Mycena Species Can Be Opportunist-Generalist Plant Root Invaders 2 3 Christoffer Bugge Harder1,2, Emily Hesling3, Synnøve S

bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436563; this version posted March 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-NC-ND 4.0 International license.

1 Mycena species can be opportunist-generalist plant root invaders 2 3 Christoffer Bugge Harder1,2, Emily Hesling3, Synnøve S. Botnen1,4, Bálint Dima5,6, Tea von 4 Bonsdorff-Salminen7, Tuula Niskanen5,8, Susan G. Jarvis9, Kelsey E. Lorberau10, Andrew 5 Ouimette11, Alison Hester12, Erik A. Hobbie11, Andy F.S. Taylor3,12, Håvard Kauserud1 6 7 1Department of Biosciences, University of Oslo, Box 1066 Blindern, 0316 Oslo, Norway, 2Lund 8 University, Department of Microbial Ecology, Sölvegatan 37, 223 62 Lund, 3School of Biological 9 Sciences, University of Aberdeen, Aberdeen, UK, 4Oslo Metropolitan University, PO Box 4 St. 10 Olavs plass, NO-0130 Oslo, Norway, 5Plant Biology, Department of Biosciences, University of 11 Helsinki, P.O. Box 56, Helsinki FI-00014, Finland, 6Department of Plant Anatomy, Institute of 12 Biology, Eötvös Loránd University, Pázmány Péter sétány 1/C, H-1117 Budapest, Hungary, 13 7Botany Unit, Finnish Museum of Natural History LUOMUS, P.O. Box 7, FI-00014 University of 14 Helsinki, Finland 8Jodrell Laboratory, Royal Botanic Gardens, Kew, Surrey TW9 3AB, UK, 9 UK 15 Centre for Ecology & Hydrology, Lancaster, LA1 4AP, UK, 10Department of Arctic and Marine 16 Biology, UiT - The Arctic University of Norway, Tromsø, Norway, 11Earth Systems Research 17 Center, University of New Hampshire, Durham, New Hampshire, 03824, USA, 12The James 18 Hutton Institute, Aberdeen, UK. 19 20 Corresponding author: Christoffer B: Harder, Phone: +46704565065, e-mail: 21 [email protected]. Originality significance statement: This is the first study to apply a dual 22 approach of systematic metabarcoding of plant roots and stable isotope signatures on dried 23 field material to the large and common saprotrophic fungal genus Mycena. This is significant as 24 it shows that members of this genus, normally not expected to be found inside plant roots at all, 25 are in fact associated eith multiple plant hosts. The study furthermore shows that species in this 26 genus may occupy different ecological roles in the field besides being saprotrophic. That a large 27 and common fungal genus known to be a quantitatively important litter decayer can be an 28 opportunistic root invader and interact with host plants is of interest to all mycologists and 29 ecologists working on plant-fungus/microb symbiosis. 30 31 Summary 32 Recently, several saprotrophic genera have been found to invade/interact with plant roots in 33 laboratory growth experiments, and this coincides with reports of abundant saprotrophic fungal 34 sequences in plant roots. However, it is uncertain if this reflects field phenomena, and if reports 35 on coincidentally amplified saprotrophs are simply coincidental. 36 We investigated root invasion by presumed saprotrophic fungi by focusing on the large genus 37 Mycena in 1) a systematic analysis of the occurrence of saprotrophic fungi in new and bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436563; this version posted March 23, 2021. 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-NC-ND 4.0 International license.

38 previously published ITS1/ITS2 datasets generated from roots of 10 mycorrhizal plant species, 39 and 2) we analysed natural abundances of 13C/15N stable isotope signatures of fungal/plant 40 communities from five comparable field locations to examine the trophic status of Mycena 41 species. 42 Mycena was the only saprotrophic genus consistently found in 9 of 10 plant host roots, with high 43 within-host variation in Mycena sequence proportions (0-80%) recovered. Mycena carpophores 44 displayed isotopic signatures consistent with published 13C/15N profiles of both saprotrophic or 45 mutualistic lifestyles, with considerable intraspecific variation, resembling the patterns seen in 46 growth experiments. These results indicate that multiple Mycena species opportunistically 47 invade the roots of a range of plant species, possibly forming a spectrum of interactions. This 48 potentially challenges our general understanding of fungal ecology. 49 50 Introduction 51 Among ecologists, a consensus is emerging that the classical assignment of fungal species into 52 single trophic groups with a mycorrhizal, saprotrophic or pathogenic lifestyle may be too 53 restrictive (Baldrian and Kohout, 2017; Selosse et al., 2018). Some otherwise free-living fungi 54 have been shown to invade plant roots and exist as either asymptomatic endophytes (neither 55 harmful nor beneficial), and then switch from endophytic into becoming pathogenic (e.g. 56 Fusarium graminearum, Lofgren et al., 2018), or into forming AM mycorrhizas (Piriformospora 57 indica, Weiß et al., 2016) or ericoid mycorrhizal associations (Meliniomyces spp., Martino et al., 58 2018). 59 A wide range of saprotrophic fungal genera have been screened for their ability to colonise 60 Pinus sylvestris and Picea abies seedling roots in vitro (Smith et al., 2017), and several, 61 including Mycena, Gymnopus, Phlebiopsis, Marasmius or Pleurotus, invaded roots apparently 62 without decomposing dead tissue in the process. However, beyond the invasion, the nature of 63 the interactions with the plant host (if any) remains unknown. 64 For Mycena, however, there are several lines of direct and indirect evidence for their ability to 65 invade and interact with living plant roots, at least in vitro. Mycena species have been identified 66 as potential orchid mycorrhizal symbionts (Ogura-Tsujita et al., 2009; Zhang et al., 2012), 67 endophytes in photosynthetic moss tissue (Davey et al., 2013) and non-mycorrhizal 68 brassicaceous plants (Glynou et al., 2018). They have also been shown to form mycorrhiza-like 69 structures in roots of Vaccinium corymbosum in growth studies (Grelet et al., 2017). Recently 70 and perhaps most importantly, Thoen et al. (2020) showed that multiple species and individual 71 strains of Mycena could colonise roots of Betula pendula seedlings in vitro, and formed a 72 gradient of interactions from harmful to neutral to beneficial, with some species/strains being 73 able to transfer nutrients to the plant host. This is significant, as prior to this Mycena sensu 74 stricto (Moncalvo et al., 2002) (henceforth simply "Mycena"), which is one the largest genera in bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436563; this version posted March 23, 2021. 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-NC-ND 4.0 International license.

75 Agaricales (over 500 species), widespread across habitats and climate zones (Kühner, 1938; 76 Maas Geesteranus, 1992; Rexer, 1994; Robich, 2003; Aronsen and Læssøe,2016), was known 77 primarily as quantitatively important litter and wood debris decomposers (Boberg et al., 2008; 78 Baldrian et al., 2012; Kyaschenko et al., 2017). The spectrum of interactions seen in Thoen et 79 al. (2020) is further noteworthy in the light of the "Waiting room hypothesis" (van der Heijden et 80 al. 2015) on mycorrhizal evolution, which suggests that the mycorrhizal habit evolves from 81 saprotrophs gradually via neutral endophytic intermediate states. This hypothesis has remained 82 controversial even though it is accepted that the mycorrhizal habit has evolved on numerous, 83 independent occasions from saprotrophic ancestors (Tedersoo and Smith 2013, Kohler et al. 84 2015). Thus, the genus Mycena may represent a promising research model for studying both 85 ecological versatility in fungi and the possible ongoing evolution of fungi traditionally believed to 86 be purely saprotrophic en route to developing mycorrhizal abilities. 87 Most of this evidence for trophic versatility in Mycena originates from in vitro studies, 88 and it is uncertain to what extent this translates to the field. To investigate the trophic mode of 89 fungi in natural environments, analysis of the natural abundance of 13C:12C and 15N:14N ratios 90 (isotope ratios, expressed as d13C and d15N values relative to known standards) can be applied 91 directly to fungal carpophores and other field material. Mycorrhizal fungi are generally more 92 enriched in 15N and depleted in 13C than saprotrophic fungi (Taylor et al., 1997; Kohzu et al., 93 1999; Hobbie et al., 1999; Hobbie et al., 2001; Griffith, 2004; Mayor et al., 2009). Based on 94 comparisons between fungi of known trophic status, natural abundance of 15 and 13C can also 95 give strong indications of the nutritional mode of fungi with unknown trophic status. Thus, 96 Halbwachs et al. (2018) recently used this approach to strongly suggest that Hygrocybe, 97 another genus traditionally believed to be saprotrophic, was most likely biotrophic with plants. 98 The occurrence and abundance of Mycena sequences retrieved from wild plant roots also 99 suggests interactions with plant roots in situ. There are scattered reports of Mycena identified 100 from inside wild plant roots, particularly in the Arctic plants, including Bistorta vivipara, Cassiope 101 tetragona, Dryas octopetala, and Salix polaris (Blaalid et al., 2014; Botnen et al., 2014; 102 Lorberau et al., 2017). In some cases, Mycena sequences comprised >30-50% of the total 103 reads, suggesting that they are not simply casual colonisers. It has been suggested that the 104 harsh and oligotrophic Arctic environments stimulate otherwise free-living fungal genera 105 (including Mycena) to explore new ecological niches (Jumpponen and Trappe, 1998; Ryberg et 106 al., 2009; Ryberg et al., 2011; Timling et al., 2012; Botnen et al., 2014; Lorberau et al., 2017). 107 Nevertheless, Mycena reads have also been recovered in high quantities from inside living 108 Picea abies roots in temperate environments (Kohout et al., 2018). In this case, Mycena 109 species were present in the roots prior to felling but then became dominant post-felling. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436563; this version posted March 23, 2021. 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-NC-ND 4.0 International license.

110 Overall, however, current information on the occurrence of Mycena and other saprotrophs in 111 roots unsystematic and too scattered to identify any clear patterns of their occurrence and 112 abundance. 113 High throughput sequencing (HTS) studies of fungal communities in plant roots are generally 114 targeting mycorrhizal fungi (Buee et al., 2009; Tedersoo et al., 2010; Bahram et al., 2011; 115 Blaalid et al., 2014; Vasar et al., 2017; Kaur et al., 2019), and the workflow requires the 116 annotation of tens of thousands of OTUs/clusters of fungi into ecological guilds. Most studies 117 tend to favour more conservative taxonomic ecological generalisations based at the genus 118 level, which is traditionally considered the most relevant level for separating fungal taxa by 119 nutritional mode (Fries and Mueller, 1984; Molina and Trappe, 1994; den Bakker et al., 2004; 120 Tedersoo and Smith, 2013; Garnica et al., 2016). Ecological annotation software (Nguyen et 121 al., 2016) is largely based on this view. Thus, in studies of mycorrhizal fungi in roots, fungal 122 genera identified as being saprotrophic may be at best briefly mentioned and/or reported as one 123 lumped ecological group (Menkis et al., 2012; Tedersoo and Smith, 2013), or simply dismissed 124 as accidental contamination (Liao et al., 2014). This means that the ecological understanding of 125 root ecosystems may become oversimplified, and large quantities of potentially informative data 126 are left unanalysed and erroneously ignored. 127 Here, we present a dual analysis on the occurrence of Mycena in roots of wild plants in a range 128 of ecosystems, and an investigation of the potential trophic versatility of Mycena in the field by 129 presenting: 1) a systematic analysis of data from 10 plant species from Arctic and temperate 130 regions (Information on plants and studies given in Table 1) from previously published and 131 newly generated ITS1/ITS2 HTS data sets from living plant roots, and 2) a comparison of the 132 natural abundance of 13C and 15N in carpophores of Mycena with other fungi, soils and 133 mycorrhizal host plants from 253 fungal collections, host plants and soils from five field 134 locations (See Fig S1-S2 for a map of sampling sites). 135 We investigated four main research questions: 1) Are Mycena species (and other 136 supposedly saprotrophic genera) systematically found inside living plant roots in significant 137 quantities? 2) If so, are these more prevalent in Arctic/Alpine environments? 3) Are there 138 indications of host preferences/specificity among invasive Mycena species? 4) Do the data from 139 13C and 15N abundances support the view that Mycena species may form mutualist association 140 with plants? 141 142 Results 143 144 HTP-sequencing data summary bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436563; this version posted March 23, 2021. 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-NC-ND 4.0 International license.

145 Our dataset consisted of 3 species amplified with ITS1 primers and 2 species amplified with 146 ITS2 primers from previously published data reanalysed here. 5 new species (from three 147 separate 454 datasets amplified with ITS2 primers) represent new data (Table 1). 148 After quality sorting, a final dataset of 889,290 ITS1 sequences were clustered into 1193 3% 149 OTUs (n≥10, henceforth simply OTUs) for the three plant species where the ITS1 marker were 150 used, and an ITS2 dataset of 992890 sequences and1032 3% OTUs in the seven datasets with 151 ITS2 (Table 2). For a detailed list of the sequence sorting steps on the ITS1 and ITS2 data and 152 the respective counts for each host species, see Supplementary data and Tables S1-6. 153 Applying the "coverage/completeness" method for assessing saturation (Chao and Jost, 2012), 154 111 samples failed to meet the 97% coverage cutoff value and were discarded. Though the 155 iNEXT (Hsieh et al., 2016) extrapolations of observed species richness suggested that some 156 slight undersampling remained in some samples (Fig S5), none of the ten species showed 157 correlations between Mycena infection levels (all Ps>0.05, table S7) with sampling depth. 158 We analysed the datasets with both amplicon sequence variants (ASVs/”zotus”, aimed at 159 capturing the haplotype diversity) and 3%-OTUs, aimed at capturing species. As expected, 160 there were higher numbers of ASVs than OTUs. However, using 3% OTUs or ASVs made little 161 difference to the taxonomic composition of the datasets, as seen by the near-identical Mycena 162 shares in OTUs and ASVs . There were no clear signs of potential host specialisation by 163 Mycena at either the finer ASV-(“haplotype”) scale or at the coarser 3% OTU scale (Tables S8- 164 9). Thus, the further analyses focused on the 3% OTU datasets. 165 For ITS1, 606 of 1193 OTUs (78.3% of the sequences) could be identified to genus level by 166 SINTAX at the threshold of BPP >0.6; for ITS2, this number was 513 of 1032 ITS2 OTUs 167 (84.5% of sequences). 168 The same SINTAX classification identified 13 Mycena OTUs (1.5% of all ITS1 sequences) in 169 the ITS1 dataset, and 14 Mycena OTUs in ITS2 (12.6% of all ITS2 sequences). However, in a 170 second identification step especially targeting Mycena where representative sequences of all 171 OTUs were clustered with the Mycena ITS database 576 sequences (described below), an 172 additional 7 ITS1 OTUs and 7 ITS2 OTUs not identified as Mycena by SINTAX at BPP >0.6 173 formed clusters (at 97%) with Mycena species in the database. Thus, in total 20 ITS1 OTUs 174 (2.1% of sequences) and 21 ITS2 OTUs (15.8% of sequences) could be identified as Mycena 175 (s. str) with these two combined methods. (3 ITS1 OTUs identified as Mycena by the 8.2 utax 176 eukaryote reference database represented taxa now classified as Phloeomana and Atheniella 177 (Redhead, 2013) and were excluded from detailed analysis). 178 Of other (non-Mycena) taxa traditionally considered to be saprotrophic/endophytic, we found 179 Sebacinales in the four Arctic host plants, the zygomycete Mortierella in most Scottish hosts, 180 and Phialocephala in B. vivipara and Clavulinopsis/Clavaria in C. tetragona (Fig. 2a-c). No other 181 saprotrophic/endophytic genera formed more than 0.5% of the sequences in any of the host bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436563; this version posted March 23, 2021. 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-NC-ND 4.0 International license.

182 plants. The high and very variable infection levels and frequency patterns of Mycena were not 183 found in any other saprotrophic/endophytic taxa. 184 185 Mycena infection levels 186 187 In nine out of ten host plants, Mycena infection levels reached 25-80% of all reads in individual 188 samples (Fig. 2a-c), with considerable intraspecific infection variation (Fig 1, Fig. S3). For the 189 ITS1 data, Mycena average read content for all 519 B. vivipara samples was significantly lower 190 (than for S. polaris (n=20) and D. octopetala (n=22) (Fig.1a-b, Table S10)). However, the S. 191 polaris and D. octopetala data sets came from only one locality (Botnen et al., 2014), and when 192 comparing them only with the B. vivipara dataset (n=19) from the same locality, no significant 193 differences were observed (1-way ANOVA, df=2, F=1.36, p=0.263). Without considering the 194 differences in sample sizes (Table 1), ITS2 host species could be roughly divided into three 195 groups based on average Mycena infection level - 1) P. sylvestris with virtually no Mycena, 2) 196 an intermediate group (median values about 5-10%) with S. herbacea, A. alpine, B. nana, and 197 A. uva-ursi, and 3) B. pubescens and C. tetragona with median Mycena infection levels above 198 20%. While all species (except P. sylvestris) harboured individual samples with few if any 199 Mycena and some with >30%, there were still significant differences between these three rough 200 categories. 201 (Fig. 1 d-e, Table S11)). 202 203 Environmental influences on Mycena 204 Disparities in sample size and study metadata only permitted limited testing of environmental 205 influences on Mycena infection to three host species. In C. tetragona, there were no difference 206 in Mycena infection levels between samples derived from drought and control plots applied by 207 Lorberau et al. (2017)(two-tailed t-test, unequal variances, p=0.57). In A. uva-ursi, the level of 208 Mycena infection decreased significantly with increasing altitude (65-805 m above sea level) 209 (R2=0.2, p<0.0001, data not shown), which is contrary to the assumption (question 2) that 210 increasingly stressful environments facilitating infection with Mycena/saprotrophs. 211 In B. vivipara, no correlations between Mycena infection level and annual mean temperature, 212 latitude nor mean temperature of the wettest quartal were found (all adjusted R2<0.01, all 213 P>0.25, see Table S12). A very weak correlation between decreasing Mycena species richness 214 in roots and increasing mean temperature of the wettest quartal (R2=0.01, P=0.04) disappeared 215 when the Austrian outlier samples (which contained no Mycena) were excluded. 216 A chi-quare test on the observed vs. expected prevalence of Mycena in 222 B. vivipara host 217 plants from 44 patches (a patch constituted multiple plants collected in close proximity) showed bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436563; this version posted March 23, 2021. 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-NC-ND 4.0 International license.

218 a significant, non-random association (c2=65.22, df=43, p=0.01) of Mycena infections in host 219 plants, suggesting that Mycena-infected B. vivipara were distributed in clumps. 220 221 Mycena phylogenetics of OTUs and species diversity 222 Among the 20 ITS1 and the 21 ITS2 OTUs that were identified as Mycena, we found no 223 phylogenetic signal suggesting that root invasion might be linked to certain clades (Fig. 3). 224 Many of the same Mycena species were found in both the ITS1 and ITS2 datasets with 12 225 ITS1-ITS2 pairs of OTUs clustered with >90% probability to the same branches. There were no 226 indications of host specialisation by Mycena species on particular host species, with large 227 individual variations in all host plants (again except P. sylvestris) between which Mycena 228 species that were found (Fig. S3), Several Mycena species such as M. epipterygia or M. 229 leptocephala occurred in infection levels of >10% in at least one individual of 6 of 10 host 230 species or more (Fig. S3). Indeed, the two OTUs were the only OTUs shared between C. 231 tetragona from Svalbard and all Scottish host species (except P. sylvestris). 232 233 Mycena database 234 We compiled 576 new and previously published full-length Mycena ITS Sanger sequences 235 representing 137 identified species level into a database (see Experimental procedures 236 below).They clustered into 156 and 139 ITS1 and ITS2 3% OTUs, respectively. 237 For both regions, some OTUs contained two or more species (such as Mycena galericulata + 238 M. megaspora and M. olivaceomarginata, M.citrinomarginata and M. albidolilacea), while other 239 species were split into multiple OTUs (e.g. M. pura and M. epipterygia). Average intraspecific 240 variation was 3.6% (ITS1) and 2.7% (ITS2) (Fig. S4). 241 242 Stable isotope data 243 On average, carpophores values of 15N and 13C placed Mycena among the saprotrophs. They 244 were higher in δ13C and lower in δ15N than the average of the remaining non-Mycena 245 saprotrophs (Fig. 4a-e). The δ13C values of all saprotrophic species for all regions were 246 between -26 and -22‰, except for a Phloeomana speirea at Finse (Fig. 4a) at -27.1‰, and one 247 Mycena metata collection from Svalbard (Fig. 4c) at -26.9‰. However, there were striking 248 anomalies (and intraspecific variations) in the δ15N values for certain individual collections of 249 Mycena, particularly M. pura, which varied between 1.7‰ for M. pura in Gribskov to 12.6‰ for 250 M. pura1 at Finse. A t-test showed the M. pura1 and M. pura2 collections at Finse to be strongly 251 and significantly higher in δ15N than the average for the other Mycena at Finse (p < 0.0001), 252 and slightly but still significantly higher in δ13C (p = 0.02). bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436563; this version posted March 23, 2021. 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-NC-ND 4.0 International license.

253 In the stepwise regression of δ15N, genera were separated by up to 12‰ into five groups (Table 254 S13). Relative to the mean, Mycena grouped at -3‰ with the litter decay fungi Calvatia, 255 Lycoperdon, and Rhodocollybia. The overall adjusted r2 of the regression model was 0.56, with 256 site accounting for 8.1% of variance and the remaining 48.0% accounted for by genus. 257 In the stepwise regression of δ13C, genera were separated by up to 5‰ into eight groups 258 (Table S14). Relative to the mean, Mycena grouped at +1‰ with both the ectomycorrhizal 259 Rhizopogon, the partly saprotrophic/ectomycorrhizal Ramaria and the litter decay fungi 260 Calvatia, Lepista, and Rhodocollybia. The overall adjusted r2 of the regression model was 0.66, 261 with site accounting for 11.7% of variance, nitrogen concentration (%N) for 2.8%, and the 262 remaining 51.4% accounted for by genus. 263 The δ15N values for host plants in all 5 regions were all below 0‰, which is significantly lower 264 than not only carpophores, but also the soil (Fig.4). Soil δ15N values were below 2‰, with lower 265 overall N contents but higher δ15N values for deeper soil depths, in line with earlier studies 266 (Evans, 2007; Seitzman et al., 2011, Clemmensen K, 2013; Halbwachs et al., 2018). The δ15N 267 and δ13C values differed significantly between the five regions, but the amount of variance for 268 both measures explained by region in the mixed linear model was <20% for both isotopes and 269 well below that explained by sample type and genus/sample type in combination (> 75% for 270 both). 271 Overall, the other fungal genera had isotopic profiles that matched their expected nutritional 272 mode. 273 274 Discussion 275 276 This study constitutes the first systematic analysis of Mycena in wild plant roots, and the results 277 clearly indicate that Mycena species are frequent root colonisers of a taxonomic range of 278 mycorrhizal host plants, although infection levels are very variable. The isotopic data here 279 suggest that they could have several potential ecological functions inside the roots. They could 280 be endophytes, as has been suggested for Sebacinales (Blaalid et al., 2014; Botnen et al., 281 2014; Lorberau et al., 2017) or dark septate endophytes (Newsham, 2011) in many Arctic 282 plants. However, here we found Mycena infection to be widespread in Arctic/alpine hosts as 283 well as in temperate hosts, and the general lack of host-specificity in Mycena was also 284 universal. The ability to colonise living roots appears to be a widely shared trait across the 285 Mycena phylogeny, consistent with the findings of Thoen et al. (2020). 286 Mycena infections displayed a qualitatively remarkably similar pattern in 9 of 10 host plants: 287 present in many samples and varying from little or no infection up to >40-50% of the recovered 288 reads i.e:. The complete lack of Mycena (and saprotrophs in general) in P. sylvestris (Fig.1) is bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436563; this version posted March 23, 2021. 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-NC-ND 4.0 International license.

289 noteworthy, as Mycena were frequent root invaders in P. sylvestris seedlings in in vitro growth 290 experiments by Smith et al. (2017). Pines have strongly heterorhizic root systems and only the 291 final feeder roots are not either suberised or metacutinised, which will severely limit colonisation 292 by fungi. In addition, under field conditions ectomycorrhizal colonisation levels of the feeder 293 roots will be close to 100%, so the available surface for colonisation by non-mycorrhizal fungi 294 will be very limited. 295 However, Kohout et al. (2018) found Mycena to be widespread in roots of mature (+80y) 296 stands of conifers (P. abies) in forests. Furthermore, the Mycena infection patterns in B. 297 pendula seedlings in vitro (Thoen et al., 2020) were indeed consistent with our observations in 298 the close relative B. pubescens roots in the field. 299 However, the spruce in Kohout et al. (2018) originated from intensely managed and ultimately 300 clear-cut forest, and they had a high overall root fungal diversity in addition to the levels of 301 Mycena infections. The B. pubescens in this study were tree saplings of >1 m on a location 302 where they were kept low by a particularly high grazing impact from sheep and deer, and all 303 other species with high Mycena infection levels were smaller dwarf shrubs of varying sizes or 304 herbaceous plants, also known to be subject to deer/reindeer grazing (Kolari et al., 2019). In 305 contrast, the P. sylvestris in our study were in largely undisturbed stands within a national park, 306 and their near-complete dominance by one genus (Suillus, Fig. 2d) and associated very low 307 general root diversity (Fig. 1g-h) is consistent also with pre-HTP sequencing era studies of 308 undisturbed P. sylvestris roots (Jonsson et al., 1999). 309 It is possible that differences in disturbance is an explanation for the difference in Mycena 310 infection levels, i.e. that Mycena root invasion should be seen as a largely opportunistic feature 311 of plants that are young, disturbed or otherwise vulnerable. This is also consistent with what is 312 generally known to facilitate attacks from known fungal parasites (Walters, 2011). We speculate 313 that this disturbance effect could also apply within species: e.g. grazing by herbivores passing 314 by at one location would be likely to impact multiple individuals close by each other. The 315 clumped (uneven) distribution of Mycena infections levels in B.vivipara can thus be interpreted 316 as offering some support for this theory. 317 An important issue (question 4) is what is the basis of interaction when Mycena invades a host 318 root. It was not possible to determine i the Mycena carpophores sampled for analysis of stable 319 isotopes were free-living or from mycelia associated with a plant, Although the average value of 320 Mycena suggested a saprotrophic lifestyle, there were several individual collections with profiles 321 that suggested an alternative mode of nutrition. Most clearly, two collections of Mycena pura in 322 Finse have isotopic profiles that resemble that of mycorrhizal fungi., Interestingly, in the study 323 by Thoen et al., (2020) the culture of M. pura which could transfer 32P to its plant host was 324 grown from the M. pura1 sample from Finse. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436563; this version posted March 23, 2021. 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-NC-ND 4.0 International license.

325 In addition, the isotopic profiles of M. pura and its close relatives M. rosea2 and M. diosma of 326 the section (Calodontes) from Gribskov (in Denmark) are not far from those of the 327 ectomycorrhizal Russula or Cortinarius.(Fig. 4 However, the M. pura from Svalbard and 328 Vettakollen (in mainland Norway) and a M. rosea1 from Gribskov had isotopic signatures closer 329 to the expected profiles for saprotrophs. The members of the Mycena section Calodontes are 330 notably hard to grow in culture which has previously led to speculations about their nutrition 331 (Perreau et al., 1992; Boisselier-Dubayle et al., 1996; Harder et al., 2010; Harder et al., 2012). 332 However, it must be noted that Mycena pura was rarely found in the root samples and only 333 constituted a significant (>10%) fraction of the root community in one single B. vivipara 334 individual (Fig. S5). None of our collections of M. galopus displayed similar intraspecific 335 variations and/or mycorrhizal-like patterns in their isotopic profiles as one might have 336 expected based on Grelet et al. (2017) or Thoen et al. (2020). Whether this is a coincidence for 337 this study or not must be left for further research to explore. 338 Overall, the variable isotopic patterns of Mycena from the field are broadly consistent with the 339 interspecific and intraspecific variation observed in the interactions between Mycena and birch 340 roots in the growth experiments by Thoen et al. (2020), where different species and conspecific 341 strains displayed harmful, neutral/endophytic, or beneficial interaction phenotypes in vitro. In 342 the light of the emerging discoveries of variable ecologies among several fungal genera, these 343 findings highlight the need for more targeted organism-level research on multiple individuals of 344 fungal species to obtain a more comprehensive picture of the possible ecological versatilities. If 345 fungal ecology is versatile not only below the genus, but also below the species level, then this 346 may lead to reconsideration of the high importance ascribed to nutrition as a decisive taxon- 347 delimiting trait (as in Serpulaceae (Skrede et al., 2011) or Clavariaceae (Birkebak et al., 2013)). 348 Redhead et al. (2016) proposed to split the monophyletic Amanita sensu lato into 349 ectomycorrhizal Amanita sensu stricto and a new saprotrophic Saproamanita, precisely to make 350 ecological annotation in molecular studies easier, but this would be unnecessary with greater 351 appreciation of ecological versatility. This has important implications for the widely applied 352 approach in HTP-sequencing/metabarcoding plant root studies where annotating ecology to a 353 sequence with genus-level based taxonomy could be misguided. 354 A question was raised by Vohník (2020) concerning the high numbers of Mycena/Clavaria 355 sequences recovered from C. tetragona by Lorberau et al. (2017) suggesting that they might be 356 explained by a lack of root cleaning/washing. While this is an issue that should not be ignored, 357 all root samples included in the present study were either serially washed and/or surface 358 sterilised as standard, including (Lorberau et al. (2017). It is therefore very unlikely that the 359 recovery of large numbers of Mycena reads (and other saprotrophs) is purely due to mycelia 360 living commensally on the root surface. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436563; this version posted March 23, 2021. 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-NC-ND 4.0 International license.

361 Until now, most data on the occurrence of saprotrophic fungi inside plant roots has arisen as a 362 by-product of other research, and the sampling for these metabarcoding datasets here were not 363 originally designed to investigate this question or to be analysed together. The differences (Fig 364 1) in average Mycena infection levels between 9 of the 10 host plants in our sample should be 365 interpreted with the caution warranted by differences in sample sizes and site variations, and in 366 comparing two ITS regions (Harder et al., 2013). The reverse primer (ITS2_r) of the ITS1 primer 367 set has a terminal mismatch with 99% of all Mycena species (Tedersoo and Lindahl, 2016), 368 which suggests that Mycena content in the ITS1 dataset could be underestimated. If true, this 369 would merely strengthen our overall conclusions about Mycena as an overlooked but significant 370 root invading genus; however, more studies directly targeting supposedly saprotrophic or 371 endophytic (non-mycorrhizal) fungi in roots are certainly desirable. 372 To test our hypothesis that Mycena (or other saprotrophic) root infections are a result of 373 opportunistic invasion under disturbance-related circumstances, future targeted metabarcoding 374 root studies should directly analyse roots of multiple host species of different age and 375 disturbance levels in the field in order to identify particular factors that may affect root invasion. 376 Annotation databases should be continuously updated to reflect our best taxonomic knowledge, 377 and further efforts should be undertaken to identify fungal OTUs or ASVs beyond the genus 378 level, which may require more attention to detail than relying on SINTAX/RDP classification 379 based on even the best possible databases. 380 Most importantly, more studies on direct ecological interactions between particular hosts and 381 known fungal species are needed; both resynthesis host-fungus experiments with studies of 382 nutrient and C transfer between the symbionts, and stable isotope studies in the field that 383 specifically target saprotrophic taxa. 384 385 Conclusions 386 387 The investigation of the trophic status of genus Mycena using sequence data from wild plant 388 roots and 15N and 13C stable isotope signatures yielded the following: 1) In nine of ten 389 analysed herbaceous and ericaceous plants and tree mycorrhizal host plants from temperate, 390 alpine and arctic environments, Mycena was consistently present in living plant roots across 391 species and in different environments, but other saprotrophic taxa were only occasionally 392 present; 2) Mycena infections were not generally more prevalent in Arctic environments or at 393 higher altitudes, but we hypothesise that infection may be more prevalent under conditions of 394 disturbance; 3) The ability to invade living plant roots is a feature of multiple Mycena species 395 that do not discriminate between plant hosts, and 4) The stable isotopic data on carpophores 396 suggested that, although the genus Mycena is indeed mostly saprotrophic, strains of bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436563; this version posted March 23, 2021. 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-NC-ND 4.0 International license.

397 certain Mycena species can display an ecological versatility in the field and exchange nutrients 398 with plants, consistent with previous results from in vitro resynthesis experiments. 399 The evidence that fungal trophic modes may be variable on the species level, and that within a 400 large genus such as Mycena there may be several potential trophic options in addition to pure 401 free-living saprotrophy, raises intriguing questions about the general understanding and study of 402 fungal ecology. More research directly targeting root-associated fungi with unclear or unknown 403 ecologies is required to resolve these questions. This study highlights the importance of 404 continued detailed studies on interactions among organisms at the species level in order 405 enhance data usage from broad, environmental metabarcoding approaches to community 406 characterisation. 407 408 Experimental procedures 409 410 Sample site and sample descriptions 411 Betula pubescens roots were collected at the RSPB Nature Reserve at Corrimony in north-west 412 Scotland in August 2008. The trees were regenerating saplings at a maximum 1 m in height, 413 growing on moorland within heather-dominated vegetation on a site previously browsed by 414 sheep and deer. Roots samples (supporting 100-200 ECM tips) were taken from the trees by 415 direct tracing fine roots from the main laterals. Roots from 5 trees from within a block were 416 pooled to give one single sample. 417 Roots from Salix herbacea, Betula nana, Arctostaphylos. alpina, and 8 A. uva-ursi from an 418 original biogeography study were collected from mountains across Scotland (Fig S1) (Hesling 419 and Taylor 2013). 420 The remaining 68 sample of A. uva-ursi roots were from an altitudinal gradient study (and 9 421 additional P.sylvestris samples in addition to those from Jarvis et al. (2015) in this study), 422 collected June-July 2011 in the Invereshie-Inshriach National Nature Reserve in the north-west 423 of the Cairngorms National Park in Scotland (Figs. S1, S2). Samples came from 9 elevation 424 transects from 450-850 masl on a Calluna-Arctostaphylos subalpine heath with scattered Scots 425 pine trees up until the tree limit at ~650 masl. This was in close proximity to the P. sylvestris 426 forest studied in Jarvis et al. (2015) 427 The previously published datasets of B. vivipara, S.polaris, D. octopetala and C.tetragona were 428 all collected in Arctic and Alpine tundra above the treeline in Arctic Norway, Iceland and Austria, 429 and from grassland below the treeline in Scotland. For more details on the previously published 430 data, we refer to the original publications. A more detailed description of the plant species 431 targeted and the sample sites for the new data can be found in the supplementary data. 432 433 Preparation of roots and old amplicon libraries for previously published ITS2 datasets bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436563; this version posted March 23, 2021. 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-NC-ND 4.0 International license.

434 For the three new ITS2 datasets/454 runs representing 5 of 7 host species (see 435 bioinformatics below), roots were sampled and cleaned under a dissection microscope to 436 remove visible soil debris, woody and non-target species’ roots, then lyophilized in 2 ml tubes 437 and milled using a steel bead in a mixer mill (RETSCH, Düsseldorf, Germany). Dry weight for 438 DNA extraction was adjusted for each sample so that extracted mass was proportional to total 439 sample dry weight: extract weight (mg) = (42.50 × total sample dry weight (mg)) + 47.98. DNA 440 was extracted using 96 well, DNeasy Plant Minikits (QIAGEN, Hilden, Germany).

441 PCR amplification of the ITS2 region was conducted on a 2720 Thermal Cycler (Life 442 Technologies, Carlsbad, CA, USA) in 10 µl reactions: 5 µl diluted template; 40 µM of each

443 nucleotide; 0.55 mM MgCl2; 40 nM ITS7A primer (Ihrmark et al. 2012); 40 nM ITS 4 primer with 444 a 3’ 8 bp tag (unique by ≥2 bp between samples); and 0.005 U/µl polymerase (DreamTaq 445 Green, Thermo Scientific, Waltham, MA, USA) in buffer. Cycling parameters were: 94 °C for 5 446 min then 25, 30 or 35 cycles at 94 °C for 30 s; 57 °C for 30 s; 72 °C for 30 s; with a final 447 extension of 72 °C for 10 min. PCR products were checked using gel electrophoresis 448 (dilutions/cycles adjusted if products were out with the range 1-10 ng µl-1), then purified using 449 AMPure 96 (Beckman Coulter, Brea, USA). DNA concentrations were established using a Qubit 450 2.0 fluorometer (Invitrogen, Paisley, UK), samples combined in equal molar proportion, further 451 purified using GeneJET PCR Purification (Thermo Scientific, Waltham, USA) and lyophilized. 452 Adaptor ligation, 454-sequencing and sequence adapter trimming were performed by the NERC 453 genomics facility (Liverpool, UK) on one pico-titre plate using the GL FLX Titanium system 454 (Roche, Basel, Switzerland). 455 456 Mycena database 457 458 For identification of Mycena sequence data to species level, we first generated 151 new 459 sequences from herbarium specimens and personal collections, using the ITS1F/ITS4 primers 460 and the PCR protocol of Gardes and Bruns (1993). All Mycena ITS full-length sequences from 461 GenBank and from the UNITE database (1099 sq) were extracted. Sequences not identified to 462 species level, which did not cover the regions amplified by the ITS1F-2/ITS3-4 primer target 463 regions, and which were not inside the Mycena sensu stricto clade (Fig. 3), or duplicates 464 between both databases were discarded. Additionally, 14 complete Mycena sequences in 465 GenBank were also discarded, as these were deemed to be misidentified (see table S15), most 466 of those from (Hofstetter et al. 2019). The final database comprised 576 high-quality sequences 467 with 136 named Mycena species, 89 of which with ≥2 sequences. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436563; this version posted March 23, 2021. 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-NC-ND 4.0 International license.

468 Sequences were aligned with the FFT-NS-i algoritm in MAFFT v 5 (Katoh and Standley, 2013). 469 The complete (628 bp) and annotated M. pura EU517504 sequence was used to identify the 470 ITS1 and ITS2 regions in the alignment. 471 472 Bioinformatics 473 474 From the previously published studies of B. vivipara, a high-quality dataset of 119 054 475 sequences was compiled from Blaalid et al (2012), 191,099 from Yao et al. (2013),157 181 from 476 Botnen et al. (2014), 244 523 from Blaalid et al. (2014) 272 595 sequences from Mundra et al. 477 ,(2015) 249 888 from Davey et al. (2015), and 132912 from Botnen et al. (2019), making a total 478 of 1095997 ITS1 sequences for clustering into OTUs/ASVs. 479 For the ITS2 dataset, we first analysed the two previously published studies of Jarvis et al 480 (2015) and Lorberau et al (2017), and obtained 175829 and 1952314 sequences, For the three 481 unpublished ITS2 454 runs, 327480 raw reads were obtained on a run with 104 Betula 482 pubescens samples; 494187 raw reads on a run combining altitude and biogeography 483 samples, and 232125 for a run with 16 1st year biogeography samples. After denoising, 484 chimera check, length, primer/base pair match and quality controls, 121587, 326380 and 485 154121 high-quality reads remained, respectively. In total, this amounted to 2730231 high- 486 quality ITS2 sequences of all fungal ecological groups. These were then used for clustering into 487 OTUs/ASVs. 488 The OTUs/ASVs were classified taxonomically with the non-Bayesian SINTAX classifier (Edgar, 489 2016) using the 8.2 utax eukaryote reference database (Abarenkov et al., 2020). 490 QIIME (Caporaso et al., 2010) 1.9.1 pipeline was employed for the three unpublished 454 runs 491 through the same steps as in Jarvis et al., (2015) until the OTU clustering step. We retained 492 those with a sequence length 200-550 bp, only 100% match to in primer/tag sequences, passed 493 chimera check in UCHIME(Edgar et al., 2011), a sliding window quality check of 50 bp applied 494 to identify low-quality regions (average Phred score < 25). The resulting fasta files from the 495 individual ITS1 and ITS2 runs were combined, and clustered first into OTUs at 97% similarity 496 using vsearch (Rognes et al., 2016) and its usearch_global command function, and then into 497 ASVs using the standard settings in UNOISE (Edgar, 2016). The R decontam package (Davis 498 et al., 2018) with the default settings to remove likely contaminants based on the negative 499 controls on a per sample basis for each of the 6 different datasets in the ITS1 part, and on the 500 single negative control samples in the Cassiope tetragona ITS2 dataset (no negative controls 501 were sequenced in Jarvis et al. (2015) nor in any of the new ITS2 datasets). Non-fungal 502 sequences (P<0.95), and OTUs and ASVs with respectively <10/<8 counts were removed. 503 Finally, sampling saturation was assessed with the iNEXT package (Hsieh et al., 2016), (see bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436563; this version posted March 23, 2021. 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-NC-ND 4.0 International license.

504 also Fig. S5), and all samples not meeting a coverage-based completeness (Chao and Jost, 505 2012) of 97% were discarded. 506 507 Collection of samples for isotope analysis: Fungal carpophores, host plants and soil were 508 collected from 5 different regions: Svalbard (arctic Norway); Finse/Hardangervidda (alpine 509 central Norway), Vettakollåsen (boreal forest) in southeastern Norway; Solhomfjell National 510 Park (boreal forest) in South Norway, and Gribsø /Gribskov (North Zealand, Denmark), in a 511 beech-dominated broadleaf forest patch (Fig. S1) in 2015 and 2016. For more information and 512 geographic coordinates of the field locations, see Fig. S1 and legend. In Svalbard, the collection 513 sites spanned several similar valleys on the southern banks of Isfjorden, with the sites 514 separated by up to ~60 km (Fig. S1); for the other four remaining collection sites, samples were 515 taken from an area that extended over no more than 1 km2. 516 Fungal carpophores, soil samples and plants were dried with continuous airflow for 12-36 517 hours at 70 °C until dry. Plants and fungi were identified morphologically, and Mycena 518 furthermore by ITS sequences. For Svalbard, some additional fungal samples were taken from 519 dried mushroom specimens kept at the herbarium at Tøyen at the Natural History Museum in 520 Oslo. It was assumed that individual carpophores collected within a distance of <50 centimeters 521 between them originated from the same mycelium. Conspecific Mycena carpophores sampled 522 from larger distances were treated as separate samples. Whenever possible, collections from a 523 given site were triplicated or at least duplicated, using separate fruit bodies from the same 524 collection. Fungi were divided into the three categories "ectomycorrhizal", "saprotrophic" or 525 "Mycena". For every 36 samples analysed, internal replicates of material from two samples from 526 the same fruitbody was used to verify consistent machine functioning. Soil samples were taken 527 from top-soil (A horizon, 0 cm) and from mineral soils in ~50 cm depths. We sampled soil from 528 three different locations on the different sites. Plant samples were all taken from leaves. 529 530 Stable isotope analysis: Dried samples (plants, fungi, soil) were ground by hand, weighed 531 (see supplementary table TS4) into 5 x 9 mm tin capsules (Sercon), closed and compressed. 532 Samples consisted of 5 mg of fungi/plant, 10 mg of topsoil, or 20 mg of 50 cm depth soil. 533 Samples were analysed for δ13C, δ15N, % C, and % N by continuous flow with a Costech 534 ECS4010 elemental analyser (Costech Analytical Technologies Inc, Valencia, California) 535 coupled with a DELTAplus XP isotope ratio mass spectrometer (Thermo Scientific, Bremen, 536 Germany) at the University of New Hampshire Stable Isotope Laboratory. All carbon and 537 nitrogen isotope data are reported in delta notation according to this equation: δX = 13 15 13 12 15 14 13 538 [(Rsample/Rstandard) - 1] × 1000 where X is C or N and R is the ratio C/ C or N/ N. All δ C 539 and δ15N values were normalised on VPDB (δ13C) and AIR (δ15N) reference scales with the bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436563; this version posted March 23, 2021. 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-NC-ND 4.0 International license.

540 following internationally calibrated standards and values: IAEA CH6 (210.45%), CH7 541 (232.15%), N1 (0.4%) and N2 (20.3%). Laboratory working standards included NIST 1515 542 (apple leaves), NIST 1575a (pine needles) and tuna muscle, as well as a Boletus quality 543 control. 544 545 Statistics/graphics 546 Stepwise multiple regression models of fungal δ15N and δ13C were analyzed with genus, site, 547 and %N as the independent variables. Because of the declining δ13C of atmospheric carbon 548 dioxide, year was also included as a continuous factor in the δ13C regression. Genus and site 549 were categorical variables and year and %N were continuous variables. These statistical 550 analyses were carried out in JMP 13 Pro (SAS Institute, Middleton, Massachusetts, USA). 551 Models that minimized the Bayesian Information Criterion (BIC) were selected. The variance 552 inflation factor (VIF) of each model factor was also calculated, which measures multicollinearity. 553 This approach allowed a test of whether Mycena generally grouped with saprotrophic or 554 ectomycorrhizal genera without a priori setting up specific contrasts among Mycena, 555 saprotrophic genera, and ectomycorrhizal genera. 556 All other statistics were done in R using'phyloseq' 1.19.1 R package (McMurdie and Holmes, 557 2013) for combining and rearranging OTU tables and taxonomy information, and the heatmap.2 558 function from the 'gplots' package (Warnes et al., 2016) for visualising heatmaps. We applied a 559 sequential ANOVA for Fig. 1a-d at the 0.05 significance threshold with the Scheffe post-hoc test 560 correction for multiple comparisons, using the 'agricolae' package (de Mendiburu, 2020). 561 562 Phylogenetics 563 564 The ITS phylogeny (Fig. 3) was constructed by first aligning a selected high-quality subset of 565 89 ITS full-length sequences with the Q-ins-i algoritm in MAFFT(Katoh and Standley, 2013) for 566 a final alignment of 1502 positions (gaps included), and then running a maximum likelihood with 567 1000 bootstrap replications in RaxML (Stamatakis, 2014), saving branch lengths. Then, the 20 568 + 21 Mycena ITS1 and ITS2 OTUs were added the, the Q-ins-i alignment redone, and the 569 OTUs mapped to the branches using the EPA algoritm (Barbera, 2019). The tree was visualised 570 in FIGTREE v. 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree). 571 572 Sequences and data 573 MiSeq/454 files are found at the respective sources listed in Table S16. Sanger sequences can 574 be accessed through GenBank/UNITE, see Table S17 for accession numbers. R scripts and 575 downstream analysis files can be obtained from C.B. Harder upon request. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436563; this version posted March 23, 2021. 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-NC-ND 4.0 International license.

576 577 Acknowledgements 578 We thank Karl-Henrik Larsson and Arne Aronsen for provisions of specimens from the Natural 579 History Museum of Oslo, and for help with identification of field specimens from Svalbard. We 580 further thank Cecilie Mathiesen and Mikayla Jacobs for technical assistance in the laboratory, to 581 Brendan J. Furneaux for a valuable input to the R script, and to the curators of H, TUR and 582 OULU. The Mycena ITS sequences originating from the specimens deposited in H, TUR and 583 OULU were produced as part of the Finnish Barcode of Life Project (FinBOL) funded by the 584 Ministry of Environment, Finland (YM23/5512/2013), Otto A Malm's Donationsfond, and the 585 Kone Foundation. 586 We thank the European Commission (grant no. 658849) and the Carlsberg Foundation (grant 587 no. CF18-0809) for grants to C.B. Harder that made this research possible. 588 589 Conflicts of interest 590 The authors declare no conflicts of interest. 591 592 References 593 Abarenkov, K., Zirk, A., Piirmann, T., Pöhönen, R., Ivanov, F., Nilsson, R.H., and Kõljalg, U. 594 (2020) UNITE USEARCH/UTAX release for eukaryotes. Version 04.02.2020. UNITE 595 Community. 596 Aronsen, A., and Læssøe, T. (2016). The genus Mycena s.l. Fungi of northern Europe, vol. 5. 597 Danish Mycological Society. 598 Bahram, M., Polme, S., Koljalg, U., and Tedersoo, L. (2011) A single European aspen (Populus 599 tremula) tree individual may potentially harbour dozens of Cenococcum geophilum ITS 600 genotypes and hundreds of species of ectomycorrhizal fungi. FEMS Microbiology Ecology 75: 601 313-320. 602 Baldrian, P., and Kohout, P. (2017) Interactions of saprotrophic fungi with tree roots: can we 603 observe the emergence of novel ectomycorrhizal fungi? . New Phytologist 215: 511-513. 604 Baldrian, P., Kolarik, M., Stursova, M., Kopecky, J., Valaskova, V., Vetrovsky, T. et al. (2012) 605 Active and total microbial communities in forest soil are largely different and highly stratified 606 during decomposition. ISME J 6: 248-258. 607 Barbera, P., Kozlov, A. M., Czech, L., Morel, B., Darriba, D., Flouri, T., & Stamatakis, A. (2019) 608 EPA-ng: massively parallel evolutionary placement of genetic sequences. Systematic biology 609 68: 365-369. 610 Birkebak, J.M., Mayor, J.R., Ryberg, K.M., and Matheny, P.B. (2013) A systematic, 611 morphological and ecological overview of the Clavariaceae (Agaricales). Mycologia 105: 896- 612 911. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436563; this version posted March 23, 2021. 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-NC-ND 4.0 International license.

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833 834 835 836 837 838 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436563; this version posted March 23, 2021. 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-NC-ND 4.0 International license.

a) ITS1 ITS2 0.8 a) OTUs e) x OTUs 0.6 xy x yz 0.4 x x yz y 0.2 y z 0.0

fraction 0.8 b) ASVs f) ASVs 0.6 x 0.4 xy x yz

Mycena yz y 0.2 y x x z 0.0 150 c) OTUs g) OTUs 100 x x x x x 50 x x x y y 0 x 150 d) ASVs x ASVs 100 x x x 50 x x x y y

OTU/ASV OTU/ASV richness 0 h) a r ris ris est va−ursiB.nana ragona v S.pola A.alpine B.vivipa B.pendula A.u P.syl S.herbacea D.octopetala C.tet Fig.1. Mycena infection levels (fraction of read shares) and species richness at 97% coverage corrected for sampling depths (Chao and Jost 2012) for the ITS1 (a-d) and ITS2 (e-h) datasets. Very little differerence between the OTU and the ASV approaches were found. Letters x-y-z denote host species «significance groups» as found by ANOVA + Scheffes multiple comparisons test for a significance at the P< 0.05 level. Species sharing one identical letter (x, y or z) do not significantly differ from each other in mean Mycena infection level/overall species richness (at 97% coverage). bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436563; this version posted March 23, 2021. 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-NC-ND 4.0 International license.

Fig.2. Heatmap of Mycena species occurrence (a-c) and overall genus occurrence (b-d). Note the slightly different colour bars in each plot. Only Mycena species that made up >1% of at least one sample were included as separate rows in a-c. In b-d, only genera that comprised >5% of reads as an average in at least one host species had its own separate row. Besides these criteria, we also included Helotiales that could not be identified to generic level, but might still conceivably harbour ericoid mycorrhizal fungi or dark septate endophytes (as Phialocephala). but might. ERM=Ericoid mycorrhiza, ECM= Ectomycorrhiza, Sap=Saprotroph, End=Endophyte. Note the near-complete dominance of ectomycorrhiza (particularly Suillus) in P. sylvestris. (Ps). bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436563; this version posted March 23, 2021. 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-NC-ND 4.0 International license.

ITS1_OTU_631 M. galopus ITS2_OTU_10 M. galopus 99 FIMY09013 H6036831 M. leucogala ITS1_OTU_124 M. albidolilacea/olivaceo-/citrinomarginata + ITS2_OTU_73 M. albidolilacea/olivaceo-/citrinomarginata 99 FIMY04112 H6032614 M. galopus 47 FIMY10613 H6036847 M. haematopus 73 100 HK47/15 M. olivaceomarginata ITS1_OTU_314 M. metata + ITS2_OTU_17 M. metata 32 JF908377 M. albidolilacea 50 FIMY26114 F076595 M. cf citrinomarginata FIMY17013 H6039063 M. viridimarginata 20 FIMY01612 H6030712 M. niveipes ITS1_OTU_614 M. sp. + ITS2_OTU_358 M. alexandri 91 FIMY11313 H6036854 M. aetites 20 52 FIMY21814 TUR168636 M. tristis ITS2_OTU_948 M. rubromarginata 22 FIMY20314 TUR182370 M. cf algeriensis FIMY14313 H6038555 M. zephirus ITS2_OTU_536 M. capillaripes 74 79 JN198391 M. plumbea FIMY02512 H6032598 M. abramsii ITS1_OTU_1828 M. cinerella + ITS2_OTU_20 M. cinerella FIMY17513 H6039068 M. rubromarginata 53 71 CBHHK001 MT153132.1 M. filopes ITS1_OTU_118 M. pasvikensis + ITS1_OTU_201 M. sp. + ITS2_OTU_33 M. pasvikensis 96 CBHHK153 MT153140.1 M. metata 44 CBHHK201 MT153126.1 M. alexandri 29 17 100 FIMY17113 H6039064 M. vitilis ITS2_OTU_185 M. latifolia FIMY18613 H6039079 M. polygramma FIMY25314 F074461 M. aff capillaripes FIMY07513 H6036816 M. clavicularis ITS1_OTU_90 M. epipterygia1 + ITS2_OTU_4 M. epipterygia1 57 MF926553 M. cf. cinerella ITS1_OTU_618 M. epipterygia2 + ITS2_OTU_883 M. sp. 93 FIMY28314 F043625 M. cineroides 25 FIMY06413 H6018219 M. cinerella 31 27 EU669223 M. tenax ITS2_OTU_743 M. septentrionalis + ITS2_OTU_1061 M. sp. 92 KU861557 M. pasvikensis 97 FIMY05412 H6033552 M. latifolia ITS1_OTU_21 M. strobilinoidea/aurantiomarginata + ITS2_OTU_127 M. strobilinoidea/aurantiomarginata 88 FIMY11813 H6036859 M. erubescens ITS2_OTU_723 M. sp. 23 69 FIMY09813 H6036839 M. vulgaris FIMY16413 H6039057 M. rosella 22 92 ITS1_OTU_543 M. leptocephala KP406534 M. epipterygia 33 100 HM240533 M. epipterygia Mycena sensu stricto ITS2_OTU_6 M. leptocephala 13 CBHHK145 MT153131.1 M. epipterygia 99 FIMY02012 H6032424 M. aurantiomarginata ITS1_OTU_1204 M. sanguinolenta + ITS2_OTU_13 M. sanguinolenta JF908393 M. strobilinoidea 54 FIMY15113 H6012651 M. tintinabulum ITS1_OTU_762 M. galericulata/megaspora + ITS2_OTU_400 M. galericulata/megaspora FIMY21514 TUR157124 M. maculata 93 ITS1_OTU_274 M. concolor 36 FIMY05312 H6033551 M. plumipes 99 15 JF908440 M. strobilicola ITS1_OTU_505 M. sp. FIMY06813 H6029112 M. stipata 85 HK5/15 M. leptocephala 52 FIMY09313 H6036834 M. leptocephala 75 32 FIMY05512 H6033553 M. sanguinolenta ITS1_OTU_1169_Mycena_rebaudengoi + ITS1_OTU_448 M. sp. FIMY04712 H6004009 M. silvaenigrae 100 FIMY09213 H6036833 M. megaspora 92 FIMY03712 H6032610 M. galericulata ITS1_OTU_426 M. pura FIMY25614 F018012 M. concolor 80 FIMY19614 TUR168621 M. cf mirata ITS2_OTU_194 M. amicta 75 100 FIMY16213 H6038561 M. pterigena 96 FIMY22714 F073581 M. rebaudengoi 35 FIMY20014 TUR171435 M. cf rapiolens FIMY12813 H6036869 M. laevigata 46 100 FIMY13713 H6008519 M. rosea 62 FIMY19013 H6039083 M. pura FIMY20114 TUR194167 M. picta 99 FIMY12913 J24082010 M. cyanorhiza 88 FIMY11013 H6036851 M. amicta 95 91 FIMY07713 H6036818 M. adscendens 60 95 FIMY08913 H6036830 M. mucor FIMY24914 F073091 M. stylobates 67 FIMY19714 TUR173933 M. aciculata 90 FIMY16813 H6039061 M. tubaroides FIMY18413 H6039077 M. polyadelpha FIMY20914 TUR136996 M. rorida 52 93 JF908413 M. sp. 47 FIMY17313 H6039066 Phloeomana speirea JQ666641 M. maurella 38 FIMY00512 H6029500 M. oregonensis ITS1_OTU_51 M. stylobates1 + ITS1_OTU_598 M. stylobates2 + ITS2_OTU_139 M. stylobates 78 FIMY10013 H6036841 M. olida Mycena sensu lato 98 100 FIMY21414 TUR197323 M. alba 95 FIMY10313 H6036844 M. hiemalis ITS2_OTU_232 M. aciculata FIMY11913 H6036860 Phloeomana speirea 100 AF145448 Tricholoma matsutake 96 MF958448 Laccaria bicolor 37 95 SH1543386 Atheniella adonis 100 FIMY03512 H6032608 Atheniella flavoalba FIMY27514 F016971 M. amabilissima 87 FIMY12213 H6036863 Atheniella adonis 100 Mycena sensu lato 100 FIMY08213 H6036823 Atheniella acicula 100 SH1527282 Atheniella floridula 79 FIMY28914 F079668 M. alphitophora 71 UDB015592 Hemimycena cucullata 99 UDB023649 Hemimycena lactea 99 SH1515402 08FU Mycenella trachyspora SH1515403 08FU Mycenella bryophila 90 SH1557736 Panellus serotinus SH1612273 Clitocybe subditopoda SH1611711 Arrhenia retiruga

Fig.3. RaxML complete ITS phylogeny of 64 Mycena s.s. sequences, with 25 outgroups from "Mycena s.l." and other Agaricales. Bootstrap supports indicated about each branch. 20+21 OTUs from ITS1 and ITS2 are superimposed upon the branches with the best fit. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436563; this version posted March 23, 2021. 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-NC-ND 4.0 International license.

Finse(Subarctic tundra) Solhomfjell(coniferous forest) Vettakollen(coniferous forest) M. pura1 Tricholoma saponaceum Hydnum Hydnellum ferrugineum rufescens 10 10 10 R. subrubens Suillis bovinus C. obtusus Suillus bovinus M.pura2 Inocybe acuta Rhizopogon obtextus Ramaria R.cessans 5 5 Collybia 5 stricta C. croceus tuberosa L. pseudomucidus Lepista flaccida H. marginatum G. paludosa Laccaria laccata L. trivalis BS Ma Marasmiellus afer BS Helvella arctica BS Ma A. vaginata R. emetica Mf Mp Laccaria laccata TS 0 Vu H. myosotis 0 0 En Vm Rhodocollybia butyracea Mg S. herbacea Do Mf1 M.alexandri Mf H. elongatum Calvatia sp. TS Vm Vv Juniperus S. phylicifolia M.rubromarginata M.epipterygia G. tibiicystis communis TS Cv S. cinerea Sorbus sp. Mf2 Bp Vu MuMg1 −5 −5 Mr −5 Bp Vm M.cinerella Pinus nigra Ps Ps Mr Bv Picea abies Mr2 Lycoperdon perlatum Vv Quercus G. cerina M.vulgarisMg2 S.reticulata Empetrum nigrum Strobilurus esculentus Naucoria sp. Microcephale perforans robur −10 −10 Picea abies −10 S. lapponum Phloeomana speirea Ma Cystoderma aminanthina −30 −25 −30 −25 −30 −25 Svalbard(Arctic tundra) Gribskov(Beech forest) Sample type Abbreviations δ15N‰ a BS=Soil ~50cm depth Agaricus aristocratus Host plant TS=Topsoil C.polaris a Mycena Bp=Betula pendula Laccaria 10 10 a Mycorrhiza Bv=Bistorta vivipara R.chloroides laccata Cv=Calluna vulgaris a Saprotroph Do=Dryas octopetala L. pseudouvidus2 M.rosea2 M. leptocephala C. sp (Telamonia) Ps=Pinus sylvestris L. pseudouvidus1 a Soil S=Salix 5 5 Vm=Vaccinium myrtillus R. nana C. alpinus R. ocholeuca G. pseudomycenopsis H. myosotis M.diosma Vu=Vaccinium uliginosum Vv=Vaccinium vitis−idea BS Mp TS Mp A. rubescens G. clavata2 Mu G=Galerina Mo3 R. emetica H=Hypholoma 0 G. clavata1 0 M.rosea1 S.polaris M.metata2 M.atroalboides Laccaria BS H. fasciculare Bv R=Russula Mo2 ametysthima TS M.metata L=Lactarius Do M.sp 59/15 Bp Hygrophoropsis C=Cortinarius aurantiaca A=Amanita −5 −5 Pa Mo1 Fagus Trametes Ma=Mycena amicta M.metata1 sylvatica Pluteus versicolor Mf=M.filopes cervinus M.crocata Mg=M.galopus M.vitilis Cassiope tetragona Mo=M.olivaceomarginata −10 −10 Mp=M.pura Mr=M.rosella Mu=M.galericulata −30 −25 δ13C‰ −30 −25

Fig.4. Biplots of stable isotopes 15N and 13C for soil, host plants, and the three simplified categories Mycena, Mycorrhiza and saprotrophs. Overall, saprotrophs will be found predominantly bottom right, mycorrhizal fungi top left. Note the deviant particular M. puras at Finse, upper left.

bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436563; this version posted March 23, 2021. 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-NC-ND 4.0 International license.

ITS1 Samples1 Locality Description of Title of study original purpose Blaalid et al. (2012) Bistorta vivipara Finse(Nor Succession (n=59) way) Yao et al. (2013) Bistorta vivipara Finse(Nor Ridge-to-snowbed (n=51) way) gradient Blaalid et al. (2014) Bistorta vivipara 32 Spatial/bioclimatic (n=146) localities variation on Svalbard Botnen et al. (2014) Bistorta vivipara Blomsterd Host specificity (n=19), Dryas alen, octopetala (n=22), Svalbard Salix polaris (n=20) Mundra et al. (2015) Bistorta vivipara Isdammen, Temporal variation (n=84) Svalbard Davey et al (2015) Bistorta vivipara Svalbard, Succession/glacial (n=103) Finse(Nor chronosequence way) gradient Botnen et al. (2019) Bistorta Scotland, Biogeography vivipara(n=57) Austria, Iceland, Jan Mayen ITS2 Title of study Jarvis et al. (2015) Pinus sylvestris Scotland Altitude (n=32) Lorberau et al (2017) Cassiope tetragona Endalen, Drought (n=15) Svalbard Biogeography project Arctostaphylos Scotland Biogeography (new data) alpine(n=10), Arctostaphylos uva- ursi(n=8) Betula nana (n=8) Salix herbacea (n=7)

Altitude project (new Arctostaphylos uva- Scotland Altitude data) ursi(n=68), Pinus sylvestris (n=9) Birch (new data) Betula pendula Scotland Grazing effects (n=81)

Table 1.Overview of all species and samples included in this study. 1Number represent samples which ended up being included in the final analyses. Our plant samples consisted of the herbaceous ectomycorrhizal Arctic Bistorta vivipara, subshrub Dryas octopetala, and the dwarf shrub Salix polaris (Blaalid et al., 2012; Yao et al., 2013; Blaalid et al., 2014; Botnen et al., 2014; Davey et al., 2015; Mundra et al., 2015); the Arctic ericaceous Cassiope tetragona (Lorberau et al., 2017) and the ectomycorrhizal conifer Pinus sylvestris (Jarvis et al., 2015) from Scotland; and new data also from Scottish (temperate) plants: the arbutoid mycorrhizal bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436563; this version posted March 23, 2021. 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-NC-ND 4.0 International license.

Arctostaphylos alpina and A. uva-ursi, ectomycorrhizal dwarf shrubs Betula nana and Salix herbacea, and the ectomycorrhizal trees Betula pubescens and additional Pinus sylvestris. The Betula pubescens samples all came from saplings of < 1 m kept low by deer/sheep grazing; the other host plants collected were mature.

ITS1 B. vivipara (n=519)1 D. octopetala (n=22) S.polaris (n=20)2 OTUs 803649 39880 30069 (n=1193) ASVs 908621 43286 33141 (n=2272) ITS2 A.alpine A.uva-ursi B.nana B.pendula P. S. C. tetragona (n=10) (n=76) 1 (n=8) (n=81)2 sylvestris herbacea (n=15) (n=41) (n=7) OTUs (n=1032) 59829 241418 49927 90214 212577 25298 313627 ASVs (n=1559) 61703 247091 51182 93429 219124 25497 385763

Table 2. Total sequence counts for OTU/ASVs for each host species.2Numbers represent samples included in the final analyses.