FEMS Microbiology Ecology, 91, 2015, fiv012

doi: 10.1093/femsec/fiv012 Advance Access Publication Date: 6 February 2015 Research Article

RESEARCH ARTICLE Diverse ecological roles within fungal communities Downloaded from https://academic.oup.com/femsec/article-abstract/91/3/fiv012/436629 by guest on 06 August 2020 in decomposing logs of Picea abies Elisabet Ottosson1, Ariana Kubartova´ 1, Mattias Edman2,MariJonsson¨ 3, Anders Lindhe4, Jan Stenlid1 and Anders Dahlberg1,∗

1Department of Forest Mycology and Plant Pathology, BioCenter, Swedish University of Agricultural Sciences, Uppsala SE-750 07, Sweden, 2Department of Natural Sciences, Mid Sweden University, SE-851 70 Sundsvall, Sweden, 3Swedish Species Information Centre, Swedish University of Agricultural Sciences, Uppsala SE-750 07, Sweden and 4Armfeltsgatan 16, SE-115 34 Stockholm, Sweden ∗ Corresponding author: Department of Forest Mycology and Plant Pathology, BioCenter, Swedish University of Agricultural Sciences, Uppsala SE-750 07, Sweden. Tel: +46-70-3502745; E-mail: [email protected] One sentence summary: A Swedish DNA-barcoding study revealed 1910 fungal species in 38 logs of Norway spruce and not only wood decayers but also many mycorrhizal, parasitic other saprotrophic species. Editor: Ian C Anderson

ABSTRACT

Fungal communities in Norway spruce (Picea abies) logs in two forests in Sweden were investigated by 454-sequence analyses and by examining the ecological roles of the detected taxa. We also investigated the relationship between fruit bodies and mycelia in wood and whether community assembly was affected by how the dead wood was formed. Fungal communities were highly variable in terms of phylogenetic composition and ecological roles: 1910 fungal operational taxonomic units (OTUs) were detected; 21% were identified to species level. In total, 58% of the OTUs were ascomycetes and 31% basidiomycetes. Of the 231 337 reads, 38% were ascomycetes and 60% basidiomycetes. Ecological roles were assigned to 35% of the OTUs, accounting for 62% of the reads. Wood-decaying fungi were the most common group; however, other saprotrophic, mycorrhizal, lichenized, parasitic and endophytic fungi were also common. Fungal communities in logs formed by stem breakage were different to those in logs originating from butt breakage or uprooting. DNA of specific species was detected in logs many years after the last recorded fungal fruiting. Combining taxonomic identification with knowledge of ecological roles may provide valuable insights into properties of fungal communities; however, precise ecological information about many fungal species is still lacking.

Key words: boreal forest; decaying wood; fungal diversity; fungal ecology; Norway spruce; pyrosequencing

INTRODUCTION dia, our knowledge about the distribution and ecology of these fungi is based primarily on fruit body observations (Stokland, Dead wood is pivotal for species diversity and for certain ecosys- Siitonen and Jonsson 2012). However, for most of its life, a tem processes in forests (Stokland, Siitonen and Jonsson 2012). wood-inhabiting fungus is cryptic and difficult to study, and as In northern Europe, more than 7500 species, mainly fungi yet the diversity, spatio-temporal dynamics and factors regu- and insects, are dependent on wood (Siitonen 2001; Stokland, lating the presence of fungal species in wood are poorly un- Siitonen and Jonsson 2012). Although more than 2500 wood- derstood. However, novel high-throughput sequencing methods inhabiting fungal species have been reported in Fennoscan- and bioinformatics tools have enabled new insights into fungal

Received: 14 December 2014; Accepted: 26 January 2015 C FEMS 2015. All rights reserved. For permissions, please e-mail: [email protected]

1 2 FEMS Microbiology Ecology, 2015, Vol. 91, No. 3

phylogenetics and community ecology (Hibbett et al., 2011;Lin- succession (spatiotemporal dynamics) of wood-inhabiting fungi. dahl et al., 2013; Bohmann et al., 2014). Wood-decaying fungi are typically unit restricted, and selection Studies using high-throughput sequencing methods have re- for combative long-lived mycelia has implications for species fit- ported a remarkably high and largely cryptic species richness of ness and the degree of species turnover during the decomposi- fungi in environmental samples from litter, soil and wood (e.g. tion of wood. Buee´ et al., 2009; Kubartova´ et al., 2012; Clemmensen et al., 2013; Early establishing fungi may significantly affect subsequent Meiser, Balint and Schmitt 2013; Vorˇ´ıskovˇ a´ and Baldrian 2013; fungal community development in wood by capturing space Tedersoo et al., 2014). The number of operational taxonomic and modifying the wood (Renvall 1995; Fukami et al., 2010; units (OTUs) that can be identified to species level or to a higher Ottosson et al., 2014). Field observations, field experiments and taxon level is steadily increasing as databases with reliable se- time-series analyses of fruit bodies suggest that primary species quenced fungal taxa continue to grow. Identified sequences can may cause priority effects and non-random co-occurrence pat- be used to characterize communities phylogenetically, and can terns of wood-inhabiting fungal communities (Renvall 1995; also be linked to species distributions and life strategies to gain Pouska et al., 2011;Lindneret al., 2011; Ottosson et al., 2014;see Downloaded from https://academic.oup.com/femsec/article-abstract/91/3/fiv012/436629 by guest on 06 August 2020 deeper insights into fungal ecology and fungal-mediated pro- also Stokland, Siitonen and Jonsson 2012). Coarse dead wood cesses, as recent studies of wood-inhabiting fungi have demon- in Norway spruce forests occurs as a result of different distur- strated (Kubartova´ et al., 2012; Rajala et al., 2012;Ovaskainen bance agents, such as strong winds and heart-rot fungi, which et al., 2013; van der Wal, Ottosson and de Boer 2015). is likely to affect the fungal communities that develop in the Molecular phylogenetics has been widely used to determine coarse wood (Johannesson and Stenlid 1999;Edman,Jonsson¨ whether the phylogenetic relatedness of interacting species may and Jonsson 2007; Kuuluvainen and Aakala 2011). However, as help to explain mechanisms behind the coexistence of species yet, few studies have related community composition to the ori- and the functional consequences of phylogenetic relatedness gin of the decaying wood. in communities (Webb et al., 2002; Vamosi et al., 2009;Pausas In an earlier study, we detected 1910 fungal OTUs in an eval- and Verdu 2010). Phylogenetically closely related species tend uation of the fine-scale distribution of fungal mycelia within to be ecologically more similar than distantly related species and among decaying Norway spruce logs (Kubartova´ et al., 2012). (McGuire et al., 2010). This could, in theory, result in strong com- We concluded that in addition to the wood decayers, fungi with petition among closely related lineages due to niche overlap. Al- other ecological roles also appeared to be significant in wood. ternatively, functional complementarities may evolve, resulting Here, we present an in-depth species level analysis of the in coexistence between distinct evolutionary lineages due to re- phylogenetic diversity and ecological roles, or nutritional strate- duced competition and enhanced ecosystem functioning (Ma- gies, of the wood-inhabiting fungal communities reported in our herali and Klironomos 2007;Rinconet al., 2014). However, evi- earlier study (Kubartova´ et al., 2012). To achieve this, we linked dence that these assumptions apply to fungal communities is the identities of fungal taxa detected in wood by 454 sequencing limited (but see Maherali and Klironomos 2007;Rinconet al., with their known ecological roles as compiled from the scientific 2014). literature. We also combined the molecular data with long-term The relationship between fruit bodies and mycelia in wood fruit-body inventories of the same sites to examine our expec- is not simple because many species are predominantly or only tation that mycelia may be present despite the absence of fruit present as mycelia (e.g. Allmer´ et al., 2006; Kubartova´ et al., 2012; bodies and that mycelia may still be present many years after Monard, Gantner and Stenlid 2013), which has also been found the production of fruit bodies has ceased. Finally, we used the to be the case in studies of soil fungal communities (e.g. Porter, data from one of the sites where the logs had been formed as a Skillman and Moncalvo 2008; Hibbett et al., 2011; Clemmensen result of different types of stem breakage to explore whether the et al., 2013). Indeed, the majority of fungal OTUs detected in cause of tree death may affect the resulting fungal community wood to date represent either taxa that cannot be named ow- composition. ing to a lack of reference sequences or taxa that are as yet un- described (e.g. Kubartova´ et al., 2012). However, typically, a small MATERIALS AND METHODS proportion of the OTUs, whose taxonomic identity and ecologi- cal role is generally known, form a significant proportion of the The work reported in the present study was a continuation of sequence reads, implying that these OTUs perform a relatively a study of the fine-scale distribution of fungal mycelia within large proportion of the fungal functions in wood. 38 logs at two forest sites reported by Kubartova´ et al. (2012). A Fruit-body analyses of wood-inhabiting fungal species can summary of the study sites and the methods are given here; for provide insights into their ecologies and largely cryptic life: for a more detailed description, see Kubartova´ et al. (2012). example, a recent study of dead Norway spruce (Picea abies) wood reported a high correlation between the detection of mycelia Study sites by high-throughput sequencing and the observation of fruit bodies, although the correlation was variable among species We studied Norway spruce (P. abies) logs at two sites with con- (Ovaskainen et al., 2013). There was a strong correlation be- trasting forest histories, vegetation type (hemiboreal and boreal, tween the detection of mycelia and fruiting bodies through- respectively) and quality of logs (experimentally cut 14 years ago out successional stages of decay by the generalist species Fomi- and naturally fallen of older age, respectively). topsis pinicola, whereas there was a long time delay between The hemiboreal site, Fageron¨ Nature Reserve (hereafter re- the point of mycelial establishment by the specialist species ferred to as site F), is located on an island along the Baltic coast in Phellinus nigrolimitatus and the formation of fruit bodies. In southern Sweden (60◦12 N, 18◦27 E, 10 m above sea level). Mean general, the mycelia of species with perennial fruit bodies were annual precipitation 700 mm; mean monthly temperature range detected more frequently in wood than species that formed an- from −3◦CinJanuaryto+15◦C in July with an annual mean of nual fruit bodies, which probably reflects differences in the in- +5◦C (Swedish Metrological and Hydrological Institute, records vestment needed for reproduction and in mycelial longevities. 1961–1990). The area has been extensively used for selective cut- Mycelial longevity is an important factor in understanding the ting, charring and pasturage for several centuries until the early Ottosson et al. 3

20th century. The forest within the study area is a mixed, un- (Edman and Jonsson 2001; Berglund, Edman and Ericson 2005; managed forest with Norway spruce dominating the canopy. In Edman, Jonsson¨ and Jonsson 2007;Jonsson,¨ Edman and Jonsson 1994, an experiment was established that involved cutting Nor- 2008). The fieldwork was undertaken once a year between Au- way spruce trees to produce 3-m long freshly cut logs on the gust and October when most annual wood fungi produce fruit ground (Lindhe, Asenblad˚ and Toresson 2004). A total of 12 of bodies (Halme and Kotiaho 2012). The majority of fruit bodies, these logs were randomly selected for this study. especially among the polypores, were identified to species level The boreal study site, Norra Gardfjallet¨ Nature Reserve (site in the field. However, samples of some fruit bodies were col- G), borders subalpine forest in northern Sweden (65◦25 N, 16◦06 lected, particularly among the corticoids, for later identification E, 550 m above sea level). Mean annual precipitation 700 mm; to species level in the laboratory using microscopic characteris- mean monthly temperature range from −11◦CinJanuaryto tics. +11◦C in July with an annual mean of −1◦C (Swedish Metrologi- cal and Hydrological Institute, records 1961–1990). The area con- DNA extraction, PCR amplification and sequencing sists of old-growth forest dominated by Norway spruce and is Downloaded from https://academic.oup.com/femsec/article-abstract/91/3/fiv012/436629 by guest on 06 August 2020 mainly regenerated by an internal gap-phase dynamic without One and a half cubic centimetres of each sawdust sample was evidence of either fire disturbance or logging activities (Jonsson placed in a 2-mL screw-cap tube together with glass beads and and Dynesius 1993). In an area of 6 ha, all 684 downed logs with homogenized using a FastPrep shaker (Precellys 24 Bertin Tech- a base diameter of >10 cm were mapped in 1997 (Edman and nologies) to form a fine powder. After this, DNA was extracted Jonsson 2001). A total of 26 of these logs were randomly sampled using a CTAB protocol (Ihrmark et al., 2002), and DNA concen- for this study. The logs were in the middle to late stages of de- tration was measured using NanoDropTM (Thermo Scientific). − cay, corresponding to decay stages 4–6 based upon the 1–8 decay Samples with a DNA concentration above 15 ng μL 1 were pu- stage classification system devised by Soderstr¨ om¨ (1988). The in- rified using a Jet Quick purification kit (Genomed GmbH). Ap- dividual ages of the logs was not known. However, for this site, proximately, 5–7 ng of DNA from each sample was amplified. the volume of downed logs in decay class 1–6 has been reported PCR was performed in two steps. In the first step, a fungal- to be 44.8 m3 ha−1, the addition of logs (snags not included) has specific primer combination of the internal transcribed spacer been calculated to be 1 m3 ha−1 yr−1, the half-life of the logs has ITS1-F (Gardes and Bruns 1993) and ITS4 (White et al., 1990)was been calculated to be about 42 years and, after approximately used. In the second PCR step, the ITS primers included a 454 60 years, 90% of the logs have passed beyond decay stage 6 (Ed- adaptor together with a sample-specific 5-bp-long tag. The final man, Jonsson¨ and Jonsson 2007;Fraveret al., 2008). PCR products were purified using a QIAquickR Gel Extraction kit (Qiagen) and the final concentration of the PCR products was  Field sampling measured using Qubit R fluorometer (Invitrogen). The final py- rosequencing sample was obtained by pooling 5 ng of DNA per In August 2008, samples were collected from the logs according sample and then run on a Genome Sequencer FLX 454 (Roche to a standardized scheme: a more detailed sampling was per- Applied Biosystems) at the Royal Institute of Technology, Stock- formed at site F (see Kubartova´ et al., 2012). At site F, sampling holm, Sweden. was conducted at a distance of 0.5, 1.25, 1.75 and 2.5 m from the end of the coarsest part of the log in four of the cut logs and at a Bioinformatics and assignment to ecological roles distance of 1.25 m in the remaining eight logs. At each position, samples were taken from four holes, which were drilled opposite Sequencing from the ITS4 adaptor yielded approximately each other. At site G, samples were taken where the logs were 550 000 sequences covering the ITS2 region (200–250 bp). Se- 18, 30 and 42 cm in diameter, if possible, with two drill holes at quences were quality filtered and clustered into OTUs that each position along the side of each naturally fallen log. At both approximately corresponded to the species level (1.5% dis- sites, subsamples from the outer and inner parts of the logs on tance single linkage) using the bioinformatics pipeline SCATA the lower and upper parts were obtained by drilling into the log (Sequence Clustering and Analysis of Tagged Amplicons) down to a depth of 12 cm, or to the log centre, using cleaned (http://scata.mykopat.slu.se/; Clemmensen et al., 2015). This 8-mm diameter drill bits. Prior to drilling, bark and other litter process involved removal of short sequences (<200 bp) and se- were carefully removed with a knife. The inner and outer 6 cm quences with poor read quality, primer dimers and homopoly- of the wood sample were kept separate at all drilled holes. Each mers. After quality filtering, 90 000 sequences from site Fand drill-dust sample comprised on average approximately 3 cm3 of 140 000 sequences from site G remained. The most common wood. The drill-dust samples were collected in plastic zip bags genotype within a cluster was used to represent each OTU. For and frozen until extraction of DNA. In total, 192 samples were clusters that contained two sequences, one sequence was ran- obtained from the 12 logs at site F and 180 samples from the domly chosen and single read clusters were omitted from the 26 logs at site G. The samples were kept at −21◦C and extracted data. The clusters were then brought together into a matrix within 2 months. with cluster absolute and relative abundance versus sample identities. Fruit-body inventory Each OTU was taxonomically identified in the SCATA pro- gram via a combined comparison with the curated databases We combined data from earlier studies of fruit-body invento- UNITE (Abarenkov et al., 2010) and SAF (spruce-associated fungi) ries with data collected during the drill-dust sampling. At site F, (Ovaskainen et al., 2010), together with a batch BLAST search logs had been monitored annually in October between 1995 and against GenBank (NCBI) (Altschul et al., 1997). The results from 2008 (except in 1999) for fruit bodies of basidio- and ascomycetes the three databases were then compared. The ITS homology for (Lindhe, Asenblad˚ and Toresson 2004; Anders Lindhe unpub- defining taxa in GenBank were fixed to at least 97–100% ande- lished observations). At site G, polypore and corticoid fruit bod- values below e−100 for species, 90–97% and e−90 for genus level, ies were surveyed in September and October in 1997 and 2003 and 80–90% and e−80 at the order level, corresponding to at least 4 FEMS Microbiology Ecology, 2015, Vol. 91, No. 3

90% of the sequence length. We did not use a rarefying approach 100 in order to optimize the use of the dataset and taxa with low fre- quencies and abundances (McMurdie and Holmes 2014). The identified fungal taxa were categorized as follows based on their ecological role, which was assigned on the basis ) 10 of their assumed nutritional strategy: (1) mutualists, these 10 fungi were further divided into (1a) lichens and (1b) mycor- rhizae; (2) saprotrophs, of which certain taxa could be classi- Site F fied as (2a) wood-decaying fungi, (2b) other saprotrophic species; Site G (3) parasites, including mycoparasites, plant parasites as well 1 as fungi combining saprophytic and parasitic abilities (e.g. Het- Abundance (Log erobasidion parviporum); (4) endophytes; and (5) unknown role, which included fungi that we were unable to assign a definite Downloaded from https://academic.oup.com/femsec/article-abstract/91/3/fiv012/436629 by guest on 06 August 2020 ecological role as well as fungi that we were unable to clas- 0.1 sify to an adequate taxonomic level. The categorization was 0 50 100 150 200 250 300 based on information acquired from Hallingback¨ and Aronsson OTU rank (1998) and other published literature, and complemented with searches of the Nordic Saproxylic Network database described Figure 1. Rank abundance curves showing the OTU richness and evenness at in Stokland and Meyke (2008) and available upon request from both sites, based upon the presence of OTUs in the samples. the Swedish Species Information Centre (www.artdata.slu.se). In certain cases, a genus or even a whole order was known to only include species that had the same nutritional strategy. tation test with 999 permutations to test for the level of signif- Such OTUs were also assigned an ecological role. For instance, icance of the different log variables. The multivariate analyses all OTUs belonging to the Dacrymycetales were considered to were performed in CanocoTM (Microcomputer Power). be wood-decay fungi. The taxonomic identity, ecological role, Data on mycelial presence at site G were combined with data frequency and sequence read counts for the OTUs are shown on the causes of tree mortality (up-rooting, butt breakage and in Table S1 (Supporting Information) and references supporting stem breakage) in order to infer their conceivable effect on the the ecological classification are shown in Table S2 (Supporting fungal community in the 22 analysed logs (Table S4, Supporting Information). Information).

Data analyses and statistics RESULTS As a comparative description of the dataset from the two sites, Taxonomic diversity of fungal communities in Norway OTU richness per sample and absolute abundances (number of spruce logs sequences of each OTU per sample) were calculated based on the output matrix from the SCATA pipeline. OTU richness per sam- We recovered 1910 OTUs from the two sites. Species accumu- ple and absolute abundances were used to calculate the number lation curves showed that more samples would have generated of OTUs in the different taxonomic groups and the OTU accumu- more OTUs at both sites but that the sequencing depth of the lation curves, which were performed using the species accumu- samples was satisfactory (see Fig. S1, Supporting Information). lation function ‘specacc’ in the vegan package (Oksanen et al., The majority of the OTUs were detected in a few samples only, 2009) in R. Species accumulation curves for the four extensively but a greater number of rare OTUs were found at site G (Fig. 1). sampled logs at site F, based on number of samples taken per Although the majority of the fungal taxa (1109 OTUs, 58%) be- log, are presented in Kubartova´ et al. (2012). longed to the Ascomycota, the Ascomycota accounted for only We also included data on the fruit bodies present on the sur- 38% (87 456) of the sequence reads. Basidiomycetes made up veyed logs at site G (Edman, Jonsson¨ and Jonsson 2007;Jonsson,¨ nearly a one third (586 OTUs, 31%) of the OTUs but accounted Edman and Jonsson 2008) to determine whether there was any for 60% (138 605) of the sequence reads (Fig. 2a and b). A total correlation with the molecular detection of mycelia. A matrix of of 42% of the OTUs were identified down to the level of family Bray–Curtis distances was calculated using the read abundance or lower, of which 21% (400 OTUs) were assigned to species level data for the 100 most abundant OTUs in samples taken at the (Table S1, Supporting Information). Of these, 169 OTUs belonged 18-cm diameter position in the logs at site G to account for the to the Basidiomycota (Fig. 2a), which accounted for 9% of the to- different sizes of the logs. Such samples were available for 22 of tal number of OTUs, but accounted for almost half (43%) of the the 26 logs. These OTUs corresponded to on average 93% of all sequence reads (100 348) (Fig. 2b). reads (Table S3, Supporting Information). In order to display the The phylogenetic compositions of the logs were similar overall dissimilarities of the fungal communities, a detrended between the sites, although only 24% of the OTU identities over- correspondence analysis (DCA) was performed (Oksanen et al., lapped. The majority of OTU sequence reads belonged to the 2009). Based upon the long axis in the DCA, we expected that Basidiomycota whereas the majority of OTUs with unknown the OTUs would have a unimodal response to gradients in the ecological roles belonged to the Ascomycota (Fig. 3). Basid- data. To test the null hypothesis that there was no difference in iomycete fungi belonging to the Polyporales, Hymenochaetales, fungal community composition among logs, a canonical corre- Russulales and Dacrymycetales were among the most abundant spondence analysis (CCA) was performed with the type of tree OTUs, and ascomycete OTUs belonging to the order Helotiales breakage and the earlier records of fruit bodies on at least three were also common at both sites (Table 1). At site G, taxa belong- of the 22 analysed logs as predictor variables (Table S4, Support- ing to the Lecanorales, Hypocreales and Coniochaetales were ing Information). CCAs were followed by a Monte Carlo permu- more common than at site F (Fig. 4aandb). Ottosson et al. 5

(a) (b)

Higher 467 22 Ascomycota 239 Higher 19 Basidiomycota 202 Order Order 17 97 14 52 Family Family 6 16 2 166 Genera Genera 16 65 3

Species 222 Species 26 169 100

0 250 500 04080120Downloaded from https://academic.oup.com/femsec/article-abstract/91/3/fiv012/436629 by guest on 06 August 2020 No of OTUs No of reads x 103

Figure 2. Bar charts showing the taxonomic level of identification for OTUs from both sites as(a) the relative proportion of OTUs (n = 1695) and (b) the relative proportion of sequence reads (n = 226 061). Grey bars represent OTUs assigned to Ascomycota; black bars represent OTUs assigned to Basidiomycota. OTUs assigned to the Glomeromycota and Mucoromycota and taxa that were amplified with the fungal-specific primers but could not be aligned to any fungal taxonence inrefer databases (number of OTUs = 215, reads = 5276) are not shown.

Ascomycota Basidiomycota (a)OTUs (b) OTUs unknown 50 50 wood decayers other saprotrophs mycorrhizae lichens 25 25 parasites endophytes % of OTUs % of OTUs 0 0 FG FG FG FG FG FG FG FG FG FG

(c)Reads (d) Reads 50 50

25 25 %of reads %of reads

0 0 FG FG FG FG FG FG FG FG FG FG

mutualists parasites mutualists parasites saprotrophs endophytes saprotrophs endophytes unknown unknown

Figure 3. Assumed ecological roles used by the OTUs detected in the logs at site F (F) and at site G (G). The percentage comparison is based on the total number of OTUs, n = 973 at F and n = 1406 at G (a, b), and the total number of sequence reads, n = 90 114 at F and n = 141 223 at G (c, d). These parameters are shown separately for Ascomycota (a, c), and for Basidiomycota (b, d). Saprotrophs refers to wood-decaying fungi and other saprotrophs, mutualists refers to mycorrhizal fungi and lichens. Glomeromycota, Mucoromycotina and fungi with unidentified affiliation are not shown (10.1% and 11.5% of OTUs at sites F and G, respectively, and2.6%and2.1%of reads at sites F and G, respectively).

Ecological roles of fungal communities in Norway in the logs (Figs 4 and 5a and b). Only a small proportion of the spruce logs OTUs belonging to the Ascomycota were wood-decay species (Fig. 3a and b), such as species in the Helotiales and Xylariales In total, 647 OTUs (34%) were assigned to an ecological role, cor- orders (Fig. 4a and b). In addition to a saprotrophic role, several responding to more than half (145 748, 63%) of the sequence ascomycete OTUs were also classified as putatively parasitic, in reads. However, in most cases, particularly with regards to the particular OTUs in the orders Hypocreales and Helotiales (many ascomycete taxa, published information about the ecology of of the species belonged to orders that include known mycopar- the species in question was scarce or ambiguous (Fig. 3aandc, asites). Basidiomycete parasitic species were found mainly in Table S1, Supporting Information). Among the OTUs that were the Tremellomycetes, which is a class that contains species that assigned to an ecological role, basidiomycetes with wood- may grow both as yeasts and hyphae and can be either myco- decaying abilities were the most abundant (Fig. 3b and d). In par- parasitic or saprotrophic. Other yeasts, such as species within ticular, OTUs that belonged to the orders Hymenochaetales and the basidiomycete genera Rhodotorula and the ascomycete gen- Polyporales contributed to these patterns (Fig. 4aandb). era Candida, Debaromyces, Aureobasidium and Coniosporium,were Several other ecological groups were also present in the also recovered from the dead wood (Fig. 4aandb). wood. Within the Basidiomycota, mycorrhizal OTUs were rep- Wood-decaying fungi, other saprotrophic fungi and fungi resented in six orders at site F and in five orders at site G with other ecological roles constituted 23, 35 and 42%, respec- (Fig. 4b). Mycobionts of lichenized ascomycete fungi were found tively, of the total taxa identified but 57, 17 and 26%, respectively, 6 FEMS Microbiology Ecology, 2015, Vol. 91, No. 3

Table 1. The 20 most common OTUs found in the log samples.

OTU number Group Taxonomic rank Identification Ecology No. of sequences No. of samples

Site F 1 B Polyporales F. pinicola wd 10 510 84 5 B Hymenochaetales Resinicium bicolor wd 9306 71 3 B Russulales H. parviporum wd 8133 64 2 B Hymenochaetales Hyphodontia pallidula wd 5784 67 8 B Hymenochaetales K. alutacea wd 3903 60 6 A Sordariomycetes Phialophora sp. unk 2997 97 25 B Polyporales A. serialis wd 1824 27 18 A Helotiales Helotiales sp. unk 1790 54 26 B Polyporales Phlebia livida wd 1521 18 15 B Hymenochaetales Hyphodontia alutaria wd 1385 15 Downloaded from https://academic.oup.com/femsec/article-abstract/91/3/fiv012/436629 by guest on 06 August 2020 32 - Unidentified fungus unk 1172 34 10 B Tremellales Tremellales sp. unk 1134 17 43 B Incertae sedis Phlebia subcretacea wd 1030 5 42 B Thelephorales Thelephorales sp. my 997 16 19 B Dacrymycetales Calocera viscosa wd 903 69 61 A Diaporthales Phomopsis theicola unk 879 3 50 A Saccharomycetales Candida paludigena unk 838 45 57 B Dacrymycetales Dacrymycetales sp. wd 835 20 66 B Thelephorales Sarcodon glaucopus my 763 20 28 B Trechisporales Trechispora farinacea sa 639 25

Site G 4 B Hymenochaetales P. nigrolimitatus wd 10 068 75 2 B Hymenochaetales Hyphodontia pallidula wd 5249 76 7 A Helotiales Leptodontidium elatius p 4603 116 9 B Russulales Conferticium ochraceum wd 4174 19 6 A Sordariomycetes Phialophora sp. unk 3938 102 11 B Agaricales Mycena purpureofusca sa 3406 70 10 B Tremellales Tremellales sp. unk 2922 17 14 A Sordariomycetes Sordariomycetes sp. unk 2809 26 16 A Chaetothyriales Chaetothyriales sp. unk 2726 85 13 A Helotiales Hymenoscyphus sp. unk 2708 91 12 A Helotiales Hy. hymeniophilus p 2707 86 17 A Coniochaetales Coniochaetaceae sp. sa 2484 63 21 A Unidentified Ascomycete sp. unk 2232 13 22 B Hymenochaetales Hymenochaetales sp. unk 2028 27 24 B Atheliales Tylospora fibrillosa my 1999 39 23 B Polyporales Physisporinus vitreus wd 1939 22 20 B Unidentified Basidiomycete unk 1895 46 15 B Hymenochaetales Hyphodontia alutaria wd 1731 15 29 B Agaricales Stropharia hornemannii sa 1663 10 19 B Dacrymycetales Calocera viscosa wd 1603 25

Abbreviations: A, Ascomycota; B, Basidiomycota; my, mycorrhizae; OTU, operational taxonomic unit; p, parasite; sa, saprotroph; unk, unknown; wd, wood decayer. of the sequence reads (Fig. 3 and Table S1, Supporting Informa- At site G, 114 fruit bodies from 31 species were recorded be- tion). tween 1997 and 2008 (see Table S6, Supporting Information). In 2008, 31 fruit bodies belonging to 13 different species were Relationship between presence of fruit bodies recorded. For six of these species, mycelial DNA was detected and mycelia in logs inthreeormorelogs(Table3). Except for Antrodia serialis and Columnocystis abietina, DNA was detected in all logs where fruit At site F, 427 fruit bodies belonging to 64 different species were bodies had been recorded. In most cases, mycelial DNA was observed on the 12 logs between 1995 and 2008 (see Table S5, also recorded in some logs where fruit bodies had not been Supporting Information). In 2008, 21 fruit bodies belonging to observed: for example, although fruit bodies of P. nigrolimitatus nine different species were recorded. For eight of these species, were recorded on only 10 logs, the mycelial DNA was found in mycelial DNA was detected in three or more logs (Table 2). In 20 of the 26 logs investigated. Fruit bodies of Coniophora olivacea all cases where fruit bodies were recorded, the DNA of the fun- and H. parviporum were last recorded in 1997 but mycelial DNA gal species was also detected in the logs. However, DNA of all was still present in these logs in 2008. species was also detected in logs where fruit bodies had not been recorded (Table 2): for example, although fruit bodies of Relationship between cause of tree death H. parviporum were only observed on four of the logs, mycelial and subsequent fungal community development DNA of H. parviporum was found in all 12 logs. Mycelial DNA from Kneiffiella alutacea was identified in a log that had not been ob- Of the 22 logs that were analysed, 5 logs were formed as a re- served to produce a fruit body for five years. sult of uprooting, 3 were formed as a result of butt breakage and Ottosson et al. 7

(a) OTUs Reads

Mucoromycotina (3, 40) Mortierellales (8, 27)

Erythrobasidiales (6, 13) Pucciniomycotina (14, 115) Leucosporidiales (2, 12)

Sporidiobolales (3, 36) Ustilagomycotina (2, 3) Malasseziales (3, 23)

Tremellomycetes (40, 1652)

Agaricomycotina Dacrymycetales (10, 1963) (8, 935) Sebacinales (3, 8) Downloaded from https://academic.oup.com/femsec/article-abstract/91/3/fiv012/436629 by guest on 06 August 2020

Cantharellales (4, 926)

Trechisporales (2, 644)

Hymenochaetales (12, 11444)

Corticiales (5, 630)

Polyporales (29, 25194)

Thelephorales (15, 1820)

Russulales (10, 8196)

Agaricales (28, 1652)

Atheliales (7, 557)

Boletales (7, 514)

Taphrinales (2, 3) Saccharomycotina (1, 288) Saccharomycetales (11, 1015) Orbiliales (3, 232) Dothideomycetidae (25, 753) Pleosporales (10, 70) Botryosphaeriales (4, 112) Pezizomycotina Chaetothyriales (43, 2055) (41, 5641) Verrucariales (4, 898) Mycocaliciales (2, 5) Eurotiales (11, 414) Lecanoromycetidae (20, 337) wood decayer Ostropomycetidae (8, 476) other saprotrophs Helotiales (86, 5499) mycorrhiza Rhytismatales (5, 84) lichen Phyllachorales (2, 21) parasite endophyte Xylariales (2, 26) unknown lifestyle Hypocreales (28, 374) Chaetosphaeriales (10, 852) Coniochaetales (5, 1133) Ophiostomatales (5, 507) < 5 % 5-10 % > 10 % Sordariales (4, 182)

Figure 4. Fungal taxa detected at (a) site F and (b) site G, identified down to order level or lower. The number of OTUs assigned to each order and the corresponding number of sequence reads is shown in parentheses. The corresponding distribution of ecological groups within each order is depicted by pie charts, showingboththe number of OTUs and the number of sequences reads. The relative number of OTUs or sequence reads of all OTUs found at each site is highlighted in white, grey and dark grey, indicating <5% of the total OTUs, 5–10% of the OTUs and >10% of the OTUs, respectively. The division of the OTUs into orders follows Hibbett et al. (2007). 8 FEMS Microbiology Ecology, 2015, Vol. 91, No. 3

(b) OTUs Reads

Mucoromycotina Mucorales (3, 8)

Mortierellales (15, 200)

Pucciniomycotina Erythrobasidiales (7, 47) Leucosporidiales (2, 93)

Sporidiobolales (2, 4) Ustilagomycotina Malasseziales (2, 2)

Tremellomycetes (35, 3405)

Agaricomycotina Dacrymycetales (8, 2615) Downloaded from https://academic.oup.com/femsec/article-abstract/91/3/fiv012/436629 by guest on 06 August 2020 Sebacinales (10, 570)

Cantharellales (9, 1057)

Trechisporales (4, 1290)

Hymenochaetales (15, 20161)

Corticiales (12, 2057)

Polyporales (36, 9411)

Thelephorales (15, 824)

Russulales (20, 6372)

Agaricales (40, 11268)

Atheliales (12, 3586)

Taphrinales (3, 8) Saccharomycotina Saccharomycetales (6, 204) Orbiliales (3, 21) Pezizales (4, 340) Pezizomycotina Dothideomycetidae (20, 738) Pleosporales (9, 180) Botryosphaeriales (2, 140) Chaetothyriales (43, 6389) Verrucariales (4, 1356) Eurotiales (9, 584) Onygenales (2, 7) Umbilicariales (4, 114) Lecanorales (45, 2961) Ostropomycetidae (16, 677) wood decayer Helotiales (192, 19197) other saprotrophs Rhytismatales (7, 1586) mycorrhiza Thelebolales (2, 8) lichen Xylariales (5, 311) parasite Hypocreales (44, 1256) endophyte Microascales (2, 5) unknown lifestyle Chaetosphaeriales (6, 1164) Coniochaetales (13, 2994) Diaporthales (2, 5) Ophiostomatales (5, 96) < 5 % 5-10 % > 10 % Sordariales (3, 60)

Figure 4 – continued Ottosson et al. 9

Stem breakage Str hor Xer cam Vel abi Pha lae Tre far Phe nig Phellinus nigrolimitatus Hyp alu Fom pin Phellinus viticola Coniophora olivacea (B) Kne alu Stereum sanguinolentum Veluticeps abietina (B) Downloaded from https://academic.oup.com/femsec/article-abstract/91/3/fiv012/436629 by guest on 06 August 2020 Antrodia serialis (B) Hyp asp Axis 2 Trichaptum abietinum Het par

Asc cyl uprooted Myc pur Butt rotted Uprooted log Bot sub Pau pea butt-rotted Hyp pal log

Con och Pen pra stem broken log Hyp alb -4 3

-4 Axis 1 6

Figure 5. CCA ordination diagram showing the relationship between the wood-decaying fungi detected as DNA (names abbreviated, full species names are given in Table S1, Supporting Information) in logs at site G and the different types of trunk breakage (stem, butt and root breakage) and the earlier presence of fruiting wood-rot fungi. Only the 30 most abundant OTUs identified to species level are shown. The first and the second axes are shown, explaining 14.9% of the variabilityn i species data, corresponding to differences in tree fall and primary species, respectively. Significant canonical axes primary species, T. abietinum (P = 0.033) and tree fall due to stem breakage (P = 0.031) are highlighted in black. Logs that are closer together have a more similar OTU composition. ‘(B)’ indicates brown-rot fungi.

Table 2. Compilation of species that were recorded both as fruit bodies and DNA from the experimentally cut logs at site F (n = 12) between 1995 and 2008 (no inventory in 1999). For each species, the table shows the annual and total number of unique logs with fruit-body records, the number of logs where the DNA of the fungal species was detected in 2008 and the number of logs where both fruit bodies and DNA were recorded. Only species detected from DNA in at least three logs are shown.

No. of logs with fruit bodies year−1

Total no. No. of of logs logs with with fruit DNA in ∗

Species 1995 1996 1997 1998 2000 2001 2002 2003 2004 2005 2006 2007 2008 bodies 2008

A. serialis 3354554313 5 8 Botryobasidium subcoronatum 11 6 F. pinicola 133355466666 6 10 H. parviporum 211 21111 4 12 K.alutacea 1111 Phlebia livida 111 1 5 Resinicium bicolor 132244364 8 11 Trechispora farinacea 115

∗DNA of the fungal species was detected in all logs with records of fruit body formation.

14 logs were formed as a result of stem breakage (Table S4, clustered together in stem-broken logs (permutation test, F = Supporting Information). In a study of all the 684 downed logs 1.35, P = 0.031) (Fig. 5). Uprooted and butt-broken logs had dis- at site G, 7% were formed as a result of uprooting, 49% as a tinct communities, including the root-rot pathogen H. parvipo- result of butt breakage and 44% as a result of stem breakage rum. Furthermore, the fungal communities in logs colonized (Edman, Jonsson¨ and Jonsson 2007). The fungal communities by the white-rot fungi Trichaptum abietinum and Stereum san- found in logs that were formed by stem breakage were dif- guinolentum were different to the communities found in logs ferent to those found in logs that originated from butt break- that had been colonized by brown-rot fungi and the white-rot age or uprooting. The CCA ordination showed that the white- species P. nigrolimitatus (permutation test, F = 1.45, P = 0.033) rot species P. nigrolimitatus and the brown-rot species F. pinicola (Fig. 5). 10 FEMS Microbiology Ecology, 2015, Vol. 91, No. 3

Table 3. Compilation of species that were recorded both as fruit bodies and DNA from the naturally fallen logs at site G (n = 26) between 1997 and 2008. For each species, the table shows the annual and total number of unique logs with fruit-body records, the number of logs where DNA of the fungal species was detected in 2008 and the number of logs where both fruit bodies and DNA were recorded. Only species detected from DNA in at least three logs are shown.

No. of logs with fruit bodies year−1

Species 1997 2003 2008 Total no. of logs with fruit bodies No. of logs with DNA in 2008∗

A. serialis 1 C. abietina 12 3 6 3(3) C. olivacea 114(1) F. pinicola 21 2 3 14(3)

H. parviporum 1120(1)Downloaded from https://academic.oup.com/femsec/article-abstract/91/3/fiv012/436629 by guest on 06 August 2020 P. nigrolimitatus 9 10 10 10 20 (10)

∗The number of logs where both DNA was detected in 2008 and fruit bodies were recorded between 1997 and 2008 are shown in parentheses.

DISCUSSION wood-inhabiting fungi (Robinson et al., 1993; Wells and Boddy 2002). Primary delignification by wood-decay fungi may also pro- In this study, we analysed the wood-inhabiting fungal commu- vide access to cellulose compounds that would otherwise be nities identified by 454 sequencing from a phylogenetic, taxo- inaccessible to non-wood-decay fungi (Fukasawa, Osono and nomic and ecological perspective. We were only able to iden- Hiroshi 2011). Several of the non-wood-decay fungi detected, tify one third of the 1910 OTUs to fungal species or genus level, such as Hyphodiscus hymeniophilus, have putative parasitic roles but these taxa were the most frequently detected and accounted in fungi and in plants. Mycoparasitic fungi may parasitize the for almost two thirds of the sequence reads. Based on ecologi- mycelium and fruit bodies of other fungi, and some species cal knowledge of the identified fungi, we could assign ecological have been proposed as biocontrol agents of wood-decay fungi, roles to more than half of the amplified DNA sequences. As ex- partly based on their ability to produce fungal-growth-inhibiting pected, wood-decaying basidiomycetes (i.e. the identified poly- antibiotics (Verma et al., 2007). We also identified OTUs with pore and corticoid fungi within the Agaricomycetes) were the mutualistic roles: ectomycorrhizal (i.e. Russula and Cortinarius most abundant in terms of sequence reads despite accounting spp.) and potentially ericoid mycorrhizal species (i.e. Sebacina for less than 10% of the OTUs. However, the identified fungal spp.), as well as lichenized fungi (i.e. Cladonia spp.). Several stud- communities also consisted of other saprotrophic fungi, which ies have reported the presence of ectomycorrhizal fungi in de- may include taxa with as yet unreported wood-decaying capac- caying wood and an increasing prevalence as decay proceeds ities, as well as several mutualistic, parasitic and endophytic (e.g. Harvey, Larsen and Jurgensen 1979; Tedersoo et al., 2003; fungi that are not usually referred to as wood-inhabiting fungi. Rajala et al., 2011, 2012). Nutrient transfer between ectomycor- Although we do not have precise ecological information about rhizal and wood-decay fungi has been reported in laboratory ex- many of the identified species taxa and lack information about periments (Lindahl, Stenlid and Finlay 2001), and some ectomy- all the unidentified OTUs, it is clear that in addition to wood- corrhizal species may decompose lignocellulose, but their sig- decay fungi, the fungal communities in decaying wood largely nificance in decomposition is unclear (Baldrian 2009;Bodeker¨ comprise fungal species with a range of other ecological roles. et al., 2009; Cullings and Courty 2009; Courty et al., 2010). The The prospects of linking identity with ecology are more promis- composition of fungi in logs and their ecological role undergoes ing for fungi than for bacteria and archaea because a key issue a transition from wood decaying to other saprotrophic and my- confronting bacterial taxonomists is the challenge of delimiting corrhizal life traits as wood decay proceeds, similar to the tran- species due to horizontal gene transfer (Hibbett and Taylor 2013). sition seen in the composition of fungi in aboveground forest Ecological classifications of fungi need to be viewed with litter from saprotrophic to mycorrhizal fungi as the forest lit- some caution because ecological roles may vary with the eco- ter decomposes and moves into the soil (Lindahl et al., 2007). logical context. Much of the information about fungal ecologi- DNA of lichenized fungi was found in wood fairly frequently, cal roles is limited, imprecise or ambiguous and, for many taxa, which raises questions about the biology and the life cycle of particularly among the ascomycetes, has yet to be determined. lichenized fungi. The mycobionts of certain lichens may poten- Some mycorrhizal fungi, such as Cortinarius spp. have, for exam- tially have free-living stages; however, as yet, there has only ple, certain abilities to decompose organic substrates (Dighton, been limited investigation of the potential of free-living myco- Thomas and Latter 1987;Bodeker¨ et al., 2009; Courty et al., 2010). bionts in wood, despite being suggested to occur in three species Similarly, wood-decaying fungi have been isolated from inside of Stictidaceae (Wedin, Doring¨ and Gilenstam 2004). The possi- mycorrhizal roots and have been shown to colonize fine roots bility that the DNA of the lichen genera Cladonia detected inside of conifers, suggesting some kind of mutualistic mode under the logs examined in this study could have originated from veg- certain conditions (Menkis et al., 2005; Vasiliauskas et al., 2007). etative propagules or dispersed thallus fragments could not be Despite these limitations, this approach of linking the results excluded (Tuovinen et al., 2015). of large-scale high-throughput sequencing methods with the Many taxa could not be named. These OTUs may repre- known ecological roles of the fungi that have been identified sent not only unknown species but also described species that may provide new perspectives on the patterns and processes of are not yet represented by any sequence in GenBank (Hibbett fungal communities. et al., 2011). Our ability to identify species or taxa of higher The non-wood-decay fungi primarily use simple organic taxonomic rank from environmental sequences by critically compounds such as carbohydrates released during the process comparing them to published sequences could be improved by of decomposition (Rayner and Boddy 1988; Osono and Takeda taxonomic mycological research and increased bioinformatic 2001; Botha 2011) or that leach during interactions between efficiency (Ovaskainen et al., 2010). However, even though the Ottosson et al. 11

identification is crucial, the most critical aspect for functional and fruit bodies and the variation among species supports simi- analysis is to obtain more information about the ecological roles lar findings in other studies of fungal communities in woodand and traits of the identified taxa (Crowther et al., 2014). Labora- in soil (e.g. Allmer´ et al., 2006; Porter, Skillman and Moncalvo tory and field studies of fungi show that differences in resource 2008). The fungal species richness detected as mycelia in the use are correlated with genetic distance (Hanson et al., 2008, study logs is remarkably high given that the boreal forest is con- McGuire et al., 2010). According to the theory of niche conser- sidered to be a relatively species-poor biome (Bernes 2012)and vatism, which refers to the tendency of species and clades to that the total number of known wood-inhabiting fungi reported maintain their niches and traits over time, communities com- in Fennoscandia is 2500 (Stokland, Siitonen and Jonsson 2012). prising closely related species may result from environmental ThenumberofOTUsfromsiteFwasmorethanoneorderof habitat filtering, whereas communities made up of distantly magnitude higher than the number of species detected by taking related species may indicate antagonistic interactions where annual inventories of the fruit bodies growing on the logs over closely related species with overlapping niches have been ex- 14 years, and two orders of magnitude higher than the number cluded by competition (Webb et al., 2002). According to this rea- of species that had formed fruit bodies at the time of sampling. Downloaded from https://academic.oup.com/femsec/article-abstract/91/3/fiv012/436629 by guest on 06 August 2020 soning, the phylogenetic distance might serve as a proxy for Our study indicates that the way in which the logs were the ecological differences between co-occurring species in con- formed (i.e. different types of stem breakage) may affect subse- trast to neutral models where differences between species are quent fungal community composition. The fungal community not considered to influence the community assembly process. in stem-broken logs was different to the fungal community de- However, our analysis of the wood-inhabiting fungal communi- tected in logs that were uprooted or butt-broken. In an earlier ties at two sites indicates that this assumption may differ be- study, it was concluded that even though windfall was the ul- tween and within fungal orders. Within some orders (i.e. Hy- timate downing factor at study site G, the presence of different menochaetales, Lecanorales and Polyporales) the ecological role pioneer-rot fungi was the main reason that the trees were sus- (i.e. wood decay) is phylogenetically conserved, even though the ceptible to wind and broke in different ways (Edman, Jonsson¨ species may decay the wood using different processes, which and Jonsson 2007). Furthermore, trees exposed to butt- and root- will affect their fitness. By contrast, species of, for example, rot often result in whole-tree logs (Oliva et al., 2008), whereas Agaricales, Helotiales and Russulales represent many different stem-broken trees often lose their top part. The major determi- ecological roles, suggesting that certain species with different nants of fungal succession in wood are the biological, chemical niches within these orders might be more prone to co-exist and physical conditions (Rayner and Boddy 1988); the presence compared with species with a similar ecological role. This type of a long-lived fungal species with a large mycelium can alter of analysis needs to be conducted at a lower taxonomic level these conditions. Therefore, long-lived species can potentially in fungi: for example, mycorrhizal fungi have evolved repeat- have a strong impact on the sequential community, and this can edly in several fungal lineages and from saprotrophic ancestors be seen as a priority effect (Ottosson et al., 2014). Experimental (Hibbett, Gilbert and Donoghue 2000). laboratory- and field studies have demonstrated that the inocu- Interestingly, we detected mycelia in almost all logs where lation of different primary fungal species may have pronounced the same species had previously been recorded as fruiting, in effects on the composition of secondary established fungal com- some cases up to 11 years after fruiting was last recorded in munities (Lindner et al., 2011;Dickieet al., 2012). Priority effects annual inventories. Given that only a few wood samples were may thus ultimately affect ecosystem properties such as the de- analysed, corresponding to minute parts of the wood volume in composition of organic matter even though these effects may the individual logs, our ability to detect these species suggests attenuate at higher scales of ecological organization and over that they can be long lived and have a large distribution within longer time scales (Dickie et al., 2012). the logs. This idea is supported by observations of long-lived sporocarps: for example, 71-year-old sporocarps of P. ro bus tus SUPPLEMENTARY DATA (Jeppson 2007) and sporocarps of P. igniarius that are more than 60 years have been reported (Niemela¨ 2005). The post-fruiting Supplementary data is available at FEMSEC online. occurrence of a species may represent a senescence of the in- dividual mycelium, but the fungus might also fully revert to ACKNOWLEDGEMENTS mycelial growth after fruiting (Moore et al., 2008). It is also pos- sible that fruiting might resume after a prolonged pause, but We thank Maria Jonsson for laboratory work, Bengt-Gunnar Jon- this is likely to be relatively uncommon. The allocation of re- sson for kindly permitting the use of data from Gardfjallet,¨ Hans- sources for reproduction is reflected in the different life histories Goran¨ Toresson for species inventories at Fageron,¨ Leif Carlsson of fungi (Rayner and Boddy 1988). Most wood-decaying species for advice about drilling wood and Karl-Henrik Larsson for help are unit restricted and compete for space: competitively superior with taxonomic issues. long-lived species may develop mycelia throughout large parts of the log, influence the fungal community throughout the de- FUNDING cay process and distribute their resources for fruiting over an This work was supported by a grant from the Swedish University extended period, potentially throughout the existence of the log. of Agricultural Sciences to J.S. A prolonged non-reproductive occupation of the substrate may enable the resident fungus to control resources that otherwise Conflict of interest statement. None declared. could have supported the spore production of other fungal indi- viduals. 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