 4 20
291
431 Alternaria alternata 228 164 26 4 38
Aureobasidium pullulans 190 141 19 3 30
244
Cladosporium fulvum 174 98 48 2 28
493 Aspergillus clavatus 107 93 4 0 10
190
183 Aspergillus flavus 186 128 14 0 44
220 Arthroderma benhamiae 26 20 6 0 0
Usnea florida 65 43 18 0 4
357 228
Xylona heveae 34 28 6 0 0
Tuber magnatum 52 40 5 0 7
60
Tuber aestivum 52 38 6 0 8
63
Tuber melanosporum 49 39 5 0 5
64
Tuber borchii 48 38 5 0 5
112
Choiromyces venosus 105 80 7 0 18
178 Morchella importuna.C 159 122 5 0 32
160
173
Morchella importuna 159 121 5 0 33
187 Gyromitra esculenta 161 125 5 0 31
252 90 66 5 0 19
Caloscypha fulgens
222 Wilcoxina mikolae 65 48 8 0 9
68
91 Trichophaea hybrida 56 39 9 0 8
153 Pyronema confluens 77 63 5 1 9
243
Sarcoscypha coccinea 143 106 8 0 29
37
Kalaharituber pfeilii 78 68 6 0 4
82
130
Terfezia boudieri 43 35 5 1 3
Ascobolus immersus 114 102 6 0 6
Monacrosporium haptotylum 179 125 9 0 45
183
19 Arthrobotrys oligospora 142 108 6 0 28
Wickerhamomyces anomalus 19 18 1 0 0
16
20
Saccharomyces cerevisiae 13 12 0 0 1
Candida tanzawaensis 15 14 0 0 1
Taphrina deformans 16 14 2 0 0
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, the number of PCWDEs in
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
6 NATURE COMMUNICATIONS | (2020)11:5125 | https://doi.org/10.1038/s41467-020-18795-w | www.nature.com/naturecommunicationsNATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18795-w ARTICLE 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 (Hysterangiales)31, have diverse PCWDEs acting on cellulose, PCWDE to MCWDE repertoires (Figs. 4 and 5, Supplementary 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 com- GH7 cellobiohydrolases. Thus, a robust suite of PCWDEs appears pounds trapped in SOM (e.g., chitin) by selectively using these to be a characteristic of both saprotrophs and ectomycorrhizal hydrolytic enzymes in addition to oxidative mechanisms32. 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- report the first genomes, both have highly reduced complements opmental processes, such as ectomycorrhiza and sporocarp of PCWDEs. The two Thelephorales species (Thelephora terrestris formation10,11,33. 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 NATURE COMMUNICATIONS | (2020)11:5125 | https://doi.org/10.1038/s41467-020-18795-w | www.nature.com/naturecommunications 7
ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-18795-w
AA 1
AA 1
AA 2
AA 1
AA 1
AA 2
C
C
G
G
BM
G
G
BM
G
G
C
C
AA
AA
AA
AA
AA
AA
H
H
1_
1_
1_
1_
1_
1_
H
H
H
H
E1
E1
45
45
1
9
3
2
6
7
1
9
3
2
6
7
Chalara longipes 12 24 4 6 8 1 2 9 2 7 Laccaria amethystina 1 2 5 1
Meliniomyces variabilis 11 18 2 7 5 1 2 8 4 4 Laccaria bicolor 1 4 1 9 1 1
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
Leotiomycetes
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
Agaricales
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
Sclerotinia sclerotiorum 16 8 4 1 2 2
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
Dothideomycetes
Schizophyllum commune 5 19 2 1 2 1 7
Glonium stellatum 13 16 1 1 13 5 2 3 3 5
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
Xerocomus badius 3 9 1
Ascomycota
Aureobasidium pullulans 4 4 1 1 4 2 1 3 2
Boletus edulis 2 4 1
Cladosporium fulvum 2 1 8 2 2 2
Boletus edulis P
Boletales
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
Usnea florida 2 1 4 1 Suillus luteus 2 16 1
Suillus brevipes 2 14 1
Basidiomycota
Xylona heveae 1 2 2
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 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
Pezizomycetes
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
Sarcoscypha coccinea 14 18 1 2 1 2 2 1 Sistotremastrum niveocremeum 48 21 1 9 14 4 3 2 2
Russulales
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
Terfezia claveryi 2 2 1 1 Lentinellus vulpinus 20 9 1 6 11 1 3 1 2
Russula emetica 1 9 2 4
Tirmania nivea 5 5 1 1
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
Candida tanzawaensis 1 Sphaerobolus stellatus 45 34 2 14 41 4 8 2 5
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
Cantharellales
Mucoromycota
Rhizophagus diaphanus 3 Cantharellus anzutake 1 7 7 1 1 3
Rhizophagus irregularis 5 Sistotrema sp. 49 18 1 2 4 3 2
Glomerales
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
Rhizoctonia solani 34 31 11 7 3 8 5
Rhizophagus irregularis A5 8
Tremella mesenterica 2
Rhizophagus irregularis B3 4 1
Dacryopinax primogenitus 1
Rhizophagus irregularis C2 7 Wallemia sebi 8
Ectomycorrhiza Orchid mycorrhiza Ericoid mycorrhiza Arbuscular mycorrhiza
Saprotroph Pathogen Parasite Endophyte Wood 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 Data 6a,
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
PS5
Cenococcum geophilum 78 (16%) 358 (73.5%) 129 (26.5%) 487 (100%)
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
Sarcoscypha coccinea
PS2
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
Emergence of mycorrhizal symbiosis PS13 Marasmius fiardii
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
PS10
Postia placenta 6% 67% 20%
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 9ARTICLE 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 (cluster VI). Most of 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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