Supplementary Information for Mycena Genomes Resolve the Evolution of Fungal Bioluminescence

Home , Galerina marginata, Laccaria amethystina, Mycena, Mycena chlorophos, Mycena sanguinolenta, Neonothopanus gardneri

Supplementary Information for

Mycena genomes resolve the evolution of fungal bioluminescence

Huei-Mien Ke, Hsin-Han Lee, Chan-Yi Ivy Lin, Yu-Ching Liu, Min R. Lu, Jo-Wei Allison Hsieh, Chiung-Chih Chang, Pei-Hsuan Wu, Meiyeh Jade Lu, Jeng-Yi Li, Gaus Shang, Rita Jui-Hsien Lu, László G. Nagy, Pao-Yang Chen, Hsiao-Wei Kao and Isheng Jason Tsai

Huei-Mien Ke [email protected] and Isheng Jason Tsai [email protected]

This PDF file includes: Supplementary text Figures S1 to S19 Tables S1 to S13 Legends for Datasets S1 to S9 References for SI reference citations

Other supplementary materials for this manuscript include the following: Datasets S1 to S9

1 www.pnas.org/cgi/doi/10.1073/pnas.2010761117 I. SUPPLEMENTARY INFORMATION TEXTS

Materials and Methods

Strains and fungal materials M. kentingensis, M. venus (1), M. sanguinolenta, M. indigotica and M. chlorophos were isolated from fruiting bodies collected from forest in Taiwan. M. indigotica was isolated from basidiospores. The mycelia were grown and maintained on potato dextrose agar (PDA) plates at 25°C. To identify the pattern of bioluminescence, a piece of mycelium from each species was inoculated in the centre of a sheet of sterilized dialysis cellulose membrane (8030-32, Cellu Sep T-Series) on a 3 cm PDA agar plate at 25°C. The diameter of the mycelium was measured and its bioluminescence was recorded with a Glomax 20/20 luminometer (Promega BioSystems Sunnyvale, Inc., USA) for seven days (Dataset S9). The taxonomic status of species was reconfirmed by sequencing the internal transcribed spacer (ITS) with the primer pair SR6R(5’-AAGWAAAAGTCGTAACAAGG-3’)/ITS4(5’- TCCTCCGCTTATTGATATGC-3’). Using the other available Mycena ITS sequences, all sequences were aligned by MAFFT(2) (ver. 7.310) and trimmed by trimAl(3) (1.2rev59; with option -automated1). The ITS phylogeny was constructed by IQ-TREE(4, 5) (ver. 1.6.10; with option -bb 10000 -alrt 1000).

Genomic DNA extraction and sequencing Genomic DNA was extracted using the traditional CTAB and chloroform extraction method. Briefly, 0.1 g mycelium was grinded with liquid nitrogen and then mixed with CTAB extraction buffer (0.1 M tris, 0.7 M NaCl, 10 mM EDTA, 1% CTAB, 1% Beta- Mercaptoethanol). After incubating at 65°C for 30 min, an equal volume of chloroform was added, then the mixture was centrifuged at 8000 rcf for 10 min. The supernatant was mixed with an equal volume of isopropanol and the DNA was precipitated. After washing with 70% EtOH, the DNA was dissolved with nuclease free water. Genome sequencing was carried out in two platforms. First, paired-end libraries were constructed using the KAPA LTP library preparation kits (#KK8232, KAPA Biosystems). All libraries were prepared in the High Throughput Genomics Core at Biodiversity Research Center, Academia Sinica and sequenced on an Illumina HiSeq 2500 platform. A total of 51.6 Gb of 150- or 300-bp read pairs were generated. Second, Oxford Nanopore libraries were prepared using SQK-LSK108 and sequenced on a GridION instrument. Basecalling of Nanopore raw signals was performed using Guppy (ver. 3.2.4) into a total 67.7 Gb of raw sequences at least 1 kb or longer. A summary of the sequencing data is shown in SI Appendix, Table S1.

RNA extraction and sequencing Bioluminescent mycelia were collected in two ways. i) For M. chlorophos and M. kentingensis, a piece of mycelium was inoculated at the centre of a sheet of sterilized dialysis cellulose membrane (8030-32, Cellu Sep T-Series) on PDA agar plates at 25°C. The plates were cultured for 10 and 14–18 days for M. chlorophos and M. kentingensis, respectively. For M. kentingensis, bioluminescence was detected by camera (Nikon D7000, Sigma 17-50mm ISO100 f2.8 with 16 min exposure time) (Fig. 6B). The mycelia with low or high bioluminescent intensities which occurred spontaneously were collected from two separated plates inoculated on the same day. In M. chlorophos, bioluminescence was detected by luminometer. Mycelium with low bioluminescence showed the intensity of 7-14 Relative Light Unit (RLU)/mg, and the mycelium with high bioluminescence showed the intensity of 5,000-10,000 RLU/mg (Dataset S1). Three replicates were collected. ii) For M. sanguinolenta and M. venus, a piece of mycelium was inoculated at the centre of a sheet of sterilized dialysis cellulose membrane on PDA agar plates at 25°C. The plates were cultured for 13–17 days, and the bioluminescent features were detected by CCD camera; the tissues were collected and their luminescence intensity was recorded with a Glomax 20/20 luminometer (Promega BioSystems Sunnyvale, Inc., USA). A total of 12 samples with different bioluminescence intensities were collected (Dataset S1). After homogenizing 5–10 mg of tissues by liquid nitrogen, total RNA was extracted using the Direct-zol RNA Miniprep (Zymo Research). Concentrations were measured by Qubit fluorometer (Invitrogen USA), and quality was assessed by the BioAnalyzer 2100 RNA Nano kit (Agilent, USA) with RIN values higher than 8.0. The paired-end libraries were constructed using the TruSeq Stranded mRNA library prep kit (#20020594, Illumina, San Diego, USA) with standard protocol and sequenced by Illumina HiSeq 2500 (Illumina, USA) to produce 150-bp paired-end reads.

RNA extraction and sequencing from the M. kentingensis fruiting body Fruiting body production of M. kentingensis was modified from previous studies(6, 7). Mycelia, grown on PDA for 8–15 days, was then inoculated onto sterilized commercially available peat soil mixed with 10% rice bran and 50% water in a jar. Mycelium samples were grown at 25°C for 3–4 weeks and then transferred into fresh compost. The culture was sprayed with sterilized water daily until the fruiting body formed. Four kinds of tissue were collected: (1) primordia, (2) young fruiting body (YFB, 0.5–1 cm), (3) cap and (4) stipe of mature fruiting body (> 1 cm). For each batch of culture, 15–20 primordia, 6–11 YFB, and 8–12 caps and stipes from mature fruiting bodies were pooled to measure their weight and bioluminescent intensity, and the RNA

3 was extracted using Trizol extraction and lithium chloride purification method. Three replicates were produced. The paired-end libraries were constructed using the TruSeq Stranded mRNA library prep kit (#20020594, Illumina, San Diego, USA) with standard protocol and sequenced by Illumina HiSeq 2500 (Illumina, USA) to produce 150-bp paired-end reads.

Bisulphite sequencing To construct a BS-seq library, the fragmented DNA was first ligated with a premethylated TruSeq DNA adapter (Illumina). The ligated DNA fragments were bisulfite converted using the EZ DNA methylation kit (Zymo Research), followed by PCR amplification. The BS-seq libraries were sequenced on an Illumina HiSeq 2500 sequencer. The bisulfite conversion efficiency reached approximately 99% in all of our libraries (SI Appendix, Table S13).

Identification of repetitive elements Consensus (library) sequences of repetitive elements were identified using the pipeline described in Berriman et al(8). Full LTR retrotransposons in Mycena species were defined as i) initially identified by LTRharvest(9) and ii) presence of known reverse transcriptase domains identified by Pfam(10) (ver. 31.0). Repeat contents were quantified using RepeatMasker(11) (ver. open-4.0.7). Proportions of repeat content along the scaffolds were calculated using Bedtools(12). A phylogenetic tree was built by first aligning all the putative RVT domain sequences using MAFFT(2) (ver. 7.310; --genafpair --ep 0) and FastTree(13) with the JTT model on the aligned sequences, and were visualised using the ggtree(14) package in R.

Orthogroup inference and analysis of protein family domains CAFÉ(15) (ver. 4.2.1; lambda command) was used to predict the expansion and contraction of gene numbers of OGs based on the topological gene tree. Gene family evolutionary rate λ was estimated for the whole phylogeny as well as a separate λ since the last common ancestor of the mycenoid lineage. Simulated dataset (option -t 120) were created by the genefamily command using the two different λ and significance was assessed using a likelihood-ratio test. The phylogenetic tree was visualized by the ggtree(14, 16) package in R. Protein domains of each gene were identified by pfam_scan.pl ver. 1.6 by comparing them against Pfam ver. 32.0 db(10). To compare them to plant pathogenic fungi, the Pfam domains from Moniliophthora perniciosa FA55313 (Monpe1_1)(17) from JGI and Moniliophthora roreri (Monro) from BioProject: PRJNA279170 were also annotated. Enrichment of Pfam domain number between two sets of interest was assessed by the Wilcoxon rank-sum test (P ≤

4 0.05). We compared the Pfam copy number between six mycenoid species and the other 37 species. Gene ontology enrichments were identified for these genes using TopGO(18).

Evolution of gene families related to the luciferase gene cluster In addition to gene family identified by Orthofinder (19, 20), luciferase orthologue outside marasmioid+mycenoid were identified by the reciprocal best hits against proteomes of three bioluminescence fungi (Armillaria mellea, Neonothopanus gardneri, and Mycena kentingensis). Outgroup sequences for the luciferase phylogeny was identified by first producing a phylogeny of these luciferase sequences with top 100 non-redundant hit of M. sanguinolenta (Msan_01367500) or Fistulinahepatica (Fishe1_70153) luciferase sequence. The sequences were aligned by MAFFT (2) (ver. 7.310), trimmed by trimAl (3) (1.2rev59 ; with option -automated1) and reconstructed by IQ-TREE (4, 5) (ver. 1.6.10). Two sequences from the Ascomycota phylum basal to the clade consisting of luciferase sequences were chosen as the outgroup (Fig1D). The sequences of five orthologues in the luciferase family—hispidin-3-hydroxylase, cytochrome P450, hispidin synthase, and caffeylpyruvate hydrolase—were constructed and the sequences were aligned by MAFFT(2) (ver. 7.310) and trimmed by trimAl(3) (1.2rev59 ; with option -automated1). The protein trees were constructed by IQ-TREE (4, 5) (ver. 1.6.10; with option -bb 10000 -alrt 1000). The protein tree of luciferase was reconciled with the species tree and the branch with < 90 SH-like bootstrap threshold were rearranged using the NOTUNG software package (21, 22). The evidence for selection across gene families was tested using the HyPhy(23, 24) platform in the webserver of datamonkey(25-27). According to the recombination breakpoints analysed by the Genetic Algorithm for Recombination Detection(28) (GARD), the alignment was trimmed for analysing selection using Single-Likelihood Ancestor Counting(29) (SLAC) and Mixed Effects Model of Evolution(30) (MEME) with P<0.1.

II. SUPPLEMENTARY FIGURES

Fig. S1. Mycena ITS phylogenetic tree and bioluminescent pattern in five Mycena species. A, Bioluminescence patterns were measured for seven days after inoculation of mycelium on potato dextrose agar (PDA). The control was the PDA only. The diameter of mycelial growth is described in dataset S9. B, The ITS tree was constructed by IQ-TREE (4) with an alignment length of 666 bp. The isolates used in this study were denoted in colour. Numbers on each branch denote support values (SH-aLRT support (%) / ultrafast bootstrap support (%)).

6 x1 x3 x2 o o o c atp9rns nad2 nad3atp6 atp8 nad5 nad4L c nad6 cob nad1 nad4 rnl c M. chlorophos

x1 x3 x2 o o o c nad4Lnad5atp8atp6nad3nad2 rns atp9 c nad6 c rnl nad4nad1 cob M. indigotica

x1 x2 x3 o o o c atp6nad3nad2 nad1 cob nad6 rns rnpB rnl c nad4 atp9 c nad4Lnad5 atp8 M. kentingensis

x1 x3 x2 o o o c rns atp9 cobatp8 nad5nad4L nad4nad3nad2c nad6 nad1 atp6 rnl c M. sanguinolenta 20 kb

x1 x3 x2 o o o c rns nad2nad3 nad4 nad6 nad1 nad4Lnad5 atp8cob c atp6atp9 rnl c M. venus Fig. S2. The mitochondrial genomes of five Mycena species. Exons are denoted by red boxes, while introns are indicated with connecting lines. Green stripe denotes the tRNA.

A B

100 M. chlorophos M. indigotica M. kentingensis M. sanguinolenta 15 M. venus

e length (%) 10 v 50 e relati old length (Mb) f v ulati Scaf

m 5 25 Cu

0 0

1 10 100 1000 0 25 50 75 100 125 Scaffold index NG(X)%

Fig S3. (A) LG and (B) NG graphs showing an overview of Mycena assembly contig lengths. Coloured and dashed lines denote Mycena and published assemblies used in this study, respectively. LG is the number of scaffolds sorted by length that covered cumulative percentage of the assembly. NG was calculated according to Brandnam et al (31) where denominator was the genome size estimated from GenomeScope (32).

Fig. S4. The distribution of CG methylation levels from gene bodies of all genes in five Mycena species.

A Mycena gene 30 el (%) 25 v e 20 15 ylation l h 10

5 TSS: Transcription Start Site 0 CG met TES: Transcription End Site TSS TES B M. chlorophos DNA (1193) M. indigotica DNA (3441) M. kentingensis DNA (1658) DNA-relic (77) DNA-relic (77) DNA-relic (932) 100 100 100

LTR (19256) el (%) LTR (9150)

LTR (2227) el (%) el (%) v v v LTR-relic (722) LTR-relic (1771) LTR-relic (1600) e e e 80 80 80 LINE (144) LINE (1080) LINE (1232) LINE-relic (152) LINE-relic (254) LINE-relic (591) 60 60 60 Unknown (2168) Unknown (7197) Unknown (14301) ylation l ylation l ylation l h h h 40 40 40 20 20 20 CG met CG met CG met 0 0 0 TSS TES TSS TES TSS TES

M. sanguinolenta M. venus DNA (15305) DNA (18315) 100

100 DNA-relic (10450) DNA-relic (7273) el (%) el (%) v v LTR (27160) LTR (33087) e e 80

80 LTR-relic (13771) LTR-relic (18176) LINE (8619) LINE (6636) LINE-relic (3058) LINE-relic (3335) 60 60 Unknown (64663) Unknown (56984) ylation l ylation l h h 40 40 20 20 CG met CG met 0 0 TSS TES TSS TES

Fig S5. CG methylation levels of (A) gene, and (B) classic TEs and their relics in 5 Mycena species. Line represents a mean value of CG methylation level.

1 Panellus stiptiucs(tennessee) 1 Mycena venus 1 Mycena sanguinolenta

1 Mycena kentingensis 1 Mycena chlorophus Mycena indigotica 1 Armillaria ectypa FPL83.16 v1.0 Armillaria tabescens CCBAS 213 v1.0 1 Armillaria ostoyae C18/9 1 1 Armillaria solidipes 28-4 v1.0 0.78 1 Armillaria borealis FPL87.14 v1.0

1 1 1 Armillaria cepistipes B5 Armillaria gallica 21-2 v1.0 1 Armillaria mellea ELDO17 v1.0 Guyanagaster necrorhiza MCA 3950 v1.0 1 Cylindrobasidium torrendii FP15055 v1.0 Flammulina velutipes 1 1 Neonothopanus gardneri 0.88 1 Omphalotus olearius

1 Gymnopus luxurians v1.0 1 0.9 Rhodocollybia butyracea CCBAS 279 v1.0 1 Gymnopus androsaceus JB14 v1.0 Dendrothele bispora CBS 962.96 v1.0 Marasmius fiardii PR-910 v1.0

1 Fistulina hepatica v1.0 1 Schizophyllum commune H4-8 v3.0 1 1 Auriculariopsis ampla NL-1724 v1.0 Flagelloscypha sp. PMI_526 v1.0 1 Amanita thiersii Skay4041 v1.0 Amanita muscaria Koide v1.0 Coprinopsis cinerea 1 1 Hypholoma sublateritium v1.0 1 1 0.51 Galerina marginata v1.0 1 1 Hebeloma cylindrosporum h7 v2.0 1 1 Laccaria amethystina LaAM-08-1 v2.0 Laccaria bicolor Agaricus bisporus var. burnettii JB137-S8 Volvariella volvacea V23 Pleurotus ostreatus PC15 v2.0 1 Plicaturopsis crispa v1.0 1 Fibulorhizoctonia sp. CBS 109695 v1.0 Paxillus involutus ATCC 200175 v1.0

2.0

Fig. S6. The species phylogeny reconstructed by a coalescence of 360 single-copy orthologue trees using ASTRAL-III (33). Number on every branch represent the proportion of gene trees that support each branch

10 A. Mind_Mind_00284400 Mind_Mind_00284200 Mind_Mind_00284100 up-regulated in bioluminescent mycelium Mind_Mind_00284000 Mind_Mind_00283700 Mind_Mind_00283900 Mind_Mind_00283600 down-regulated Mind_Mind_00286100 Mind_Mind_00287700 Mind_Mind_00286200 Mchl_Mc_01326700 Mchl_Mc_01327900 no signi!cance Mchl_Mc_01327200 Mchl_Mc_01327100 Mken_Mk_00297700 Mken_Mk_00012500 * >95 bootstrap support Mchl_Mc_01325300 Mchl_Mc_01323400 Mchl_Mc_01170600 Mchl_Mc_01171500 Mchl_Mc_01323800 Mken_Mk_01401400 Mken_Mk_00688000 PANBI_PANBI_020820 Msan_Msan_00318300 Msan_Msan_00318500 Msan_Msan_00318100 Msan_Msan_00318400 Msan_Msan_00318200 Mven_Mven_01294200 Mven_Mven_02137400 Mven_Mven_02138100 Msan_Msan_00317700 Mven_Mven_02330400 Msan_Msan_02019800 PANBI_PANBI_020620 Msan_Msan_00342300 Msan_Msan_00335400 Msan_Msan_00338500 Msan_Msan_00336800 Msan_Msan_00337300 Msan_Msan_00338400 Msan_Msan_00343000 Msan_Msan_00335100 Mven_Mven_02134300 Mven_Mven_02134200 Mven_Mven_01727100 Mven_Mven_01726500 Mven_Mven_02134400 Mven_Mven_02123300 Mven_Mven_02651600 Mven_Mven_02371400 Mven_Mven_01181900 PANBI_PANBI_089880 PANBI_PANBI_089890 PANBI_PANBI_020550 Msan_Msan_01791400 Msan_Msan_01791500 PANBI_PANBI_089050 PANBI_PANBI_111580 Mven_Mven_00227500 Mind_Mind_00930200 Mchl_Mc_00155000 Mken_Mk_00589600 Gymlu1_245696 Mven_Mven_01285700 Mven_Mven_01285800 Mven_Mven_02212100 Msan_Msan_02013400 Msan_Msan_01128000 PANBI_PANBI_002760 Mven_Mven_02134700 Msan_Msan_00321600 Mind_Mind_00280600 Mchl_Mc_01323000 */* Mchl_Mc_00558400 Mven_Mven_02002500 Mven_Mven_01705600 Mven_Mven_01695200 Msan_Msan_00606200 Msan_Msan_00606000 Msan_Msan_02325300 90/* Mven_Mven_02241100 Mind_Mind_01205100 PANBI_PANBI_096430 Mven_Mven_01318300 Mven_Mven_00186300 Mken_Mk_00962600 Mven_Mven_01645400 Mven_Mven_01645900 Mven_Mven_01128200 Mven_Mven_01690300 86/79 Armosto1_262486 Armost1_1018848 Armbor1_712128 Armmel1_931283 Armga1_1072482 Armcep1_10572 hispidin-3-hydroxylase (h3h) */* Guyne1_964922 Armect1_1421328 in luciferase cluster */* Armtab1_1666463 */* Ompol1_7903 */* NEOGA_NEOGA_075410 Mven_Mven_02536300 */* Msan_Msan_01367600 PANBI_PANBI_079130 Mken_Mk_01107000 86/* */* Mchl_Mc_00780900 Mven_Mven_00291400 */* Msan_Msan_01622700 Mven_Mven_00188000 Schco3_2611288 Auramp1_487097

B. Armosto1_262484 Armbor1_1702257 Armost1_146429 Armga1_915679 Armcep1_10570 cytochrome P450 81.1/64 Armmel1_1046296 (cyp450) */* Armtab1_139423 Armect1_1382167 in luciferase cluster Guyne1_945996 Msan_Msan_01367300 */* Msan_Msan_01367700 */* Mven_Mven_02536200 // PANBI_PANBI_079120 */* Mken_Mk_01107100 Mchl_Mc_00781000

11 C. Mven_Mven_00907100 Mven_Mven_02345800 Mven_Mven_00903300 PANBI_PANBI_083780 PANBI_PANBI_083770 PANBI_PANBI_060680 Armga1_777461 Armcep1_3934 Armcep1_3935 Armmel1_1145400 Armosto1_260367 Armost1_1026031 Armbor1_1006746 Guyne1_955767 Armtab1_1316903 Armect1_1510646 Armect1_1389154 Denbi1_864690 85.3/59 Denbi1_755128 Hebcy2_372989 Galma1_232478 Hebcy2_372991 Hebcy2_372990 Hypsu1_48246 Hypsu1_72027 Hypsu1_202830 */* Hebcy2_372782 63/68 Galma1_232476 Hebcy2_372656 Mven_Mven_02536100 Msan_Msan_01367800 PANBI_PANBI_079150 */* Mken_Mk_01107200 Mchl_Mc_00781100 Armosto1_262483 Armbor1_1702387 hispidin synthase 41.2/69 Armost1_1004271 */* Armga1_1159537 Armcep1_10569 (hisps) Armmel1_1046299 Armect1_1421292 in luciferase cluster Armtab1_1666484 */* Guyne1_992677 Armbor1_1731564 */* Ompol1_5260 NEOGA_NEOGA_075420 FlaPMI526_1_368851 Armost1_902438 Amath1_145969 Rhobu1_695106 Fibsp1_1001315 Fibsp1_1037317

Armosto1_262485 D. Armbor1_711588 Armost1_935933 caffeylpyruvate hydrolase Armga1_981851 Armcep1_10571 (cph) */* Armmel1_1046293 */* Armtab1_1528987 in luciferase cluster Armect1_1373302 NEOGA_NEOGA_075411 Armga1_1012317 90.7/92 Armbor1_1891345 Armmel1_1045040 Armosto1_262813 Armcep1_10888 82.8/19 Armost1_959980 */* Guyne1_930840 Armtab1_1527355 Armect1_1460710 Plicr1_127838 PANBI_PANBI_085100 97.5/52 Rhobu1_557871 Rhobu1_208133 88.7/24 Gyman1_877746 Gymlu1_207860 Ompol1_4600 NEOGA_NEOGA_049900 Denbi1_772298 Denbi1_847618 90.1/35 Mven_Mven_01185300 93.8/54 Msan_Msan_02335400 Mchl_Mc_00998200 */* Mken_Mk_00163300 */* Mken_Mk_01107300 Marfi1_956776 Fvel_chr11_01103 Cylto1_377629 Schco3_2622574 */59 Auramp1_565074 Fishe1_64303 PleosPC15_2_1093612 Hypsu1_1072161 Hypsu1_31865 86.6/48 Galma1_236967 Hebcy2_441814 Lacbi2_656824 87/50 Lacam2_681447 Agabi_varbur_1_113217 81.6/64 Volvo1_112130 Copci1_1253 Amamu1_117624 */* Amath1_155120 FlaPMI526_1_1668112 PANBI_PANBI_099780 // Fibsp1_790691

12 Fig. S7. Phylogenies of selected gene families. The trees were constructed by IQ- TREE(4). The value on each branch denote support values (SH-aLRT support (%) / ultrafast bootstrap support (%)) Sequence name contain species abbreviations in Table S4 followed by gene ID. A, Phylogeny of OG0000706, which is the OG including hispidin-3-hydroxylase in the luciferase cluster. B, Phylogeny of OG0009696, which is the OG including cytochrome P450 (cyp450) in the Mycena and Armillaria luciferase cluster. C, Phylogeny of OG0002489, which is the OG including hispidin synthase (hisps) in the luciferase cluster. D, Phylogeny of OG0002332, which is the OG including caffeylpyruvate hydrolase (cph). For each species, the sequence id denoted the transcript ID.

Fig. S8. Synteny between Armillaria ectypa and Mycena indigotica. A, Heatmaps of single-copy orthologues shared among scaffolds of A. ectypa and M. indigotica. Numbers in cells show the orthologues percentages of M. indigotica scaffold links to an A. ectypa scaffold. B, Linkage maps of single-copy orthologue linking scaffolds of A. ectypa and M. indigotica. Fifteen M. indigotica scaffolds were assigned unambiguously (≥ 15% of pairwise orthologues) to a corresponding A. ectypa scaffold, providing strong evidence that macro-synteny has been conserved across the Marasmioid clade.

Fig. S9. Synteny between Pleurotus ostreatus and Mycena indigotica. A, Heatmaps of single-copy orthologues shared among scaffolds of P. ostreatus and M. indigotica. Numbers in cells show the orthologue percentages of an M. indigotica scaffold links to a P. ostreatus scaffold. B, Linkage maps of single-copy orthologue linking scaffolds of P. ostreatus and M. indigotica. Fifteen M. indigotica scaffolds were assigned unambiguously (≥ 15% of pairwise orthologues) to a corresponding P. ostreatus scaffold, providing strong evidence that macro-synteny has been conserved across Agaricales.

Fig. S10. The gene density, repeat density and methylation level in high- or low- synteny regions. The gene and repeat densities are calculated from the non- overlapping 10-kb window located in the core or dispensable regions. The methylation level was calculated from the mean CG methylation level in core or dispensable regions.

16 b b b 455 k 756 k 91 k 172 genes 283 genes Mycena venus scaf9 18 genes

b kb 427 k 693 Mycena sanguinolenta scaf11 172 genes 283 genes

b b 446 k 647 k Mycena kentingensis scaf1 162 genes 250 genes

b 56 k scaf6 Mycena chlorophos scaf25 24 genes scaf22

b b 99 k 382 k 26 genes 167 genes 637 kb Mycena indigotica 212 genes scaf6

old_4 Armillaria ectypa scaff

b 41 k 871 kb, 285 genes Guyanagaster necrorhiza scaf9 scaf6

Cylindrobasidium torrendii scaf49 scaf4 scaf2 scaf24

499 kb 170 genes Flammulina velutipes chr11

387 kb 111 genes 675 kb Neonothopanus gardneri scaf60 scaf5 201 genes

Omphalotus olearius scaf42 scaf28 scaf129

361 kb 171 genes 102 kb, 52 genes Marasmius fiardii scaf5 scaf22

938 kb Schizophyllum commune scaf5 394 genes 297 kb, 133 genes 161 kb, 84 genes

Coprinopsis cinerea Chr_5 530 kb, 234 genes389 kb, 172 genes 259 kb, 124 genes

161 kb, 69 genes 750 kb, 325 genes Pleurotus ostreatus scaf04 20 kb

Fig. S11. Synteny between adjacent regions of luciferase cluster in Armillaria ectypa and other species across Agaricales species. Using Armillaria ectypa as the reference, the orthologous genes in other species are denoted with same colour regardless of their orientations. The start and stop positions for each sequence fragments: (1) M. venus, 4468578–5869172 of Mven.scaff0009. (2) M. sanguinolenta, 4419874–3166494 of Msan.scaff0011. (3) M. kentingensis, 3886937–2696567 of scaff0001. (4) M. chlorophos, 322781–451127 of Mc.scaff0025, 1443132- 1451974 of Mc.scaff0022 and 904867-867954 of Mc.scaff0006. (5) M. indigotica, 3436952-2234518 of Mind.scaff0006. (6) A. ectypa, 12804877-2948226 of scaffold_4 (7) G. necrorhiza, 16830-4779 of scaffold_9 and 1845297-823119 of scaffold_6 (8) C. torrendii, 67494- 130334 of scaffold_49, 345423-381212 of scaffold_4, 297084-304272 of scaffold_2, and 180513-183495 of scaffold_24. (9) F. velutipes, 2720572-2064978 of chr11 (10) N. gardneri, 2027286-2055399 of NG_scaffold_60, 28257-1173714 of NG_scaffold_5 (11) O. olearius, 99605- 162156 of scaffold_42, 1117258-1125377 of scaffold_28, and 2738-46147 of scaffold_129 (12) M. fiardii, 59608-470702 of scaffold_5 and 1317903-1547407 of scaffold_22. (13) S. commune, 1264633-2821423 of scaffold_5. (14) C. cinerea, 2256009-943709 of Chr_5. (15) P. ostreatus, 2520611-1474541 of scaffold_04.

hisps luz h3h cyp450 cph Panellus stipticus scaffold_48//scaffold_59 Mycena venus scaff0035//scaff0009 Mycena sanguinolenta scaff0011//scaff0041 Mycena kentingensis scaff0013//scaff0001 Mycena chlorophos scaff0010//scaff0014 Armillaria ectypa scaffold_4 Armillaria tabescens scaffold_10 Armillaria ostoyae scaffold_3 Armillaria solidipes scaffold_14 Armillaria borealis scaffold_3 Armillaria cepistipes scaffold_259 Armillaria gallica scaffold_2 Armillaria mellea scaffold_5 Guyanagaster necrorhiza scaffold_6//scaffold_9 Neonothopanus gardneri scaffold_57//scaffold_22 Omphalotus olearius scaffold_83//scaffold_39//scaffold_30 20 kb Fig. S12. The similarity among genes related to bioluminescence among bioluminescent fungi and non-bioluminescent G. necrorhiza. The cph gene in some species is locate in another scaffolds. The OGs shared by at least two species are labelled with the same colour, regardless of the orientation.

Fig. S13. Synteny around cph. Most cph copies (denoted by diamond) were located in different scaffolds to luciferase cluster (denoted by triangle). A copy of cph in Armillaria ectypa (Armect1) is located outside luciferase cluster shared synteny with other Agaricales suggesting this was the ancestral copy. The links denoted single- copy orthologue between two species.

Fig. S14. Selection analysis of the genes in luciferase cluster. Normalized dN/dS from SLAC (29) and MEME (30) analysis across a multispecies alignment from those in luciferase cluster. A codon position under episodic selection or positive/diversifying selection was indicated as green and red arrow, respectively. A, 16 luz sequences with 292 sites, 7 of which are under episodic selection (green arrow), and 132 of which have been under strong purifying selection. B, sixteen h3h sequences with 554 sites, eight of which are under episodic selection and 204 of which have been under strong purifying selection. C, seventeen hisps sequences with 1893 sites, twenty-eight of which are under episodic selection, six of which are under positive/diversifying selection and 637 of which have been under strong purifying selection. D, fifteen cyp450 sequences with 567 sites, 17 of which are under episodic selection, and 131 of which have been under strong purifying selection.

22 A 0 1 10 11 12 13 14 15 16 17 18 19

0 2 20 21 22 23 24 25 26 27 28 29 3

0 30 31 32 33 34 35 36 37 38 39 4 40

0 41 42 43 44 45 46 47 48 49 5 50 51

10 log2tpm

0 52 53 54 55 56 57 58 59 6 60 61 62

0 63 64 65 66 7 8 9 MyN MyL YFB MyN MyL YFB MyN MyL YFB MyN MyL YFB MyN MyL YFB rimordia FB-stipeFB-cap rimordia FB-stipeFB-cap rimordia FB-stipeFB-cap rimordia FB-stipeFB-cap rimordia FB-stipeFB-cap P P P P P 10

5 Luciferase P450 hydroxylase Caffeopyruvate 0 Hispidin_synthase other

MyN MyL YFB MyN MyL YFB MyN MyL YFB MyN MyL YFB MyN MyL YFB MyN MyL YFB MyN MyL YFB rimordia FB-stipeFB-cap rimordia FB-stipeFB-cap rimordia FB-stipeFB-cap rimordia FB-stipeFB-cap rimordia FB-stipeFB-cap rimordia FB-stipeFB-cap rimordia FB-stipeFB-cap P P P P P P P

B Module 6 C Module 43 D Module 50

MyN MyL YFB MyN MyL YFB MyN MyL YFB FB-cap FB-cap FB-cap rimordia FB-stipe rimordia FB-stipe rimordia FB-stipe P P P

Fig. S15. The 67 co-expressed gene modules identified using the weighted correlation network analysis (WGCNA) (34, 35) across developmental stages in M. kentingensis. A, All 67 modules. Different line colour denotes the expression of different genes in the luciferase cluster: red line: luciferase (luz); green line: hispindin-3-hydroxylase (h3h); blue line: cytochrome P450 (cyp450); purple line: hispidin synthase (hisps). Yellow lines are two caffeylpyruvate hydrolase (cph) genes assigned to different modules. B, One of the cph (Mk_00163300) located in scaffold1 was assigned to module6, whereas C, the other cph (Mk_01107300) adjacent to the luciferase cluster was assigned to module43. D, The genes located in luciferase cluster including luz, h3h, cyp450, and hisps were assigned into the same module, module50.

23 0 1 10 100 101 102 103 104 105 106 11 12 15

0 13 14 15 16 17 18 19 2 20 21 22 23 15

0 24 25 26 27 28 29 3 30 31 32 33 34 15

0 35 36 37 38 39 4 40 41 42 43 44 45 15

0 46 47 48 49 5 50 51 52 53 54 55 56 15

5 log2tpm 0 57 58 59 6 60 61 62 63 64 65 66 67 15

0 68 69 7 70 71 72 73 74 75 76 77 78 15

0 79 8 80 81 82 83 84 85 86 87 88 89 15

VM RZ P1 P2-cap FB-cap 9 90 91 92 93 94 95 96 97 98 99 P2-stipeYFB-cap FB-stipeFB-gills YFB-stipe 15

VM RZ P1 VM RZ P1 VM RZ P1 VM RZ P1 VM RZ P1 VM RZ P1 VM RZ P1 VM RZ P1 VM RZ P1 VM RZ P1 VM RZ P1 P2-cap FB-cap P2-cap FB-cap P2-cap FB-cap P2-cap FB-cap P2-cap FB-cap P2-cap FB-cap P2-cap FB-cap P2-cap FB-cap P2-cap FB-cap P2-cap FB-cap P2-cap FB-cap P2-stipeYFB-cap FB-stipeFB-gills P2-stipeYFB-cap FB-stipeFB-gills P2-stipeYFB-cap FB-stipeFB-gills P2-stipeYFB-cap FB-stipeFB-gills P2-stipeYFB-cap FB-stipeFB-gills P2-stipeYFB-cap FB-stipeFB-gills P2-stipeYFB-cap FB-stipeFB-gills P2-stipeYFB-cap FB-stipeFB-gills P2-stipeYFB-cap FB-stipeFB-gills P2-stipeYFB-cap FB-stipeFB-gills P2-stipeYFB-cap FB-stipeFB-gills YFB-stipe YFB-stipe YFB-stipe YFB-stipe YFB-stipe YFB-stipe YFB-stipe YFB-stipe YFB-stipe YFB-stipe YFB-stipe

15 Module 4 Module 28 Module 33 Module 5

P1 P1 15 Module 15 Module 90 VM RZ VM RZ P2-cap FB-cap P2-cap FB-cap P2-stipeYFB-cap FB-stipeFB-gills P2-stipeYFB-cap FB-stipeFB-gills YFB-stipe YFB-stipe

5 Luciferase P450 hydroxylase Caffeopyruvate 0 Hispidin_synthase other

VM RZ P1 VM RZ P1 P2-cap FB-cap P2-cap FB-cap P2-stipeYFB-cap FB-stipeFB-gills P2-stipeYFB-cap FB-stipeFB-gills YFB-stipe YFB-stipe Fig. S16. The 107 co-expressed gene modules identified using the weighted correlation network analysis (WGCNA) (34, 35) across developmental stages in Armillaria ostoyae. Different line colour denotes the expression of different genes in the luciferase cluster: Red line: luciferase (luz); green line: hispindin-3-hydroxylase (h3h); blue line: cytochrome P450 (cyp450); yellow lines: two caffeylpyruvate

24 hydrolase (cph) genes. For the two cph, one was located in the luciferase cluster and was assigned to Ｍodule15 and the other was located outside of the cluster and was assigned to Module90.

570 806/4042 Panellus stiptiucs(tennessee) 891 1420 2144/1161 Mycena venus Expansion 703 332 1622/1638 Mycena sanguinolenta Contraction 8201 287 1293/1811 Mycena kentingensis 2200 142 1164/1354 Mycena chlorophus 588 934/1266 Mycena indigotica 122 262/1941 Armillaria ectypa FPL83.16 3506 1460/431 Armillaria tabescens CCBAS 213 229 904/1468 Armillaria ostoyae C18/9 417 459 492 733/1414 Armillaria solidipes 28-4 99 67 797 577 997/2106 Armillaria borealis FPL87.14 1208 74 1150 660 695 1064/1883 Armillaria cepistipes B5 922 216 1205 1487/949 Armillaria gallica 21-2 152 584/4369 Armillaria mellea ELDO17 3300 555/5355 Guyanagaster necrorhiza MCA 3950 165 981/856 Cylindrobasidium torrendii FP15055 5856 866/2255 Flammulina velutipes 506/1315 63 179 106 Neonothopanus gardneri 3913 160/1217 2542 2434 71 Omphalotus olearius 755 216 1494/1221 Gymnopus luxurians 302 981 791 1372/1660 Rhodocollybia butyracea CCBAS 279 411 396 279 2521/1230 Gymnopus androsaceus JB14 5312 2742/2715 Dendrothele bispora CBS 962.96 1194/4222 Marasmius fiardii PR-910 814/1498 Fistulina hepatica 10 98 718 783/432 Schizophyllum commune H4-8 508 60 1050 10849 742 956/498 Auriculariopsis ampla NL-1724 1560/2535 Flagelloscypha sp. PMI_526 229 347/1157 Amanita thiersii Skay4041 78 788 1056/800 Amanita muscaria Koide 4106 620/2060 Volvariella volvacea V23 0 773/4488 Coprinopsis cinerea 78 7 1164/1728 11154 143 188 Hypholoma sublateritium 213 1773 289 1403/636 Galerina marginata 491 451 708/1558 Hebeloma cylindrosporum h7 73 264 625 117 911/925 Laccaria amethystina LaAM-08-1 214 1239/564 Laccaria bicolor 543/5453 Agaricus bisporus var. burnettii JB137-S8 847/16618 Pleurotus ostreatus PC15 329 806/1846 Plicaturopsis crispa 142 548 2713/952 Fibulorhizoctonia sp. CBS 109695 14115 1345/2691 Paxillus involutus ATCC 200175

Fig. S17. Gene expansion along the species tree of 42 basidiomycetes. The expanded and contracted orthologous group (OG) count are coloured in red and green, respectively.

11 7 14 1 47 11 5 7 56 12 12 11 9 76 Moniliophthora roreri FA553 1 6 2 10 6 24 7 3 3 25 6 3 8 2 48 Moniliophthora perniciosa 5 44 1 0 25 19 0 26 91 33 18 14 24 104 Mycena sanguinolenta 0.8 4 17 5 0 46 17 1 47 90 25 18 14 29 153 Mycena venus 3 4 0 0 11 8 0 4 49 9 6 4 18 49 Panellus stipticus r 0.6 2 3 3 0 6 10 1 8 46 11 3 6 16 45 Mycena kentingensis 1 5 0 0 7 15 0 11 34 11 1 4 17 50 Mycena chlorophus P 1 3 9 0 15 6 0 11 31 8 0 6 10 41 Mycena indigotica 0.4 5 10 3 0 44 4 0 13 74 17 6 18 14 42 Armillaria ostoyae C18/9 5 12 3 0 41 4 0 11 75 18 8 11 14 44 Armillaria solidipes 28-4 0.2 5 12 5 0 48 4 0 13 78 21 9 21 14 45 Armillaria borealis FPL87.14 5 17 4 0 49 4 0 12 91 25 6 34 14 43 Armillaria cepistipes B5 5 16 4 0 49 5 0 16 76 23 10 22 14 41 Armillaria gallica 21-2 0 5 10 7 0 37 5 0 16 65 16 6 8 16 47 Armillaria mellea ELDO17 8 2 3 0 28 4 0 10 55 9 5 5 12 31 Armillaria ectypa FPL83.16 5 15 8 0 40 5 0 10 78 15 7 6 15 55 Armillaria tabescens CCBAS 213 5 4 2 0 30 4 0 10 50 9 3 5 14 34 Guyanagaster necrorhiza 3 2 11 0 5 13 0 2 33 13 1 4 12 24 Cylindrobasidium torrendii FP15055 2 3 20 0 13 2 0 6 27 6 1 3 13 18 Flammulina velutipes

4 15 11 0 51 5 0 12 64 14 3 8 5 55 Marasmius fiardii PR-910 5 17 2 0 45 5 0 6 88 48 9 11 13 88 Dendrothele bispora CBS 962.96 3 11 0 0 33 9 0 14 78 42 7 7 22 104 Gymnopus androsaceus JB14 6 7 0 0 77 6 0 12 74 23 5 8 11 48 Rhodocollybia butyracea CCBAS 279 8 8 0 2 45 11 0 11 82 29 9 10 10 51 Gymnopus luxurian 4 3 3 0 16 3 0 3 42 17 3 7 7 24 Omphalotus olearius 4 6 2 0 27 4 0 6 49 15 4 10 4 30 Neonothopanus gardneri

6 2 1 0 14 5 0 4 30 11 2 4 12 21 Fistulina hepatica 5 15 6 0 13 11 0 6 36 11 1 7 13 27 Schizophyllum commune H4-8 6 8 5 0 11 11 0 6 35 6 1 7 8 24 Auriculariopsis ampla NL-1724 4 2 18 4 32 4 1 2 23 2 2 7 35 55 Flagelloscypha sp. 3 4 12 0 29 2 0 2 26 3 1 6 1 26 Amanita thiersii 2 4 1 0 14 3 0 4 28 2 2 7 0 12 Amanita muscaria 2 1 5 3 11 3 0 3 34 10 1 4 4 14 Volvariella volvacea V23 5 4 1 0 34 4 0 6 33 6 2 4 3 23 Coprinopsis cinerea 2 6 3 2 36 3 0 8 32 9 4 4 7 46 Hypholoma sublateritium 1 9 2 3 60 2 0 2 37 8 5 9 6 35 Galerina marginata 1 3 3 0 19 3 0 2 26 4 3 5 5 17 Hebeloma cylindrosporum 1 3 2 2 9 1 0 2 22 5 2 9 4 15 Laccaria amethystina 1 4 2 0 13 2 0 1 28 6 2 9 4 16 2 2 2 0 20 3 0 5 31 5 3 4 0 21 Agaricus bisporus 8 4 8 1 27 3 0 8 33 10 1 4 5 29 Pleurotus ostreatus PC15 4 5 0 0 2 6 0 1 33 13 1 4 5 22 Plicaturopsis crispa 8 8 0 0 0 9 0 4 88 35 3 3 23 38 Fibulorhizoctonia sp. CBS 109695 5 2 0 2 27 3 0 1 46 13 5 10 7 37 Paxillus involutus ATCC 200175 Ce DPBB_1 NPP1 T R COeste r T A1 r r ase

Fig. S18. Protein family domain (Pfam) analysis across 44 fungal species. Some Pfam domains are related to plant pathogenicity. Moniliophthora perniciosa FA553 (17) from JGI. Monro: Moniliophthora roreri from BioProject PRJNA279170. The colours from blue to yellow denote the number rescaled between 0 to 1 (for each domain, copy number from 44 species is divided by the maximum number of copies). Number inside cells denote domain copy number. The names of protein domains are labelled in the horizontal axis and the names of species are labelled in the vertical axis. Abbreviations for Pfam domain IDs and domain functions: Cerato-platanin (PF07249;Cerato-platanin), DPBB_1 (PF03330; Expansin), Aspzincin_M35 (PF14521; deuterolysin), Aegerolysin (PF06355; Hemolysin), Hydrophobin

26 (PF01185; Hydrophobins), Thaumatin (PF00314; PR-5/Thaumatin family), NPP1 (PF05630; MpNEP1 & 2), Trp_halogenase (PF04820; Halogenase), AMP-binding (PF00501; NRPS-like synthase_1), NAD_binding_4 (PF07993; NRPS-like synthase_2), PS-DH (PF14765; Polyketide synthase), polyprenyl_synt (PF00348; Polyprenlyl Synthases), RTA1 (PF04479; RTA1_MDTM-Protein), and COesterase (PF00135; Carboxylesterase).

Fig. S19. DNA methylation levels between Nanopore long-reads and the Illumina BS-seqs in the methylomes of M. kentingensis. A total of 4,754,348 was compared with a Pearson correlation coefficient of 0.962.

28 III. SUPPLEMENTARY TABLES

Table S1. Summary of Illumina and Oxford Nanopore reads.

Number Library size Number of Depth of Species Read type Read length N50 of runs (bp) reads coverage M. chlorophos Nanopore 2 1,000-202,448 19930 9,375,866,055 965,876 184.3 M. indigotica Nanopore 3 1,000-345,966 13971 15,277,834,747 2,240,375 209.6 M. kentingensis Nanopore 3 1,000-293,280 26695 11,220,635,883 940,766 172.6 M. sanguinolenta Nanopore 2 1,000-226,007 19308 9,755,045,881 1,386,206 58.4 M. venus Nanopore 5 1,000-823,071 12272 21,979,383,148 4,187,862 135.5 M. chlorophos Illumina 3 151 N/A 5,019,692,396 33,242,996 98.7 M. indigotica Illumina 1 151 N/A 5,957,541,014 39,453,914 81.7 M. kentingensis Illumina 3 151, 301 N/A 4,118,183,612 21,304,412 63.3 M. sanguinolenta Illumina 4 151 N/A 18,276,688,774 121,037,674 109.3 M. venus Illumina 3 151 N/A 18,266,335,610 120,969,110 112.6

Table S2. Genome summaries of five Mycena species. Genome haploid length and heterozygosity of each species was calculated from Illumina reads using GenomeScope 2.0 (32)

M. chlorophos M. indigotica M. kentingensis M. sanguinolenta M. venus Abbreviation in this study Mchl Mind Mken Msan Mven Genome haploid length estimate (Mb) 58.2 80.8 62.0 148.7 149.9 Canu (36) assembly size (Mb) 75.4 73.5 97.1 263.7 265.4 Haplotype aware assembly size (Mb) 50.9 72.9 65.0 167.2 162.2 Estimated heterozygosity (%) 2.92 0.03 1.7 2.71 2.35 Genome size (Mb) 50.9 72.9 65.0 167.2 162.2 scaffold number (n) 80 30 61 155 79 N50 (Mb) 2.0 5.1 3.0 5.4 5.5 L50 9 7 8 10 10 N90 (Mb) 478,732 1,639,376 968,399 608,068 1,860,701 L90 27 15 20 43 29 Quality Value (QV) 31.1 36.3 36.8 34.0 34.2 Number of genes 14,274 14,334 15,046 25,350 26,518 Gene length (Mb) 26.4 24.5 28.5 44.0 46.0 Exon number 89,139 90,123 96,716 169,212 181,086 Exon length (Mb) 20.3 20.1 21.8 33.5 35.2 Intergene length (Mb) 24.5 48.4 36.5 123.1 116.2

Table S3. BUSCO summaries of five Mycena species based on 1,764 basidiomycete markers.

Complete Single Duplicate Fragmented Missing M. venus 92.1% 86.1% 6.0% 2.0% 5.9% M. sanguinolenta 92.8% 88.1% 4.7% 2.0% 5.2% M. kentingensis 94.2% 89.2% 5.0% 1.0% 4.8% M. chlorophos 95.3% 89.1% 6.2% 0.7% 4.0% M. indigotica 94.8% 92.7% 2.1% 0.6% 4.6%

31 Table S4. Summaries of 37 representative fungal species used in this study

Assembly Reference Assembly size Number of Species abbreviation Species version (bp) scaffolds Armosto1 Armillaria ostoyae C18/9 JGI; Sipos et al. (37) 60,106,801 106 Armost1 Armillaria solidipes 28-4 v1.0 JGI; Sipos et al. (37) 58,009,494 229 Armbor1 Armillaria borealis FPL87.14 v1.0 JGI with permission 71,689,880 864 Armcep1 Armillaria cepistipes B5 JGI; Sipos et al. (37) 75,828,441 287 Armga1 Armillaria gallica 21-2 v1.0 JGI; Sipos et al. (37) 85,336,812 319 Armmel1 Armillaria mellea ELDO17 v1.0 JGI with permission 70,856,304 474 Armect1 Armillaria ectypa FPL83.16 v1.0 JGI with permission 40,598,130 33 Armtab1 Armillaria tabescens CCBAS 213 v1.0 JGI with permission 74,875,987 659 Guyne1 Guyanagaster necrorhiza MCA 3950 v1.0 JGI with permission 53,686,691 168 Cylto1 Cylindrobasidium torrendii FP15055 v1.0 JGI; Floudas et al. (38) 31,574,086 1,149 Fvel Flammulina velutipes JGI with permission 35,642,541 11 Rhobu1 Rhodocollybia butyracea CCBAS 279 v1.0 JGI with permission 78,814,333 1,836 Gymlu1 Gymnopus luxurians v1.0 JGI; Kohler et al. (39) 66,281,680 383 Gyman1 Gymnopus androsaceus JB14 v1.0 JGI; Barbi et al. (40) 89,149,538 2,516 Ompol1 Omphalotus olearius JGI; Wawrzyn et al. (41) 28,145,093 868 NEOGA Neonothopanus gardneri Kotlobay et al. (42) 46,745,715 293 Denbi1 Dendrothele bispora CBS 962.96 v1.0 JGI; Varga et al. (43) 130,650,616 3,942 Marfi1 Marasmius fiardii PR-910 v1.0 JGI with permission 59,447,912 1,124 PANBI Panellus stiptiucs(tennessee) Kotlobay et al. (42) 39,545,155 571 Schco3 Schizophyllum commune H4-8 v3.0 JGI; Ohm et al (44) 38,670,379 25

32 Auramp1 Auriculariopsis ampla NL-1724 v1.0 JGI; Almasi et al. (45) 49,873,921 351 Fishe1 Fistulina hepatica v1.0 JGI; Floudas et al. (38) 33,847,808 588 FlaPMI526_1 Flagelloscypha sp. PMI_526 v1.0 JGI with permission 72,728,845 397 Galma1 Galerina marginata v1.0 JGI; Riley et al. (46) 59,418,196 414 Hebcy2 Hebeloma cylindrosporum h7 v2.0 JGI; Kohler et al. (39) 38,226,047 176 Hypsu1 Hypholoma sublateritium v1.0 JGI; Kohler et al. (39) 48,031,814 704 Lacam2 Laccaria amethystina LaAM-08-1 v2.0 JGI; Kohler et al. (39) 52,581,404 1,299 Lacbi2 Laccaria bicolor JGI; Kohler et al. (39) 60,707,050 55 Copci1 Coprinopsis cinerea JGI; Stajich et al. (47) 36,294,355 94 Agaricus bisporus var. burnettii JGI; Morin et al. (48) Agabi_varbur_1 32,614,401 2,016 JB137-S8 Amath1 Amanita thiersii Skay4041 v1.0 JGI; Hess et al. (49) 33,689,220 1,446 Amamu1 Amanita muscaria Koide v1.0 JGI; Kohler et al. (39) 40,699,759 1,101 Volvo1 Volvariella volvacea v23 JGI; Bao et al. (50) 35,720,033 62 PleosPC15_2 Pleurotus ostreatus PC15 v2.0 JGI; Riley et al. (46) 34,343,005 12 Plicr1 Plicaturopsis crispa v1.0 JGI; Kohler et al. (39) 34,498,416 316 Fibsp1 Fibulorhizoctonia sp. CBS 109695 v1.0 JGI; Nagy et al. (51) 95,125,689 1,918 Paxin1 Paxillus involutus ATCC 200175 v1.0 JGI; Kohler et al. (39) 58,301,126 2,681

33 Table S5. Statistics of orthologous groups (OGs) of five Mycena species. A total of 22,244 OGs were inferred in 42 species.

Species Number of OGs Species-specific OGs Singleton M. chlorophos 9048 1378 1357 M. indigotica 9055 1329 1310 M. kentingensis 9295 1587 1560 M. sanguinolenta 12345 3588 3547 M. venus 12471 3374 3348

34 Table S6. Intron length (bp) of intron-containing genes in five Mycena mitogenomes. Brackets denote of number of introns. Seven genes (nad3, nad4, nad4L, nad6, atp6, atp8 and atp9) are intron free.

M. chlorophos M. indigotica M. kentingensis M. sanguinolenta M. venus rns 1,632 (1) 2,923 (2) 0 (0) 0 (0) 0 (0) rnl 4,611 (5) 5,817 (5) 3,584 (4) 0 (0) 0 (0) cob 10,295 (6) 14,753 (8) 7,632 (6) 1,627 (1) 1,259 (1) cox1 22,578 (12) 17,250 (10) 13,223 (7) 2,832 (2) 1,348 (1) cox2 1,088 (1) 2,546 (2) 3,050 (2) 0 (0) 0 (0) cox3 3,397 (2) 3,512 (2) 1,531 (1) 423 (1) 1,295 (1) nad1 1,093 (1) 639 (1) 1,079 (1) 0 (0) 0 (0) nad2 5,116 (3) 2,892 (2) 1,563 (1) 0 (0) 0 (0) nad5 5,799 (4) 0 (0) 4,426 (3) 0 (0) 3,223 (2) sum 55,609 (35) 50,332 (32) 36,088 (25) 4,882 (4) 7,125 (5)

35 Table S7. Repeat content of five Mycena genomes

Species name SINES LINES LTRs DNA Unclassified Small Satellite Simple Low Sum (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) M. chlorophos 0.14 0.29 5.22 1.4 3.97 0 0 0.62 0.08 11.72 M. kentingensis 0 1.06 6.54 1.53 8.33 0 0.04 0.67 0.1 18.27 M. indigotica 0 0.99 19.71 2.63 5.28 0.37 0 0.43 0.07 29.48 M. sanguinolenta 0.7 1.99 9.69 3.91 21.58 0.16 0.08 0.8 0.07 38.98 M. venus 0 1.7 11.09 4.01 17.94 0.09 0.06 0.75 0.06 35.7

Table S8. Unknown and relic repeat content (%) of genomes.

Unknown LTR relic LINE DNA relic All Relic (%) (%) relic (%) (%) (%) M. chlorophos 2.73 0.91 0.16 0.04 1.11 M. kentingensis 6.85 0.70 0.36 0.32 1.38 M. indigotica 3.96 0.99 0.06 0.28 1.34 M. sanguinolenta 13.56 4.33 0.93 2.13 7.38 M. venus 10.46 4.62 0.56 1.37 6.55

36 Table S9. CG methylation in genes, TEs, relic TEs, and unknown repeats.

M. chlorophos M. indigotica M. kentingensis M. sanguinolenta M. venus Gene (%) 10.5 10.1 6.6 6.2 5.4 DNA (%) 43.3 57.9 45.4 17.0 13.1 relic DNA (%) 34.1 41.9 32.9 14.9 12.0 LINE (%) 58.2 62.9 27.9 11.6 11.8 relic LINE (%) 27.8 48.5 22.4 9.1 9.0 LTR (%) 73.0 70.0 36.5 26.9 18.0 relic LTR (%) 55.0 58.9 28.1 16.1 14.0 Unknown (%) 48.7 42.1 29.5 13.7 10.9

37 Table S10. Expanded, contracted, other gene counts in five species (from CAFÉ analysis) in high or low synteny regions. The genes that overlap in two regions were not included.

high synteny low synteny M. chlorophos expanded genes 1,621 2,852 contracted genes 315 489 other genes 5,124 3,795 M. indigotica expanded genes 1,145 3,091 contracted genes 300 433 other genes 5,288 4,046 M. kentingensis expanded genes 979 4,122 contracted genes 181 529 other genes 3,787 5,400 M. sanguinolenta expanded genes 2,985 7,333 contracted genes 389 671 other genes 7,026 6,900 M. venus expanded genes 4,008 9,001 contracted genes 230 339 other genes 6,875 6,020

38 Table S11. Annotation of gene function in the 29 OGs containing at least one upregulated gene in the four bioluminescent Mycena species.

OG number OG annotation OG0009249 luciferase OG0000706 FAD/NAD(P)-binding domain-containing protein OG0002489 hispidin synthase/polyketide synthetase OG0000386 FAD-binding domain-containing protein/reticuline oxidase/ Glucooligosaccharide oxidase OG0000288 FAD/NAD(P)-binding domain-containing/ halogenase OG0000215 FAD/NAD(P)-binding domain-containing protein/ monooxygenase OG0001192 NAD(P)-binding protein/D-xylose 1-dehydrogenase (NADP(+)) 2 OG0001818 Eukaryotic aspartyl protease/ acid protease OG0000591 Peptidase S41 family protein ustP OG0000147 ABC transporter transmembrane region OG0000398 transporter OG0000593 predicted protein (pfam:SNARE_assoc) OG0000348 predicted protein (only 4 gene have pfam annotation) OG0000253 predicted protein (no any species has Pfam) OG0000022 predicted protein (pfam:Fungal_trans +Zn_clus) OG0000345 GH79 OG0000143 GH67/alpha-L-rhamnosidase-like protein OG0000048 glycoside hydrolase/retinol dehydrogenase 12/delta-9 fatty acid desaturase protein OG0000014 carboxypeptidase s1/Uncharacterized hydrolase/acid protease

39 OG0000134 cytochrome P450/ Bifunctional P-450/NADPH-P450 reductase OG0000005 cytochrome P450 OG0000066 Zinc finger protein OG0000034 transcription initiation factor IIA gamma subunit/predicted protein OG0000026 acetyl-CoA synthetase-like protein/NRPS (nonribosomal peptide synthetase)-like enzyme OG0000007 ankyrin repeat protein OG0000006 transmembrane protein OG0000097 argonaute-like protein OG0000004 kinase-like protein/GH76/argonaute-like protein OG0000000 short-chain dehydrogenase/reductase family protein/ubiquitin family protein/glycoside hydrolase family 7 protein/MFS general substrate transporter/NAD(P)-binding protein

40 Table S12. Scaffolds containing telomeric repeats at ends.

Scaffold name Scaffold length End Repeat start Repeat end Number of copies Mc.scaff0010 1,898,893 5’ 1 189 30.8 Mc.scaff0018 1,039,458 5’ 1 160 26.8 Mind.scaff0001 6,537,710 5’ 10 117 18.3 Mind.scaff0001 6,537,710 3’ 6,537,600 6,537,710 19.2 Mind.scaff0002 6,307,876 3’ 6,307,754 6,307,876 21 Mind.scaff0003 5,994,024 3’ 5,993,915 5,994,024 19.3 Mind.scaff0006 5,133,973 3’ 5,133,894 5,133,973 14 Mind.scaff0007 5,077,951 3’ 5,077,842 5,077,951 18.8 Mind.scaff0008 5,050,887 5’ 1 97 17 Mind.scaff0009 4,505,476 3’ 4,505,343 4,505,476 22.5 Mind.scaff0011 3,413,854 3’ 3,413,723 3,413,854 22.3 Mind.scaff0013 2,685,629 3’ 2,685,517 2,685,629 19.8 Mind.scaff0015 1,639,376 5’ 8 133 21.3 Mk.scaff0001 5,940,114 5’ 1 136 23 Mk.scaff0001 5,940,114 3’ 5,939,956 5,940,114 26.5 Mk.scaff0003 4,788,751 5’ 1 165 27.5 Mk.scaff0005 4,011,168 3’ 4,011,018 4,011,168 25.2 Mk.scaff0006 3,839,597 5’ 1 144 23.7 Mk.scaff0008 3,039,429 5’ 1 159 26.7 Mk.scaff0009 2,763,528 5’ 1 165 27.8

41 Mk.scaff0012 2,261,502 5’ 1 159 26.7 Mk.scaff0013 2,164,973 3’ 2,164,823 2,164,973 25.2 Mk.scaff0014 2,112,720 3’ 2,112,566 2,112,720 26.3 Mk.scaff0016 1,974,397 5’ 2 156 26.3 Mk.scaff0017 1,721,327 3’ 1,721,151 1,721,327 29.5 Mk.scaff0019 1,349,653 5’ 1 151 25 Msan.scaff0001 17,789,092 3’ 17,789,025 17,789,090 11 Msan.scaff0025 1,760,574 3’ 1,760,504 1,760,574 11.5 Msan.scaff0027 1,650,193 3’ 1,650,124 1,650,193 11.3 Mven.scaff0003 11,118,782 3’ 11,118,677 11,118,782 17.2 Mven.scaff0004 8,657,829 3’ 8,657,762 8,657,829 11.3 Mven.scaff0007 6,670,972 5’ 1 70 11.8 Mven.scaff0008 6,224,985 3’ 6,224,891 6,224,985 15.8 Mven.scaff0011 5,452,461 3’ 5,452,365 5,452,461 16 Mven.scaff0012 5,262,452 5’ 1 125 20.7 Mven.scaff0013 4,843,808 3’ 4,843,711 4,843,808 16.3 Mven.scaff0014 4,029,179 5’ 1 100 16.7 Mven.scaff0015 3,840,008 3’ 3,839,974 3,840,008 5.8 Mven.scaff0020 3,032,789 3’ 3,032,670 3,032,789 20 Mven.scaff0028 2,074,661 5’ 1 94 15.7

Table S13. Bisulfite conversion rate from bisulfite sequences aligned to the genome assemblies of M. kentingensis.

Species Replicate Number of After PCR Number of Mappability DNA methylation level Coverage Bisulfite raw reads duplicate uniquely (%) (%) (depth) per conversion removing mapped strand (X) rate (%)* and adapter trimming CG CHG CHH M. 1 39,272,420 35,864,994 21,411,409 59.7 15 1.7 1.75 19.23 98.95 kentingensis

2 37,212,556 33,627,150 20,205,636 60.09 14.48 1.6 1.62 18.02 99.06 *The bisulfite conversion rate was estimated by spiking in unmethylated Lambda Phage DNA into the Mycena BS libraries. We mapped BS- reads to Lambda Phage genome, so any unconverted cytosines revealed the failed bisulfite conversion and were considered false positives.

43 Dataset S1 (separate file). Statistics of RNAseq reads and coverage.

Dataset S2 (separate file). OG number in 42 species.

Dataset S3 (separate file). Differentially-expressed genes between mycelia with different bioluminescent intensities in four Mycena species. For M. kentingensis and M. chlorophos, genes were identified between two tissues with contrasting bioluminescence intensity. For M. sanguinolenta and M. venus, genes were identified by significant correlation between bioluminescent intensity and expression level.

Dataset S4 (separate file). Differentially-expressed genes between the cap and stipe of the fruit body in Mycena kentingensis. The genes with red color located in luciferase cluster.

Dataset S5 (separate file). The genes assigned to module50 from the analysis of WGCNA during the developmental stages in M. kentingensis. The genes with red color located in luciferase cluster.

Dataset S6 (separate file). Molecular function of GO terms enriched in proteins of 589 OGs expanded at the origin of the mycenoid lineage.

Dataset S7 (separate file). Pfam domain number in 42 species.

Dataset S8 (separate file). 537 protein domains enriched in the mycenoid lineage. Protein domains expanded (Wilcoxon rank sum test, p < 0.01; 1-fold higher copy number) in six mycenoid species compared to the other 36 species.

Dataset S9 (separate file). The diameter of the mycelium and its bioluminescence for seven days.

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