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1 Native and invasive populations of the ectomycorrhizal death cap phalloides 2 are highly sexual but dispersal limited 3 4 Jacob Golan1, Catherine A. Adams2, Hugh Cross3, Holly Elmore4, Monique Gardes5, Sydney 5 I. Glassman6, Susana C. Gonçalves7, Jaqueline Hess8, Franck Richard9, Yen-Wen Wang1, 6 Benjamin Wolfe10, Anne Pringle1 7 8 1. Departments of Botany and Bacteriology, University of Wisconsin-Madison, Madison, 9 WI, 53706, USA 10 2. Department of Plant and Microbial Biology, University of California-Berkeley, Berkeley, 11 CA, 94720, USA 12 3. Department of Anatomy, University of Otago, Dunedin, 9054, New Zealand 13 4. Department of Organismic & Evolutionary Biology, Harvard University, Cambridge, MA, 14 02138, USA 15 5. UMR 5174 EDB UPS-CNRS-IRD, Université Paul Toulouse III, Toulouse, 31062 Cedex 16 9, France 17 6. Department of Microbiology and Plant Pathology, University of California-Riverside, 18 Riverside, CA, 92521, USA 19 7. Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, 20 Coimbra, 3000-456, Portugal 21 8. Department of Botany and Biodiversity Research, University of Vienna, A-1030, Austria 22 9. CEFE - CNRS Université de Montpellier, Université Paul-Valéry EPHE IRD INSERM 23 Campus CNRS, 1919 Route de Mende, 34293, Montpellier, France 24 10. Department of Biology, Tufts University, Medford, MA, 02155, USA. 25 26 Author for correspondence: 27 Jacob Golan 28 Tel: +1 608-890-4364 29 Email: [email protected] 30

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31 Key words: fungi, genet, global change, invasion biology, movement ecology, mutualism, 32 population genomics, spore 33 34 Summary: 200 35 Main body word count: 5,672 36 Introduction word count: 773 37 Materials and Methods word count: 2,412 38 Results word count: 1,214 39 Discussion word count: 1,273 40 Body figures: 7 (all color) 41 Body tables: 1 42 Supporting information figures: 5 (all color) 43 Supporting information tables: 3 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61

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62 Abstract 63 1. The ectomycorrhizal death cap Amanita phalloides is native to but invasive in 64 . To understand whether the spreads underground using hyphae, or above 65 ground using sexual spores, we mapped and genotyped sporocarps from European and American 66 populations. Larger genetic individuals (genets) would suggest spread mediated by vegetative 67 growth, while many small genets would suggest dispersal mediated by spores. To test whether 68 genets are ephemeral or persistent, we also sampled from the same invasive populations over 69 time. 70 2. We mapped 13 European and American populations between 2004-2007 and characterized 71 each using amplified fragment length polymorphisms (AFLP). In 2014 and 2015, we resampled 72 populations in California and added three new European populations. These populations and a 73 subset of the specimens originally collected in 2004 were characterized using whole genome 74 sequencing. 75 3. In every population and across all time points, sporocarps resolve into small, apparently 76 short-lived genets. Sporocarps nearer each other are more closely related, suggesting spores land 77 and germinate near parent sporocarps. 78 4. A. phalloides uses spores to move across landscapes. Spores travel very short distances 79 and individuals appear ephemeral. The death cap’s life history suggests yearly 80 removal as a strategy for control of this deadly fungus. 81 82 83 84 85 86 87 88 89 90 91 92

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93 Introduction 94 Invasions drive global change (Vitousek et al., 1997; Dukes & Mooney 1999; Ravi et al., 95 2009) but invasion biology often focuses on plants and animals in isolation (Shea & Chesson 96 2002), ignoring the role mutualists may play in invasion dynamics. Mutualist ectomycorrhizal 97 (ECM) fungi provide resources to plants in exchange for photosynthetically derived carbon 98 (Smith & Read, 2008), and consequently introduced or invasive ECM fungi impact local 99 ecosystems (Litchman 2010). But while the mechanisms enabling the spread of invasive plants, 100 and the roles of ECM in facilitating plant invasions, are increasingly documented (Welk et al., 101 2002; Williams et al., 2005; Barker et al., 2017), the mechanisms enabling the spread of invasive 102 ECM fungi in association with native plants are scarcely explored (Desprez-Loustau et al., 2007, 103 but see Nuñez et al., 2013). 104 Although invasions by plants are often mediated by symbiotic associations with introduced 105 or invasive ECM fungi (and vice versa; Richardson et al., 2000; Hayward et al., 2015; Teste et 106 al., 2019), introduced ECM fungi can also invade independently of introduced or invasive plants 107 (Díez et al., 2005; Pringle et al., 2009; Berch et al., 2016; Vargas et al., 2019). A fungus 108 invading on its own is likely to have an invasion dynamic distinct from a fungus associating with 109 an invasive plant (Dickie et al. 2017). However data tracking ECM invasions are limited, as are 110 data describing potential impacts. Open questions include whether these fungi persist as 111 mutualists, behave as parasites, or displace native ECM species (Chapela et al., 2001; Vellinga et 112 al., 2009). 113 The death cap Amanita phalloides is an invasive ECM fungus spreading through the endemic 114 coastal live woodlands of California (Pringle & Vellinga, 2006, Pringle et al., 2009, Wolfe et 115 al., 2010, Wolfe & Pringle 2011). Native to Europe, it now grows across the Southern 116 Hemisphere, but its invasion dynamic is best described in North America, and specifically in the 117 United States. The fungus first appeared in California and the Northeast U.S. in the mid-20th 118 century (Pringle et al., 2009), and it has since spread through California and north into 119 Washington and British Columbia (Ammirati et al., 1977; Pringle et al., 2009; Berch et al., 120 2016). In contrast, populations on the East Coast appear confined within initial points of 121 introduction, despite nearly 50 years of monitoring (Tanghe & Simons 1973; Tanghe, 1983; 122 Pringle & Vellinga, 2006).

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123 While the death cap is clearly invading California, how it spreads is unknown. Fungi 124 reproduce and disperse using vegetative fragments or spores (sexual or asexual). At one time 125 spores were assumed to mediate dispersal across continents and oceans (Peay et al., 2010; Golan 126 & Pringle 2017), but recent data suggest the sexual spores of many forming 127 Basidiomycete fungi fall just next to parent sporocarps (Galante et al. 2011; Peay & Bruns 128 2014). Steady vegetative growth can result in enormous mycelia (e.g., Armillaria spp.), but 129 whether fragmentation enables colonization of empty habitats is often unclear (Smith et al., 130 1992; Anderson et al., 2018). 131 Directly observing the dispersal of fungi in nature is challenging. Spores are difficult to track 132 and often, vegetative mycelia are hidden in substrates. But dispersal can be inferred by extending 133 the concept of a genetic individual, or genet (c.f. Harper, 1977; a term originally developed by 134 plant demographers) to include fungi. In the context of A. phalloides, we define a genet as the 135 body (or mycelium) generated by the fusion of two germinating, haploid (or monokaryotic) 136 spores, and assume the diploid body (or the dikaryon) is the dominant phase of the life cycle 137 (Rayner, 1991; Dahlberg & Stenlid, 1994; Anderson & Kohn, 1995; Booth 2014). Vegetative 138 growth would result in large genets, while frequent colonization by sexual spores would result in 139 many small genets (Dahlberg & Stenlid, 1994). Practically, genet size is measured by genotyping 140 sporocarps. Many genetically identical sporocarps scattered across a habitat characterize large 141 genets; distances among genetically identical sporocarps define the size of the genet. Genets of 142 other ECM species range from centimeters to hundreds of meters (Gryta et al., 1997; Redecker et 143 al., 2001; Sawyer et al., 2001; Bergemann et al., 2002; Dunham et al., 2003; Lian et al., 2006; 144 Rubini et al., 2011), and like these fungi, A. phalloides may propagate using either fragments or 145 spores, or both. 146 By physically mapping and genotyping sporocarps from multiple populations in European 147 and American forests, and from the same Californian sites over time, we generated data on genet 148 size to ask 1) does the death cap establish across landscapes using vegetative hyphae or sexual 149 spores, 2) can the same individuals persist in a population over time, and 3) does genet size differ 150 between native and invasive ranges? 151 152 153

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154 Materials and Methods 155 Collections and Mapping 156 Sporocarps of A. phalloides were collected from populations throughout Europe and the 157 United States (Fig. 1, Table 1, Supporting Information Table S1a,b). We define a population as a 158 group of sporocarps occurring within an area no larger than 75m by 75m. Most populations are 159 kilometers away from each other (Fig. 1), but at Point Reyes National Seashore (PRNS) in 160 California, individuals are continuously distributed along Limantour Road. Populations from 161 PRNS were delineated arbitrarily by walking across the road or at least 75m away before 162 collecting and naming a distinct population (Wolfe & Pringle, 2011). Global Position System 163 (GPS) coordinates were recorded for each location within 5 m of accuracy (Table 1). Due to 164 small-scale GPS inacuracies, we are unable to overlay the spatial maps of sporocarps from 165 different years (when we sampled over time at a single site). 166 The majority of sporocarps were collected from forests dominated by native species of 167 Fagaceae and Pinaceae. Populations from the former Centre d’Ecologie des Système Aquatiques 168 Continentaux (CESAC) surrounded a planted Cedrus libani on what is now the Marvig campus 169 of the CNRS Institute and Toulouse III University, and the population from the Escola Superior 170 Agrária de Coimbra was collected from a disturbed site on the Polytechnical Institute of Coimbra 171 campus. In New Jersey, A. phalloides grows in planted forests of native Pinus strobus (Wolfe et 172 al., 2010; Wolfe & Pringle, 2011), and in Rochester, NY, in a municipal park with Pinus strobus, 173 P. resinosa, canadiensis, and Betula spp. In California, A. phalloides were collected from 174 relatively undisturbed coast live oak woodlands in association with (Wolfe & 175 Pringle, 2011). 176 Every sporocarp in a population consisting of more than two sporocarps was carefully 177 mapped using one of three mapping methods depending on the tools available at each site. A full 178 description of each mapping method can be found in Supporting Information Methods S1. 179 180 DNA extraction and sequencing 181 Amplified Fragment Length Polymorphism (AFLP) data were generated between 2005 and 182 2007 from a total of 221 sporocarps, and genome sequence data were generated between 2015 183 and 2016 from a total of 86 sporocarps (Table 1). Approximately 50 mg of cap tissue from each 184 sporocarp was placed in a 2.0 ml microcentrifuge tube with four to five 3 mm glass beads, and

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185 macerated using a MiniBeadbeater-8 (BioSpec Products Inc., Bartlesville, OK) set at 75% speed 186 for one minute. For AFLP samples, genomic DNA was extracted from tissue using a Qiagen 187 DNeasy Tissue kit (Qiagen, Hilden, Germany) according to manufacturer’s specifications. Any 188 extractions of low quality (as measured on a NanodropTM Spectrophotometer [NanoDrop 189 Technologies, Wilmington, DE]) were re-extracted. Extraction blanks were extracted along with 190 samples and used as a control to test for potential contamination. 191 For genome samples, 700 µl of CTAB buffer (2% CTAB, 2% PVP, 100 mM Tris-HCL [pH 192 8.0], 20 mM EDTA [pH 8.0], and 1.4 mM NaCl) was added after maceration, and samples were 193 left to incubate at 60ºC for one hour. Next, 700 µl of a 24:24:1, by volume, of a 194 phenol:chloroform:isoamyl alcohol solution was added to each sample and samples were gently 195 mixed at room temperature for ten minutes, followed by centrifugation at room temperature at 196 13,000 rpm for ten minutes. The aqueous phase (~650-700 µl) of each sample was then carefully 197 transferred to a new 2.0 ml tube. 700 µl of the phenol:chloroform:isoamyl alcohol solution was 198 again added to each sample, and samples were inverted and mixed at room temperature for ten 199 minutes, followed by centrifugation at room temperature at 13,000 rpm for 10 minutes, at which 200 point the aqueous phase was again transferred to a new 2.0 ml tube. Approximately 1,400 ml of 201 100% ethanol was added to each sample, and samples were incubated at -20ºC for 45 minutes, 202 and then centrifuged at 4ºC at 13,000 rpm for ten minutes. The supernatant was discarded, and 203 the pellet dried on a ThermoSci DNA SpeedVac at room temperature for ten minutes, or until 204 dry, and finally resuspended in 400 µl of 10 mM Tris-HCL (pH 8.0) and transferred to a new 1.5 205 ml tube. To further purify genomic DNA, 12 µl of RNase A (Qiagen, Hilden, Germany) was 206 added to each sample, and each sample incubated at 37ºC for one hour. 16 µl of 5 M NaCl and 207 860 µl of 100% ethanol were then added to each tube and the solution left to precipitate at -20ºC 208 for one hour, after which each tube was centrifuged at 4ºC at 13,000 rpm for 15 minutes and the 209 supernatant discarded. A final washing was performed with 500 µl of 75% ethanol; solutions 210 were centrifuged at 4ºC at 13,000 rpm for ten minutes and the supernatant discarded. Finally, the 211 resulting pellet was resuspended in 200 µl of 10mM Tris-HCl (pH 8.0). 5 ml of an Oxygen 212 AxyPrep Mag PCR Clean-Up kit (Fisher Scientific, Pittsburg, PA, USA) was used per 213 manufacturer instructions to remove any remaining impurities. DNA was stored at -80ºC until it 214 was provided to the University of Wisconsin-Madison Biotechnology Center. 215

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216 Amplification and visualization of AFLP markers 217 The AFLP protocol is a DNA fingerprinting technique that does not require prior DNA 218 sequence information as it is based on a selective polymerase chain reaction (PCR) amplification 219 of adaptor-ligated restriction fragments formed from digested genomic DNA (Vos et al., 1995). 220 Mutations at restrictions sites result in the presence or absence of fragments of different sizes and 221 enable individuals to be distinguished from one another. AFLP data were generated for 222 sporocarps collected from 2004-2007, before genome data of A. phalloides were available, and at 223 the time AFLP was a genetic fingerprinting method of choice. 224 We adapted the AFLP protocol of the Applied Biosystems Microbial Fingerprinting Kit 225 (Applied Biosystems [ABI], Foster City, CA, USA) for use with our samples. Typically, ligated 226 fragments are amplified with increasingly selective primers in order to randomly subset the 227 number of fragments to a quantifiable number. But to generate consistent results, we first 228 amplified with no selective primers, and then proceeded to the +1 preselective (one additional 229 base pair on the primer) and +2 (two additional base pairs) selective amplifications. Moreover, 230 because A. phalloides has a smaller genome (~45 mb) than most organisms for which the ABI 231 AFLP kit is typically used, we modified the protocol to use less selective primers: we designed 232 +2 selective primers for the MseI restriction site, instead of the +3 typically used on larger 233 genomes. The primer combinations we used were: EcoRI-AC/MseI-CT (where AC are the two 234 additional base pairs on the EcoRI site and CT are the two base pairs on the MseI site), EcoRI- 235 TG/MseI-CC, EcoRI-AC/MseI-CT. Amplification products were denatured in formamide and 236 visualized on a 3730 ABI capillary sequencer. Data were analyzed using GeneMapper version 237 4.0 (ABI). Potential markers were scored using GeneMapper, and then inspected by eye. Clear 238 presence/absence patterns were recorded as 1 or 0, respectively. A total of 102 loci across 221 239 specimens were recovered and converted to a GenAlEx v6.5 format for downstream analyses 240 (Peakall & Smouse, 2012). 241 242 Genome sequencing, read filtering and variant calling 243 In 2016, genomes were sequenced using an Illumina HiSeq2500 platform at the University of 244 Wisconsin-Madison Biotechnology Center, typically with a 550 bp insert size and 251 bp paired- 245 end reads (13 specimens were prepared with 350-bp inserts; often these specimens represent 246 older collections [Supporting Information Table S1b]). Sequencing was performed using two

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247 flow cells with 48 samples and two lanes each (ten samples either failed quality control or were 248 irrelevant to this study). Mean sequencing depth of each sample ranged from 10.56 to 150.86 249 (Supporting Information Table S1b). Sequence data were filtered using Trim Galore! (v0.4.5) 250 (https://github.com/FelixKrueger/TrimGalore). Adapter trimming was set to the highest 251 stringency such that even a single nucleotide of overlap with the adapter sequence was trimmed 252 from a given read. After trimming, reads reduced to 100 bp or shorter, and those with quality 253 scores less than 30 were discarded. 254 A sporocarp from Dunas de Mira, São Jacinto, Portugal (specimen number 10511) was also 255 sequenced on four SMRTcells on a PacBio RS II Sequel platform, also at the University of 256 Wisconsin-Madison Biotechnology Center (Supporting Information Table S1b). This resulted in 257 raw coverage of 47x with N50 read length of 6,310 bp. 258 After troubleshooting of genome assembly pipelines (summarized in Supporting Information 259 Table S2), the final assembly was performed using an in-house hybrid approach. First, Illumina 260 data were subjected to a second round of filtering using Trimmomatic v0.35 (Bolger et al., 2014) 261 with the following parameters: ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10 CROP:245 262 LEADING:30 TRAILING:30 SLIDINGWINDOW:4:25 MINLEN:100. PacBio data were 263 filtered to remove sequences shorter than 500 bp and error corrected with the Illumina data using 264 FMLRC (Wang et al., 2018) with default settings. Error-corrected PacBio reads were then used 265 to simulate 20x coverage 3 kb insert size libraries with wgsim (https://github.com/lh3/wgsim) 266 and parameter setting as follows: -e 0.0 -d 3000 -s 500 -1 100 -2 100 -r 0.0 -R 0.0 -S 123 -N 267 5000000. Illumina data and simulated long-range libraries were then assembled using 268 AllpathsLG v52400 (Gnerre et al., 2011), setting HAPLOIDIFY=True. The resulting assemblies 269 were subjected to further scaffolding using error-corrected PacBio data with the software LINKS 270 v.1.8.5 (Warren et al., 2015) with -d 2000,5000,10000,15000,20000 -t 20,20,5 and a kmer value 271 of 29. Scaffolds were extended and gap-filled using PBJelly v.15.8.24 and finally polished using 272 Pilon v.1.2 (Walker et al., 2014). Polished assemblies were evaluated with QUAST (Gurevich et 273 al., 2013) and BUSCO v.2 with the database v.9 (Simão et al., 2015). 274 After genomes were assembled, single nucleotide polymorphisms (SNPs) and indels were 275 identified using the Genome Analysis Toolkit (GATK) software v3.8-0-ge9d806836 (McKenna 276 et al., 2010), following GATK best practices (Broad Institute 2019). Illumina reads from each of 277 the 86 Illumina genome libraries were first mapped to the hybrid assembly using bwa-0.7.9 (Li

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278 2013) with the following parameters: mem -M -t 8 -v 2. Mapping rates for A. phalloides 279 specimens ranged from 20.0% - 95.3%, with a median mapping percentage of 86.1% (only older 280 specimens mapped at less than 50%). The mapping rate of 10511’s Illumina reads to the 10511 281 hybrid assembly was 93.8%. Duplicate reads were marked, and the GATK program 282 Haplotypecaller was used to call variants simultaneously on all samples, using 283 MODE=DISCOVERY and type=GVCF. This procedure resulted in 212,119 indels and 284 1,580,133 SNPs. 285 To eliminate any variants that may have been called as a result of sequencing errors, the raw 286 VCF file was filtered using VCFtools-0.1.14 (Danecek et al., 2011). We took a conservative 287 approach: loci with quality scores greater than 30 (--minQ 30); with sequencing depths greater 288 than the approximate mean depth across all loci and individuals (60X, or --min-meanDP 60); 289 with a minor allele frequency (MAF) greater than 0.006 (1/2N, where N=86 diploid individuals); 290 and with no more than 50% missing data (--max-missing 0.5), were kept in our dataset. To test 291 how different filtering criteria affected the ability to distinguish genotypes, the raw VCF file was 292 subjected to a variety of alternative filtering parameters (Supporting Information Table S1b, Fig. 293 S1). To remove any remaining erroneously called variants, we removed loci deviating from 294 Hardy-Weinberg equilibrium, calculated per population using an exact test as defined by 295 Wigginton et al., (2005) (p-value cut-off 0.01; Supporting Information Table S1b). A total of ten 296 VCF files were produced: one encompassing all 86 individuals, and the remaining nine 297 containing a given population with loci deviating from Hardy-Weinberg equilibrium removed. 298 Each VCF file was then converted to a .raw file format using PLINK v1.9 (Purcell et al., 2007) 299 for subsequent analyses in R v3.4.4 (R Core Team 2013). 300 301 Intrapopulation and interpopulation patterns of polymorphism and divergence 302 Statistical analyses were performed using R v3.4.4 and all scripts are available at 303 https://github.com/jacobgolan/genets. To calculate the genetic relationships among sporocarps of 304 both the AFLP and SNP datasets, we quantified the number of AFLP or SNP differences for 305 every pair of sporocarps within a given population, and any sporocarp pair with a difference of 306 zero was considered as belonging to the same genet (Fig. 2, Fig. 3, Fig. 4). Missing data are rare 307 (no AFLP data are missing, and only 0.73% of SNP data are missing), but a SNP locus at which 308 two samples differed by missing data was counted as “not different” so as to facilitate

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309 comparisons among pairs of samples: each pairwise comparison using SNP data was scaled to 310 m(m-x)-1, where m is the total number of loci and x is the number of missing sites (Kamvar et 311 al., 2014). To understand how many markers are required to fully resolve genetic relationships 312 among AFLP and SNP genotypes, we randomly sampled 100 loci 1,000 times without 313 replacement, and calculated the number of unique genotypes for each random sample 314 (Supporting Information Methods S2, Fig. S1). Curves of these data were constructed by 315 adapting the poppr command from the package poppr v2.8.3 (Kamvar et al., 2014). 316 To understand genetic relationships among sporocarps collected across space and time, we 317 first took an ordination approach. Using ape v5.3 (Paradis & Schliep, 2018), we conducted a 318 principle coordinate analysis (PCoA) on pairwise differences of sporocarp AFLP and/or SNP 319 profiles. We plotted individual sporocarps using eigenvalues from the first two principal 320 coordinates. Axes are centered around the mean of eigenvalues per axis and scaled by the 321 standard deviation of eigenvalues per axis. The first two axes capture 27.42% and 16.27% of the 322 variance in the AFLP data, and 16.95% and 9.75% of the variance in the SNP data. Ellipses were 323 drawn to encompass a 0.95 confidence interval for each geographic region for AFLP data (Fig. 324 5), and labelled to highlight specific genetic and spatial clusters for SNP data (Fig. 6). 325 To test whether closely related sporocarps grow near each other, we performed Mantel tests 326 to test for correlations between genetic and physical distances. Analyses were performed using 327 vegan 2.5-5 on each population and then for geographic regions (e.g., California) and 328 subpopulation clusters identified from our PCoA analyses (Supporting Information Table S3; 329 Oksanen et al., 2018). To estimate the physical area of each population we first drew a polygon 330 around the perimeter of a population (as defined by sporocarps at the population’s edges) and 331 then estimated the smallest four-sided parallelogram that would completely enclose all 332 sporocarps using in-house R scripts and alphahull v2.1 (Pateiro-López & Rodríguez-Casal, 333 2010). 334 Finally, to infer population differentiation between adjacent locations sampled in the same 335 year, and between the same location sampled over time (e.g., relationships between Drake 2 and

336 Drake 3, both sampled in 2004, 2014, and 2015), we calculated FST for every pair of populations 337 and subpopulations with hierfstat v0.04-22 (Weir & Cockerham, 1984; Goudet, 2005). 338 339

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340 Results 341 AFLP and SNP data 342 Sequencing resulted in 103 AFLP bands from 221 A. phalloides genomes and 1.2 raw 343 Illumina reads (an average of 3.6 M per sporocarp) from 86 A. phalloides genomes. The 344 assembled Illumina-PacBio hybrid genome of specimen 10511 (Mira, São Jacinto, Portugal) 345 resulted in a 35.5 Mb assembly covering approximately 77% of the estimated 43 Mb genome. 346 The assembly encompasses 605 scaffolds with an N50 of 320 kb, and gene space completeness is 347 estimated at 94.4%. The 86 full genomes mapped to the 10511 assembly provided 297,133 high 348 quality SNPs. Sporocarps with identical genotypes were found exclusively in the AFLP dataset 349 (Supporting Information Fig S2a,b, Fig. S3). The two most closely related SNP genotypes were 350 collected from Dunas de Mira, São Jacinto, Portugal (specimens 10511 and 10512), and they 351 differed by approximately 24,250 SNPs (scaling for missing data; Fig. 2, Supporting Information 352 Table S1b). 353 While the 221 sporocarps of the AFLP dataset resolve into 160 unique genotypes, a plot of 354 number of loci randomly sampled versus number of genotypes (Supporting Information Fig. S1) 355 suggests 103 AFLP markers do not fully resolve sporocarp identity (Grünwald et al., 2003). SNP 356 data provided greater resolution of genotypes, and both conventional and stringently 357 conservative filtering criteria resolved each sporocarp as a unique genotype (Supporting 358 Information Fig. S1). The different datasets result in different interpretations, for example, SNPs 359 delineate each sporocarp from population Drake 3 2004 as a unique individual whereas AFLP 360 markers resolve only two genets from the same sporocarps (Fig. 2, Fig. 3). 361 362 Most sporocarps of Amanita phalloides resolve into unique genotypes 363 Regardless of genotyping strategy, the dominant pattern emerging across California, the 364 Northeast U.S. and Europe is of populations consisting of multiple genets, most of which are 365 made up of a single sporocarp. Thus, we infer that movement across landscapes is mediated by 366 sexual basidiospores, and not asexual vegetative growth and fragmentation (Dahlberg & Stenlid, 367 1994). Moreover, populations sampled over time do not transition from being composed of 368 smaller and more numerous genets to being dominated by larger and less numerous genets (Fig. 369 4). The temporal succession of genotypes suggests individuals are not long lived and reproduce 370 shortly after establishing.

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371 In fact, the majority of genets in both the AFLP and SNP datasets consist of a single 372 sporocarp (Table 1). Genets encompassing more than one sporocarp were found only within the 373 AFLP dataset. The 221 sporocarps genotyped by AFLP resolve into 160 unique genotypes, 374 72.40% represent unique genotypes and resolve into small genets consisting of either a single 375 sporocarp, or two to three (Fig. 3, Supporting Information Fig. S2a,b, Fig. S3). The 376 longest distance between two identical sporocarps was calculated from Jake’s Landing (7.51 m), 377 the longest in California was found in Heart’s Desire 2 (5.14 m), and the longest in Europe was 378 found in Serbia (3.20 m). The median length of genets consisting of more than one sporocarp is 379 1.70m (1.12m in European populations, 2.20m in East Coast populations, 1.73m in Californian 380 populations; Table 1). However, we hypothesize any approach with greater resolving power 381 would distinguish many of these sporocarps as distinct genets. 382 Only one genet was found in more than one year: an AFLP genotype found at CESAC in 383 2002 was also found at CESAC in 2006 (specimens CESAC 54 and CESAC 21, separated by 384 0.81 meters; Fig. 4). But once again, we hypothesize any approach with greater resolving power 385 would distinguish these two sporocarps. In Californian populations, genotypes did not persist 386 from year to year and genets were consistently small regardless of genotyping method. 387 388 Spores of Amanita phalloides are dispersal limited between and within populations 389 At continental scales, genotypes cluster according to geography (Fig. 5, Fig. 6). However, a 390 subset of the genotypes from Europe, the East Coast, and California appear to be closely related. 391 In particular, AFLP genotypes from the East Coast appear closely related to European genotypes 392 from southern France, and to a lesser extent, a small subset of AFLP genotypes from California 393 (especially from Heart’s Desire populations) appear closely related to genotypes from both 394 southern France and the East Coast. There is less overlap among SNP genotypes identified from 395 Europe and California; SNP genotypes from Europe were collected mainly from central 396 Portugal, with a few collections also taken from throughout northern and eastern Europe, and 397 from Sardinia and Corsica, but notably not from southern France. But whether populations of A. 398 phalloides in North America were in fact introduced from southern France is an hypothesis 399 remaining to be tested. 400 Surprisingly, closely related genotypes also cluster at local spatial scales. Populations appear 401 genetically distinct and sporocarps collected from the same physical location group together and

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402 apart from other populations. Moreover, sporocarps collected a few centimeters apart from each 403 other within a single population are often genetically more similar than sporocarps collected 404 meters apart (Fig. 5, Fig. 6). Within Drake 2 there is a clear subpopulation structure and two 405 distinct groups of genetically related sporocarps are apparent. 406 To delineate spatial autocorrelations more fully, we used Mantel tests to probe for 407 relationships between the physical and genetic distances separating sporocarps, asking whether 408 mushrooms found nearer each other are also more closely related. Correlations were significant 409 for seven of the 15 populations from which AFLP data were collected, and for two of the eight 410 populations from which SNP data were collected (Supporting Information Fig. S4a,b, Fig. S5; 411 Table S3). Mantel tests corroborate fine scale isolation by distance as a feature of multiple 412 populations (Fig. 7, Supporting Information Fig. S4a,b, Fig. S5). Moreover, when the data of 413 nearby populations were combined, for example, combining all of the data available from Point 414 Reyes National Seashore (Drake populations), Tomales Bay State Park (Heart’s Desire 415 populations), or New York (Rochester populations), correlations were also often significant 416 (Supporting Information Table S3). The physical area of a population did not influence whether 417 or not there was a significant Mantel correlation (linear regression of area versus the calculated 418 Mantel statistic: slope near zero and P >0.05 for both within and between populations). 419 Even across years, closely related genotypes often cluster in space (Fig. 6). For example, 420 individuals of Drake 2 collected in 2004, 2014 and 2015 cluster together and away from 421 individuals of Drake 3 collected in the same years. Subpopulations within single populations 422 persist through time, for example, the subpopulations identified from Drake 2 in 2014 were 423 found again in 2015 (Fig. 6). The data suggest spores fall and germinate within centimeters of 424 their parent sporocarps; even after a decade, Drake 2 and Drake 3 (which are less than 100 m 425 apart) remain genetically distinct.

426 Calculations of FST (Weir & Cockerham, 1984) confirm populations sustain a relative degree

427 of genetic differentiation across years (Hartl & Clark 2007; Branco et al., 2015; 2017). FST 428 calculated using the 2004, 2014, and 2015 data of Drake 2 ranges from 0.0054–0.0138, and for

429 Drake 3 ranges from 0.0003–0.0270. The FST statistic comparing the adjacent populations, Drake 430 2 and Drake 3, in 2004 is 0.0376 (0.021 with AFLP data), in 2014 is 0.0523, and in 2015 is

431 0.0398. FST comparing Drake 3 to either Drake 2 subpopulation (Cluster 1 or 2) reveals

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432 comparable values to those comparing Drake 2 Cluster 1 to Cluster 2 (0.0729±0.0487 vs. 433 0.0763±0.0040, respectively). 434 435 Discussion 436 The death cap uses sexual basidiospores to spread in both its native European range, as well 437 as in its introduced ranges on the East Coast and California. Vegetative fragmentation appears 438 rare. Populations consist of many small genets and typically each sporocarp is its own genet. But 439 correlations between geographic and genetic distances, even at small spatial scales (tens of 440 meters), suggest spores travel very short distances, falling near to parent sporocarps. In fact, 441 ordination analyses cluster populations in space, even across time: populations from a single 442 location sampled years apart cluster together and away from populations sampled in the same 443 year from nearby locations. Genets appear to be ephemeral, and except for a single pair of 444 sporocarps within the native range of A. phalloides (generated using AFLP data and potentially 445 an artifact), the same genotype was never recovered in more than one year (Fig. 4). Data suggest 446 frequent sexual reproduction and a high turnover of genets within populations, suggesting short 447 lifespans. 448 Small body sizes and ephemeral genets are typical of other ECM Basidiomycetes in native 449 ranges, paralleling our findings in an invasive system. The majority of genets of Hebeloma 450 cylindrosporum, Laccaria amethystina, Tricholoma terreum, and cremoricolor are small 451 and either consist of single sporocarps or are less than a square meter in size (Gryta et al., 1997; 452 Gherbi et al., 1999; Redecker et al., 2001; Huai et al., 2003). A high turnover of genets within 453 populations appears typical of R. cremoricolor and T. scalpturatum (Redecker et al., 2001; 454 Carriconde et al., 2008). 455 Genet sizes across the genus Amanita appear variable. The species A. franchetii reaches a 456 maximum size of 4.7m, but populations house many singleton genets as well; populations of A. 457 manginiana are entirely composed of singleton genets, with no genotype recovered between 458 consecutive years (Sawyer et al., 2003; Liang et al., 2005). By contrast, A. conicoverrucosa, A. 459 punctata, A. pyramidifera, and A. muscaria appear to form genets made up of multiple 460 sporocarps and may reach tens of meters in size (Sawyer et al., 2001; 2003). However 461 fingerprinting techniques with low resolution may bias inferences by binning unique genetic 462 individuals together, as we discovered by comparing our AFLP and SNP datasets.

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463 The example of A. muscaria is a particularly interesting comparison; like A. phalloides, it has 464 been introduced and is invasive outside of its native range (Richardson et al., 2000; Dickie & 465 Johnston, 2008; Dunk et al., 2012). The fungus now grows in Australia and New Zealand and is 466 invasive in Colombia (Sawyer et al., 2001; Bagley & Orlovitch 2004; Orlovitch & Cairney, 467 2004; Vargas et al., 2019). Colombian populations of A. muscaria associate with native Q. 468 humboldtii and are spreading through Colombian oak forests but Australian populations appear 469 confined to commercial plantations of introduced (Sawyer et al., 2001). In contrast 470 to the death cap, available data suggest A. muscaria is found primarily as large genets in 471 Australia and New Zealand, apparently capable of steady vegetative growth below ground 472 (Sawyer et al., 2001). Whether large genets would be detected using modern fingerprinting 473 techniques or at sites outside of Australia and New Zealand remains to be tested, but if A. 474 muscaria does grow as large genets, data would suggest frequent sexual reproduction and 475 copious spore production are not required to facilitate invasions by ECM fungi. 476 Our nascent understanding of the mode and tempo of the death cap’s invasion dynamics is 477 likely to facilitate an understanding of the species elsewhere, and of the genus Amanita as a 478 source of invasive ECM species. A. phalloides has also been introduced to South America 479 (Singer, 1953; Takacs, 1961), East and (Walleyn & Rammeloo, 1994), Australia 480 (Talbot, 1976; Shepherd & Totterdell, 1988; Wood, 1997) and New Zealand (Taylor, 1982; 481 Ridley, 1991). Other species in the genus, including A. rubsescens, A. thiersii, and A. inopinata, 482 are introduced or invasive elsewhere (Bougher, 1996; Pegler & Shah-Smith, 1997; Sawyer et al., 483 2003; Wolfe et al., 2012). The genus Amanita emerges as a developing model for work with 484 invasive ECM fungi, offering the potential for comparisons among closely related species 485 invasive in geographically distant ranges. 486 The life history of A. phalloides appears similar to the life history of many herbaceous weeds 487 (Baker, 1965; Grime, 1977; Roché & Thill, 2001). Basidiospores may give rise to relatively 488 small mycelia that persist for short periods of time before reproducing sexually. Life history 489 evolution among fungi is poorly described (a stark contrast to traditions within the plant 490 literature; Harper 1977; Grime 1977) but A. phalloides emerges in stark contrast to the 491 “humongous fungus,” epitomized by the pathogens Armillaria gallica (Anderson et al., 2018) 492 and A. ostoyae (Shaw & Roth, 1976; Ferguson et al., 2003). The death cap appears to persist in 493 habitats as a small bodied, ephemeral, potentially ruderal species.

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494 However, dispersal is clearly not the only control on A. phalloides invasion dynamics. While 495 A. phalloides uses spores to establish in both California and on the East Coast, in California A. 496 phalloides is invasive while in New Jersey A. phalloides appears confined within planted forests 497 of Pinus strobus (Thompson et al., 2000; Wolfe & Pringle, 2011). In California, A. phalloides is 498 spreading in association with a native oak, a distant relative of the in its native range (Hipp 499 et al., 2018). In New Jersey, A. phalloides grows at sites slightly outside of the southern range of 500 P. strobus. The associations of the death cap with different hosts, within and outside of hosts’ 501 ranges, may be a key influence on its spread, but any potential mechanism mediating the 502 dynamic remains unknown (Richardson et al., 2000; Dickie et al., 2017). 503 The paradox of a dispersal limited invasive fungus suggests A. phalloides will move slowly 504 as it continues to spread through its habitats in California. An earlier estimate of approximately 505 five km yr-1 is almost certainly wrong (Pringle et al., 2009). Although, establishment from spores 506 does explain the death cap’s dominance in local habitats given the magnitude of its spore 507 production: reproduction measured for multiple sporocarps during a 48 hour incubation period 508 ranged from 2.22 × 107 to 1.58 × 108 spores per sporocarp (mean: 8.66 × 107 ± 1.90 × 107; Wolfe 509 & Pringle unpublished). At the Drake sites of Point Reyes National Seashore (labeled “Drake’s 510 Landing” in Wolfe et al., 2010) sporocarps of A. phalloides average more than half (and up to 511 85%) of the total biomass of ectomycorrhizal sporocarps collected in a single season (Wolfe et 512 al., 2010). As A. phalloides spores germinate and mycelia quickly reproduce to generate more 513 spores, a local feedback likely enables populations to grow rapidly. 514 The potentially ruderal niche of the death cap also suggests an effective management strategy 515 (Dickie et al., 2016). The mushrooms of the death cap are deadly, and each year in California 516 there are poisonings (Zevin et al., 1997; Bonacini et al., 2017; Vo et al., 2017). An increasingly 517 public discussion has focused attention on whether the fungus can be eradicated from local 518 landscapes (Dickie et al., 2016; Quirós 2016; Childs 2019). In fact, our data suggest a control 519 strategy: as a sporocarp of the death cap develops, its spore-bearing surface is covered by a 520 . As the sporocarp matures, the veil ruptures to release spores. Collecting and 521 destroying immature sporocarps before veil rupture would prevent spore release and the 522 subsequent establishment of mycelia. If spores are also short lived (an untested hypothesis), 523 mycelia would be unable to germinate from a spore bank. Because spores can’t travel very far,

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524 thoroughly collecting at a site for just a few years would potentially break the life cycle of the 525 fungus and rid a habitat of an invasive and deadly poisonous fungus. 526 527 Acknowledgements 528 We thank the University of Wisconsin–Madison, Human Frontier Science Program and 529 National Science Foundation for funding (and in particular an NSF Graduate Research 530 Fellowship DGE-1747503 to Jacob Golan). Deep thanks to Benjamin Becker, Lynne Boddy, Tom 531 Bruns, Bryn Dentinger, Jeffrey Grass, Hervé Gryta, Thomas R. Horton, David Johnson, Todd 532 Mitchell, Sam Morris and his Trigonometry class at Cary Academy, Sophie Manzi, Zachary 533 Muscavitch, the Mycologists’ Association of Serbia (Branislav Uzelac, Goran Milošević and 534 Krstajic Damjan), Karla Sartor, Rod Tulloss, and Natalia Vargas-Estupiñan, for collections, 535 advice, technical assistance, and support in the field. We collected at Tomales Bay State Park and 536 Point Reyes under permits granted to Tom Bruns, Sydney Glassman, and Anne Pringle. 537 538 References 539 Ammirati JF, Thiers HD, Horgen PA. 1977. -containing mushrooms: Amanita 540 ocreata and A. phalloides in California. Mycologia 69: 1095–1108. 541 Anderson JB, Bruhn JN, Kasimer D, Wang H, Rodrigue N, Smith ML. 2018. Clonal 542 evolution and genome stability in a 2500-year-old fungal individual. Proceedings. Biological 543 Sciences 285: 20182233. 544 Anderson JB, Kohn LM. 1995. Clonality in soilborne, plant-pathogenic fungi. Annual Review 545 of Phytopathology 33: 369–391. 546 Bagley SJ, Orlovich DA. 2004. Genet size and distribution of Amanita muscaria in a suburban 547 park, Dunedin, New Zealand. New Zealand Journal of Botany 42: 939–947. 548 Baker H. 1965. Characteristics and modes of origin of weeds. In: The Genetics of Colonizing 549 Species. New York: Academic Press, 147–172. 550 Barker BS, Andonian K, Swope SM, Luster DG, Dlugosch KM. 2017. Population genomic 551 analyses reveal a history of range expansion and trait evolution across the native and invaded 552 range of yellow starthistle (Centaurea solstitialis). Molecular Ecology 26: 1131–1147. 553 Berch SM, Kroeger P, Finston T. 2016. The death cap mushroom (Amanita phalloides) moves 554 to a native tree in Victoria, British Columbia. Botany 95: 435–440. 555 Bergemann SE, Miller SL. 2002. Size, distribution, and persistence of genets in local 556 populations of the late-stage ectomycorrhizal basidiomycete, Russula brevipes. New 557 Phytologist 156: 313–320. 558 Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence 559 data. Bioinformatics 30: 2114–2120. 560 Bonacini M, Shetler K, Yu I, Osorio RC, Osorio RW. 2017. Features of patients with severe 561 due to and factors associated with outcome. Clinical

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788 789 Author contributions 790 JG, CAA, HE, BW, FR, and AP conceived and designed the experiments; JG, CAA, HE, MG, 791 SIG, SCG, FR, BW and AP collected and processed samples; HC generated AFLP data, HE, 792 YWW and JH assembled genomes and generated SNP data. JG wrote a first version of the 793 manuscript in collaboration with AP, and all authors contributed substantially to the last version 794 of the manuscript. 795 796 Figure Legends 797 Fig. 1: Map of collections. Circles are proportional to the number of sporocarps sampled. 798 Collections used to generate AFLP data are labelled in purple, and collections used to generate 799 SNP data are labelled in orange. If locations were used to generate both AFLP and SNP data, 800 labels are both purple and orange. Singleton collections are marked with an asterisk. Collections 801 are numbered as a guide to the more detailed information found in Table 1. 802 803 Fig. 2: Numbers of AFLP markers (top) or SNPs (bottom) differentiating pairs of sporocarps 804 within a given population. Genetically identical sporocarps are marked with an arrow. To 805 accommodate missing data, SNP differences are scaled up proportionally to m (m-x)-1, where m 806 is the total number of loci and x is the number of missing sites. 807 808 Fig. 3: Sporocarp maps and the genets identified in eight populations (data of all populations 809 found in Fig. S2a,b and Fig. S3). Sporocarps genotyped using AFLP data plotted as squares, 810 sporocarps genotyped using SNP data plotted as circles, and sporocarps genotyped with both 811 plotted as diamonds. Within each map, sporocarps belonging to the same genet are labelled using 812 a single color and surrounded by an arbitrarily shaped, transparent polygon of the same color. 813 Most genets are made up of a single sporocarp, regardless of technology. A dotted green polygon 814 groups sporocarps of Drake 3 2004 identified as a single genet using AFLP data but resolved as 815 distinct genotypes using SNP data. Note that the sporocarp at the top left was not included in the 816 SNP dataset and whether it would resolve into its own genet using SNP data is unknown. 817

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818 Fig. 4: Sporocarp maps and genets identified from two sites sampled over time: CESAC 819 (France) sampled in 2002 and 2006; and Drake 2 (California) sampled in 2004, 2014 and 2015. 820 Sporocarps genotyped using AFLP data plotted as squares, sporocarps genotyped using SNP data 821 plotted as circles, and sporocarps genotyped with both plotted as rhombuses. Within each map, 822 sporocarps belonging to the same genet are labelled using a single color and surrounded by an 823 arbitrarily shaped, transparent polygon of the same color. Asterisks mark a single AFLP genotype 824 from 2002 found again in 2006. 825 826 Fig. 5: Principle coordinate analysis (PCoA) of genetic relationships among sporocarps 827 genotyped using AFLP markers reveals genotypes cluster geographically, with three clusters 828 associated with populations from Europe, the East Coast, and California. Data are color coded by 829 population. Ellipses enclose genotypes from each geographic region within a 0.95 confidence 830 interval. 831 832 Fig. 6: Closely related genotypes cluster in space, even through time: for example, a 833 genotype collected from Drake 2 Cluster 1 in 2014 is more closely related to other sporocarps 834 from Cluster 1 collected in 2004, 2014, or 2015, and not to other sporocarps of Drake 2 Cluster 2 835 collected in 2014. a). principle coordinate analysis (PCoA) of genetic relationships among 836 sporocarps genotyped using genome-wide SNPs. b-c) Polygons surrounding clusters of closely 837 related genotypes from Drake 2 2014 and Drake 2 2015 in plot (a) translate to distinct clusters in 838 the physical landscape. 839 840 Fig. 7: Mantel correlations between genetic and physical distances for three geographically 841 distinct populations genotyped with AFLP (left), and three populations sampled from the same 842 location in California over time and genotyped with SNPs (right). Grey shading marks a 95% 843 confidence interval around fitted linear models. Plots list the Mantel statistic r using Pearson’s 844 correlation method, and asterisks mark significant Mantel correlations (* = p < 0.05, ** = p < 845 0.005). Data for all populations provided in Fig. S4a,b, and Fig. S5. 846 847

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Location Point on Map (Fig. 1) Pop. Name Latitude Longitude Collection Date Technology No. Mushrooms No. Individual Genets

Serbia, Obrež, Obedska Bara Nature Reserve 1 Serbia 44.745700 20.010400 2007 October 6 AFLP 14 7 Sweden, Uppland, Palsundet 2 [singleton] 59.8586 17.6389 1978 September 14 SNP 1 1 Austria 3 [singleton] N/A N/A N/A N/A AFLP 1 1 Czech Republic, Český Šternberk Na Stříbrné Nature Reserve 4 [singleton] 49.8109 14.9282 2006 September 29 SNP 1 1 Denmark, Farum, Nørreskov 5 [singleton] 55.916667 9.533333 2003 September 17 SNP 1 1 Italy, Catala, Calangianus 6 [singleton] 40.9211 9.193 2006 November 13 SNP 1 1 France, Corsica, Fango 7 [singleton] 42.0396 9.0129 n/a November 5 SNP 1 1 Spain, Girona 8 [singleton] 41.9794 2.8214 2006 December 2 AFLP 1 1 France, Arfons 9 [singleton] 43.430054 2.168282 2007 October 7 AFLP 1 1 France, Toulouse, CNRS 10 CESAC 43.578554 1.463026 2002 November 25 AFLP 25 24 France, Toulouse, CNRS 10 CESAC 43.578554 1.463026 2006 November 10-19 AFLP 37 28 England, West Sussex, Mildenhall Woods 11 [singleton] 52.3614 0.4866 2000 October 15 SNP 1 1 Scotland, Pitlochry, Black Sprout Wood 12 [singleton] 56.7044 -3.7297 1991 September 13 SNP 1 1 Northern Ireland, Fermanagh, Inishmakill 13 [singleton] 54.4795 -7.7315 2000 October 14 SNP 1 1 Portugal, Lousã, Vilarinho 14 Vilarinho 40.122204 -8.209709 2015 November 17 SNP 5 5 Portugal, Coimbra, Escola Superior Agrária de Coimbra 15 Agraria 40.2125 -8.450278 2015 November 16 SNP 2 2 Portugal, São Jacinto, Dunas de Mira 16 Mira 40.4575 -8.768611 2015 November 18 SNP 4 4 USA, NJ, Lebanon, Round Valley Resevoir 17 Round Valley 40.6179 -74.8474 2006 October 7 AFLP 11 7 USA, NJ, Dennis, Jake's Landing Rd 18 Jake's Landing 39.189940 -74.853539 2006 November 21 AFLP 44 27 USA, NY, Rochester, Durand Eastman Park 19 Rochester 1 43.233019 -77.554686 2007 September N/A AFLP 13 12 USA, NY, Rochester, Durand Eastman Park 19 Rochester 2 43.232747 -77.554917 2007 September N/A AFLP 7 5 USA, NY, Rochester, Durand Eastman Park 19 Rochester 3 43.232896 -77.554851 2007 September N/A AFLP 2 2 USA, CA, Monterey 20 [singleton] 36.600238 -121.894676 2006 December 14 AFLP 1 1 USA, CA, Point Reyes National Seashore 21 [singleton] 38.0525 -122.852778 1993 October 5 SNP 1 1 USA, CA, Point Reyes National Seashore, Limantour Rd 22 Drake 1 38.0545 -122.83343 2004 November 16 AFLP 19 12 USA, CA, Point Reyes National Seashore, Limantour Rd 22 Drake 2 38.054785 -122.833232 2004 November 16-17 AFLP SNP 13 [AFLP] / 13 [SNP] 13 [AFLP] / 13 [SNP] USA, CA, Point Reyes National Seashore, Limantour Rd 22 Drake 2 38.054785 -122.833232 2014 November 17 SNP 25 25 USA, CA, Point Reyes National Seashore, Limantour Rd 22 Drake 2 38.05471 -122.83328 2015 December 14 SNP 11 11 USA, CA, Point Reyes National Seashore, Limantour Rd 22 Drake 3 38.055212 -122.834134 2004 November 16-17 AFLP SNP 8 [AFLP] / 5 [SNP] 2 [AFLP] / 5 [SNP] USA, CA, Point Reyes National Seashore, Limantour Rd 22 Drake 3 38.055212 -122.834134 2014 November 17 SNP 9 9 USA, CA, Point Reyes National Seashore, Limantour Rd 22 Drake 3 38.05514 -122.83418 2015 December 14 SNP 3 3 USA, CA, Point Reyes National Seashore, Limantour Rd 22 Drake 4 38.054675 -122.836545 2004 November 16 AFLP 6 5 USA, CA, Point Reyes National Seashore, Johnstone Trail 23 Heart's Desire 1 38.131216 -122.88885 2004 November 18 AFLP 7 7 USA, CA, Point Reyes National Seashore, Johnstone Trail 23 Heart's Desire 2 38.12871 -122.88869 2004 November 18 AFLP 7 4 USA, CA, Point Reyes National Seashore, Johnstone Trail 23 Heart's Desire 3 38.12513 -122.88891 2004 November 18 AFLP 4 2 European Collections (Native Range) 300km

2 * 12 5 13 * * * 11 * *4 3* 10 9 1 16 * 8 7 15 * **6 14

East Coast U.S.A. Collections (Introduced Range) 100km

19

bioRxiv preprint doi: https://doi.org/10.1101/799254; this version posted October 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. California U.S.A. Collections (Invasive Range) 100km 17

18

23

22 21* 20*

AFLP Data SNP Data SNP & AFLP Data * Collection is a single mushroom bioRxiv preprint doi: https://doi.org/10.1101/799254; this version posted October 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

AFLP Data Serbia 2007 CESAC 2002 300 CESAC 2006 Jake’s Landing 2006 Round Valley 2006 Rochester 1 2007 Rochester 2 2007 200 Rochester 3 2007 Drake 1 2004 Drake 2 2004 Drake 3 2004

Count: Sporocarp Pairs Drake 4 2004 100 Heart’s Desire 1 2004 California East Coast Europe Heart’s Desire 2 2004 Heart’s Desire 3 2004

0

0 10 20 30 Number of AFLPs Differentiating Pairs of Sporocarps SNP Data

Vilarinho Agraria

30 Mira Drake 2 2004 Drake 2 2014 Drake 2 2015 Drake 3 2004

20 California Portugal Drake 3 2014 Drake 3 2015 Count: Sporocarp Pairs 10

0

0 25000 50000 75000 Number of SNPs Differentiating Pairs of Sporocarps Serbia 2007 (Serbia) CESAC 2006 (France) Jake’s Landing 2006 (NJ) 4 1.0m

10 60 3

2 5 40 meters

N meters −2 −1 0 1 0

N meters 20 5m meters -10 -5 0 5 10

bioRxiv preprint doi: https://doi.org/10.1101/799254; thisDrake version posted October 2 2014 9, 2019. The copyright(CA) holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under Drake 3 2004 (CA) aCC-BY-ND 4.0 International license. 2.5m meters 0 meters 2 N 5m meters

4 meters 0 20 40 60 1 Rochester 1 2007 (NY) N Round Valley 2006 (NJ) 0 meters meters 2.5m 0

N meters meters -4

meters -1 9 10 meters 0.5m -10 -5 0 5 -0.5 0 1.0

Heart’s Desire 2 2004 (CA)

meters AFLP Data Only 6 5 SNP Data Only 2 meters

Both AFLP and SNP Data 0 Genet with >1 Sporocarp N 3 (AFLP Data Only) -2 2.0m 2m N 0 N 0 2 4 6 2 4 6 8 0 5 10 CESAC 2002 (France) CESAC 2006 (France)

15 15

10 10

5 5 * *

0 0 N N

AFLP Data Only -5 -5 SNP Data Only Both AFLP and SNP Data meters meters Genet with >1 Sporocarp 5m -10 5m meters -10 meters (AFLP Data Only)

-10 -5 0 5 10 15 -10 -5 0 5 10 15 bioRxiv preprint doi: https://doi.org/10.1101/799254; this version posted October 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

Drake 2 2004 (CA) Drake 2 2014 (CA) Drake 2 2015 (CA)

20 10 10

N 10 0 0 N

0 -10 -10 N meters meters meters

5m meters 5m meters 5m meters -10 0 10 20 -20 -10 0 10 -10 0 10 20 bioRxiv preprint doi: https://doi.org/10.1101/799254; this version posted October 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

Serbia 2007 Austria Arfons (France) 2007 CESAC (France) 2002 Europe CESAC (France) 2006 Spain 2006 Jake’s Landing_2006 2 Round Valley 2006 Rochester 1 2007 Rochester 2 2007

East Coast Rochester 3 2007 Drake 1 2004 Drake 2 2004 Drake 3 2004 Drake 4 2004 PCoA Axis 2 [16.27%] 0 Heart’s Desire 1 2004 Heart’s Desire 2 2004 California Heart’s Desire 3 2004 Monterey CA 2006

−2

−2 −1 0 1 2 3 PCoA Axis 1 [27.42%] bioRxiv preprint doi: https://doi.org/10.1101/799254; this version posted October 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

PCoA: SNP Data Spatial Mapping: Drake 2 2014 meters

Europe Drake 3 (2004, 2014, 2015) 1 4.0 Drake 2 Cluster 1 meters (2014)

0.0 N 0 Drake 2 Cluster 1 (2004, 2014, 2015)

−4.0 Drake 2 Cluster 2 PCoA Axis 2 [9.75%] (2014) 2.5m -1 b −10 −5 0 5

Spatial Mapping: Drake 2 2015 Drake 2 Cluster 2 meters (2004, 2014, 2015)

-2 a 2.5

-1 0 1 meters PCoA Axis 1 [16.95%] Population Year Collected Drake 2 Cluster 2 0.0 (2015) European Singletons 1978-2006 (Europe) N Vilarinho 1993 (California) Drake 2 Cluster 1 Agraria 2004 (California) (2015) Mira 2014 (California) −2.5 Portugal California Singleton 2015 (California) Drake 2 2004 2015 (Portugal) Drake 2 2014 −5.0 Drake 2 2015 Drake 3 2004

California Drake 3 2014 c 2.5m Drake 3 2015 0 5 10 15 20 Genetic Distance .010 .030 4.00 3.00 2.00 1.00 0.00 .050 00 50 20.00 15.00 10.00 5.00 0.00 .02.05.075.00 50.00 25.00 0.00 0.00 10.00 20.00 0.00 5.00 10.00 15.00 20.00 0.00 3.00 6.00 9.00 certified bypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmadeavailableunder bioRxiv preprint Jake's Landing2006 CESAC 2002 Serbia 2007 doi: https://doi.org/10.1101/799254 r =0.331** r =0.773* r =265* Distance (m) ; a this versionpostedOctober9,2019. CC-BY-ND 4.0Internationallicense .050 00 50 20.00 15.00 10.00 5.00 0.00 .050 00 50 20.00 15.00 10.00 5.00 0.00

6.0e+04 9.0e+04 1.2e+05 20.00 15.00 10.00 5.00 0.00 2e+04 3e+04 4e+04 5e+04 3.5e+04 4.5e+04 5.5e+04 Drake 22004 Drake 22015 Drake 22014 . The copyrightholderforthispreprint(whichwasnot r =0.181** r =0.330* r =-0.032 bioRxiv preprint doi: https://doi.org/10.1101/799254; this version posted October 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

Supporting Information Article title: Native and invasive populations of the ectomycorrhizal death cap Amanita phalloides are highly sexual but dispersal limited

Authors: Jacob Golan, Catharine Adams, Hugh Cross, Holly Elmore, Monique Gardes, Sydney I. Glassman, Susana C. Gonçalves, Jaqueline Hess, Franck Richard, Yen-Wen Wang, Benjamin Wolfe, Anne Pringle

The following Supporting Information is available for this article:

Methods S1 Sporocarp Mapping Methods

Fig. S1 Genotype accumulation curves of AFLP and SNP data.

Fig. S2 Sporocarp maps and genets of every population genotyped using AFLP fingerprints.

Fig. S3 Sporocarp maps and genets of every population genotyped using genome-wide SNPs.

Fig. S4 AFLP data: Correlations between genetic distances and physical distances (of pairs of sporocarps).

Fig. S5 SNP data: Correlations between genetic distances and physical distances (of pairs of sporocarps).

Table S1 Metadata associated with each sporocarp, including origin, latitude and longitude at source, genome summary statistics (as appropriate) and current specimen location.

Table S2 Genome assembly summary statistics.

Table S3 Summary of Mantel tests.

bioRxiv preprint doi: https://doi.org/10.1101/799254; this version posted October 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

Methods S1 Sporocarp Mapping Methods

Mapping At CESAC (in 2002 and 2006) and Drake 2-3 (in 2014 and 2015) transects were measured from an arbitrary center point within each population (at CESAC, a planted Cedrus libani) to each sporocarp. The transect angle with respect to north and its distance from the center point were recorded and data later converted to Cartesian coordinates. Populations from Portugal (Vilarinho, Agraria, and Mira [collected in 2015]) were mapped by measuring the distance between all pairs of sporocarps. Hand drawings of each population were used to arbitrarily choose a single sporocarp as the population center. Cartesian coordinates were then calculated for each sporocarp using the spatial distances between all pairs. Populations from Drake 1-4 (in 2004), Heart’s Desire 1-3 (in 2004), Serbia (in 2007), Round Valley (in 2006), Jake’s Landing (in 2006), and Rochester 1-3 (in 2007) were mapped using transect lengths measured from each of two poles placed within each population to each sporocarp. The distance between the two poles was also measured, allowing the length of each transect to be considered as a radius in the mathematical equation of a circle. Using the transect lengths as two intersecting radii, equations for each of two circles were algebraically solved to obtain the Cartesian coordinates of each sporocarp. Using a rough sketch of each population, we identified which point was the correct physical location of any given sporocarp. After mapping, entire sporocarps were extracted from soil and stored individually in labelled bags. Each sporocarp was assigned a unique five digit specimen number (Table S1a,b) tied to an in-house database named AmanitaBASE. Within 24 hours of sampling, fresh sporocarps were stored using a variety of protocols including drying on a dehydrator, air drying at approximately 35ºC, flash freezing in liquid nitrogen and then lyophilizing, and/or cutting apart and placing in CTAB (Table S1a.b, see “Method of Preservation”). Exact protocols depended on the year, location, and the tools available at the time of sampling. Specimens are stored in the Pringle laboratory herbarium unless otherwise noted (Table S1a,b). Single sporocarps were also collected opportunistically, sent to us by colleagues or loaned from herbaria. We received permission to destructively sample herbarium specimens whose original herbarium barcodes are listed in Supporting Information Table S1a,b.

bioRxiv preprint doi: https://doi.org/10.1101/799254; this version posted October 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

Total Sporocarps

200

Maximum Number of Unique Genotypes Identified

150 Genotypes Identified

100 Number of Unique

50

0

1 10 20 30 40 50 60 70 80 90 100 Number of AFLP Loci Randomly Sampled

Maximum Number of Unique Genotypes Identified = Total Sporocarps

75

50 Genotypes Identified Number of Unique

25

SNP Loci Filter

MAF0.006, Remove loci with > 50% missing data MAF0.006, Remove any loci with missing data MAF0.006, Remove no missing data MAF0.49, Remove loci with > 50% missing data MAF0.49, Remove any loci missing data MAF0.49, Remove no missing data 0

1 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 Number of SNP Loci Randomly Sampled Fig. S1 Genotype accumulation curves are in essence rarefactions of the numbers of genetic markers needed to recover 100% of unique genotypes (Kamvar et al., 2014). One to 100 loci were randomly sampled without replacement 1,000 times, and the raw counts of genotypes observed from each random sampling used to generate means and standard errors. The curve plateaus and variance is minimized at about 90 AFLP markers. Using VCFtools (Danecek et al., 2011) we tested a variety of different VCF filters to gauge how each filter discriminated among unique genotypes. Loci with a minor allele frequency lower than 0.006 ([2*N]-1, where N = 86 diploid sporocarps), or 0.49 (as an arbitrary and overly conservative filter), with a sequencing depth below 60 (the approximate mean depth across all loci and individuals), and with missing data thresholds per locus less than 0%, 50%, and 100%, were used in different combinations to filter the raw VCF file. Dotted red lines mark the maximum number of genotypes recovered with either AFLP or SNP data; dotted grey lines mark the total number of sporocarps used to generate either AFLP or SNP datasets. bioRxiv preprint doi: https://doi.org/10.1101/799254; this version posted October 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

Serbia 2007 CESAC 2002 CESAC 2006 4 Serb 9 Serb 6 CESAC_66 CESAC_29 CESAC_65 Serb 8 CESAC_62 10 CESAC_33 Serb 7 CESAC_42 10 CESAC_36 CESAC_63 CESAC_41

3 Serb 5 CESAC_64 CESAC_61 CESAC_32 CESAC_31 CESAC_59 CESAC_58

CESAC_57 CESAC_56 CESAC_40 CESAC_19 5 5 CESAC_39 CESAC_20 CESAC_54 CESAC_55 CESAC_09 CESAC_53 CESAC_38 CESAC_21 CESAC_52 CESAC_35 CESAC_50 Distance (m) 2 Serb 10 Distance (m) Distance (m) CESAC_37 CESAC_18 CESAC_48 CESAC_27 CESAC_05 Serb 4 CESAC_51 CESAC_17 0 CESAC_47 CESAC_72 CESAC_34 CESAC_08 CESAC_28CESAC_26 CESAC_49 0 CESAC_07 Serb 3 CESAC_44 CESAC_16 CESAC_04 CESAC_03 CESAC_11 CESAC_67 Serb 1 CESAC_46 CESAC_22 CESAC_15 CESAC_06 CESAC_14 Serb 2 CESAC_68 CESAC_69 CESAC_23 CESAC_25 CESAC_24 CESAC_10 −2 −1 0 1 0 4 8 12 −10 −5 0 5 10 Distance (m) Distance (m) Distance (m) Jake’s Landing 2006 Round Valley 2006 FH:00481274 Strobus 1 10047 a 60 10040 10045 FH:00481285 10039 FH:00481266 10 FH:00481268 10043 10068 FH:00481258 10048 FH:00481281 10059 10077 40 10064 10061 10079 10031

FH:00481257 10072 10032 FH:00481253 FH:00481280 FH:00481251 10052 FH:00481272 10033 10053 10038 FH:00481265 FH:00481259 5

Distance (m) 20 Distance (m) 10069 FH:00481256

10057 10060 10066 10044 10049 10075 10050 10034 10078 10054 10067 10051 0 FH:00481250 FH:00481276 FH:00481252 10030 10080 10076 10056 0 FH:00481279 0 20 40 60 0 5 10 Distance (m) Distance (m) Roch1 2007 Roch2 2007 Roch3 2007

10131 10122 10123 7.0 10111 10137 9 12 6.5 10138 10130 10105

6 10134 10139 8 10107 6.0 10136 10133 Distance (m) Distance (m) Distance (m) 10132 10135 4 10103 3 1_13 5.5 10128 10115 10129 10124 2 4 6 8 2.5 5.0 7.5 10.0 12.5 6.4 6.8 7.2 7.6 Distance (m) Distance (m) Distance (m) bioRxiv preprint doi: https://doi.org/10.1101/799254; this version posted October 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

Heart’s Desire 1 2004 Heart’s Desire 2 2004 Heart’s Desire 3 2004 5 6 10250 10262 10266 10251 10267 10268

0 2 4 10253 10265 10252 −5 2 10254 0

Distance (m) 10255 Distance (m) 10257 Distance (m) 10263 0 −10 10256 −2 10258 10264 10259 10269 0.0 2.5 5.0 7.5 0 2 4 6 −1 0 1 2 Distance (m) Distance (m) Distance (m) Drake 1 2004 Drake 2 2004

10211 20 10225 b 10201 4 10224 10212 10223 10226 10213 10209 15 2 10208 10214 10215 10222 10216 10217 10210 0 10218 10228

Distance (m) Distance (m) 10 10227 10219 10202 10203 10207 10233 10229 −2 10204 10205 10221 10230 10231 10206 5 10232 10220 0 4 8 −5 0 5 Distance (m) Distance (m) Drake 3 2004 Drake 4 2004

2 10235 10 10243

5 1 10245

0 0 Distance (m) Distance (m) 10240 −5 10249 10241 10238 10248 −1 10242 10236 10239 10246 10237 −10 10247 −1.0 −0.5 0.0 0.5 1.0 −1 0 1 Distance (m) Distance (m)

Fig. S2 Sporocarp maps and genets of every population genotyped using AFLP fingerprints. Within each map, sporocarps of the same genotype are labeled using a single color. Black squares mark genotypes represented by a single sporocarp. Panel (a) shows European and East Coast populations, and panel (b) shows Californian populations. bioRxiv preprint doi: https://doi.org/10.1101/799254; this version posted October 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

Mira 2015 Agraria 2015 0.050 10510

0.05 0.025

0.00 10513 10512 0.000 10502 10503 Distance (m) Distance (m) -0.05 -0.025

10511 -0.10 -0.050 -2 0 2 -2 0 2 Distance (m) Distance (m) Drake 2 2004 Drake 3 2004

10242 20 10225 10241 -1.20 10224 10240 10236 10223 10226 10238 15 -1.25 10239 10222 -1.30 10228 Distance (m) 10 Distance (m) 10227 10229 -1.35 10221 10233 10230 10220 10237 5 10232 10231 -1.40 -5 0 5 0.8 0.9 1.0 1.1 Distance (m) Distance (m) Drake 2 2014 Drake 3 2014

10298 4 10292 10303 10350 10299 4 10293 10301 10347 10295 10300 3 10309 10288 10287 10356 0 10294 2 10380 10280 10355 Distance (m) Distance (m) 10283 10281 1 10282 -4 10334 10331 10304 10330 10327 10348 10329 10354 10384 10306 10349 10328 10326 0 -10 -5 0 5 -5.0 -2.5 0.0 2.5 Distance (m) Distance (m) Drake 2 2015 Drake 3 2015 0.0 10713 10719 10720

2.5 10710 -0.5 10712 10715 0.0 10711 -1.0

-2.5 10717 Distance (m) Distance (m) 10718 -1.5 10716 -5.0 10709 10708 -2.0 10721 10707 0 5 10 15 20 -0.1 0.0 0.1 Distance (m) Distance (m) Fig. S3 Sporocarp maps and genets of every population delineated using genome-wide SNPs. Black squares mark genotypes represented by a single sporocarp; no genet encompassed more than one sporocarp. bioRxiv preprint doi: https://doi.org/10.1101/799254; this version posted October 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

bioRxiv preprint doi: https://doi.org/10.1101/799254; this version posted October 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

Fig. S4 AFLP data: Correlations between genetic distances and physical distances. Grey shading marks 95% confidence intervals around fitted linear models. Each plot includes the Mantel statistic r using Pearson’s correlation method, and asterisks mark significant Mantel correlations (* = p < 0.05, ** = p < 0.005). Panel (a) shows European and East Coast populations, and panel (b) shows California populations.

bioRxiv preprint doi: https://doi.org/10.1101/799254; this version posted October 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

Fig. S5 SNP data: Correlations between genetic distances and physical distances. Grey shading marks 95% confidence intervals around fitted linear models. Each plot includes the Mantel statistic r using Pearson’s correlation method and asterisks mark significant Mantel correlations (* = p < 0.05, ** = p < 0.001).