Fungal networks and orchid distribution: new insights from above- and below-ground analyses of fungal communities

Pecoraro, L., Caruso, T., Cai, L., Gupta, V. K., & Liu, Z. J. (2018). Fungal networks and orchid distribution: new insights from above- and below-ground analyses of fungal communities. IMA , 9(1), 1-11. https://doi.org/:10.5598/imafungus.2018.09.01.01

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Key words: endophytic fungi macrofungal communities Mediterranean molecular ecology orchid-fungus relationships spatial distribution coexistence 9(1): 1–11 (2018) 1–11 9(1): · FUNGUS IMA

1 (22.5 2012). The presence and distribution et al. 2012). et al. 2015). Nearly all previous studies carried out to determine fungal to out Nearly all previousstudies carried Orchid distribution could be influenced not only by the , and Zhong-Jian Liu , and Zhong-Jian 5 (26 %), mycorrhizal fungi could mediate terrestrial orchid species mycorrhizal fungi could mediate terrestrial which The extent to coexistence, driving niche partitioning. the distribution fungal communities may determine and orchid populations (McCormick remains unclear dynamics of et al. 2012, McCormick & Jacquemyn 2014). diversity in orchid sites have been based on experimental from orchid seed-sowing and/or sequencing DNA of fungal tissues (Dearnaley 2007, McCormick & Jacquemyn 2014). Such experiments are limited in only providing information establish associations with the effectively fungi that on the (Těšitelová orchids of potentially symbiotic fungi, as well as the diversity of the associated fungal community in nature have remained unexplored (Oja alsobut fungi, mycorrhizal suitable of abundance and presence by the total diversity of associated fungi naturally occurring in direct of combinations various of possibility a is There field. the and indirect interactions between and within the fungi, and also , Vijai Kumar Gupta Kumar , Vijai 3 , Lei Cai 4 -like fungi belonging to is particularly dependent on , Tancredi Caruso , Tancredi

1, 2, 3 (2014), however, demonstratedthat (2014), however, al. et Orchids are critically dependent on fungi for seedling establishment and growth, so the

must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). You (2015) showed that the specialized the association (2015) showed that al. et did not limit the geographical the did not limit the range of Sebacina School of Biological Sciences, Queen’s University of Belfast, BT9 7BL Belfast, Northern Ireland Belfast, of Belfast, BT9 7BL University Queen’s School of Biological Sciences, Tallinn, 12618 Technology, University of Tallinn School of Science, Chair of Green Chemistry, ERA and Biotechnology, Department of Chemistry Center for Biotechnology & BioMedicine and Division of Life & Health Sciences, Graduate School at Shenzhen, Tsinghua University, 518055 University, Tsinghua Center for Biotechnology & BioMedicine and Division of Life & Health Sciences, Graduate School at Shenzhen, Beijing, China Academy of Sciences, 100101 Chinese Institute of Microbiology, of , State Key Laboratory Shenzhen Key Laboratory for Orchid Conservation and Utilization, The National Orchid Conservation Center of China and The Orchid The National Conservation Orchid China Center of and Shenzhen Key Laboratory for Orchid Conservation and Utilization, Abstract: characterised distribution and diversity of orchids might depend on the associated fungal communities. We species in three Mediterranean protected areas, using a the communities associated with eight orchid combination of above-ground analyses of sporophores and below-ground molecular analyses of orchid root samples. In three years of sporophore macrofungal belonging species to 84 genera were observed. Statistical analyses indicated correlation a collection in 25 plots around flowering orchidenvironmental of and the effect variation, regardless of between macrofungal diversity and orchid community , 268 and cloning, amplification, PCR ITS-DNA Fungal sites. orchid investigated the characterizing factors spatial sequencing revealed 2018. Accepted: 27 January 2018; Published: 12 February August 2017; Article info: Submitted: 7 Estonia as other basidiomycetes99 orchid and Sebacinaceaeas well (3.5 %), in the roots of %), ascomycetes, and plants. Mycorrhizal specificity was low co-occurringbut orchid species preferences showed orchid community contribute to may found in the sites The diverse macrofungal communities partners. for different variation without colonizing the orchid roots. Molecular analyses revealed a segregation of associated fungi, coexistence in nature. which may contribute to Mediterranean orchid Shenzhen, China Conservation and Research Center of Shenzhen, 518114 Shenzhen, China; corresponding author e-mail: [email protected] Shenzhen, China; corresponding Center of Shenzhen, 518114 Conservation and Research © 2018 International Mycological Association © 2018 International Mycological following conditions: distribute and transmit the work, under the are free to share - to copy, You Attribution: 5 2 3 4 1 Lorenzo Pecoraro Fungal networks and orchid distribution: new insights from above- and from above- insights new orchid distribution: and networks Fungal communities of fungal analyses below-ground Non-commercial: No derivative works: Any of the above conditions can be waived if you get For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. transform, or build upon this work. may not alter, You may not use this work for commercial purposes. You Nothing in this license impairs or restricts the author’s moral rights. permission from the copyright holder. widely distributed Australian orchid Pheladenia deformis. widely distributed Jacquemyn The family with INTRODUCTION fungal symbionts: orchids have obligate associations with mycorrhizal fungi, because of their initially mycoheterotrophic life-style (Leake 1994). Orchid species rely on their fungal least during the nutrients, at the provision of associates for non-photosynthetic development, and protocorm stage of in some cases throughout their life (Rasmussen 1995). Because orchids are critically dependent on fungi for seedling establishmentdistribution and and growth, the diversity of orchids might be expected to be correlated with The the composition of the associated fungal communities. relationship between orchid distribution, abundance, and spatial mycorrhizal diversity has been analysed at different scales (McCormick et al. 2009, Jacquemyn et al. 2012). Davis doi:10.5598/imafungus.2018.09.01.01 VOLUME 9 · NO. 1 Pecoraro et al.

between the plants and the fungi. Fungi forming associations cover % (coherent material), stone cover % (non-coherent with non-orchid plants may indirectly affect orchid diversity material), micro-topography (scored 1–3 related to increasing through contributing to the structure of communities, a irregularity of the ground surface), elevation above sea level, major factor influencing the establishment and growth of orchid slope %, and orientation (Table S1 Supporting Information). species (Kosaka et al. 2014).

ARTICLE In this study, we investigated abiotic and biotic factors Above-ground analysis of fungal communities influencing both putative fully photosynthetic and partially Field surveys for above-ground macrofungal sporophores mycoheterotrophic orchid diversity and distribution in more than 1 mm diam (Arnolds 1981), were carried out every three Mediterranean protected areas, across forest and week during the main basidiomata and ascomata production meadow habitats. We characterized the fungal communities seasons, essentially autumn (September-December) and associated with eight orchid species (six fully-photosynthetic spring (April-June) respectively (when rainfall occurs with meadow and two partially mycoheterotrophic forest species) suitable temperatures), from September 2006 to June in calcareous mountain sites using a combination of 2009. Sporophores were collected and identified by macro- above- and below-ground analyses. We aimed to test the and microscopic observations of fresh material. Notes on hypothesis that variation in above-ground fungal community morphological characters and ecological conditions were structure, as reflected in the sporophores produced, isa recorded in the field, and samples were taken to the laboratory predictor of the orchid species assemblage. We tested this for microscopic examination. Numbers of sporophores within hypothesis by eliminating possible effects of other major plots were recorded during every survey visit for each species covariates quantifying the environmental and geographical found. Material was dried and voucher specimens deposited characteristics of the sites, including vegetation types (e.g. in the Herbarium Universitatis Senensis (SIENA). shrub and tree cover vs. meadows). We also reversed Monitoring was for three consecutive years to provide the predictors and responses used to test the hypothesis a reasonable representation of the fungal communities that variation in the orchid assemblage was a predictor of present (Vogt et al. 1992). The structure of the macrofungal the above-ground fungal community structure. Further, we communities within each plot was expressed by assembling also examined associated below-ground fungal taxa using all the data on species identity and productivity. molecular tools to determine whether orchid species identity was a predictor of the associated root fungal community. Below-ground analyses of orchid root fungal associates

MATERIAL AND METHODS Orchid sample collection In early summer of 2007 and 2008, during the flowering Study sites and species period, roots were collected from 11-15 adult individuals of the The study was conducted in three geographically distinct eight orchid species, resulting in 99 samples from the three protected areas in central , the ‘Monte Cetona’, ‘Monte study areas (Table S2 Supporting Information). Roots were Penna’ (specifically on ‘Monte Rotondo’), and ‘Cornate di rinsed in water and carefully scrubbed with a brush to remove Gerfalco’ Nature Reserves. The areas consist of calcareous most soil debris. They were then treated in an ultrasonic bath mountains characterized by Mediterranean montane dry (three cycles of 30 s each) to remove any remaining soil grasslands with Bromus erectus dominant among herbaceous particles and to minimize the risk of detecting all microscopic plants. These meadow habitats are surrounded by forests with organisms adhering to the root surface. Thin cross sections of various broad-leaved trees including species of Acer, Fagus fresh root samples were checked for mycorrhizal colonization sylvatica, ornus, Ostrya carpinifolia, and Quercus by light microscopy. Root fragments exhibiting high fungal cerris, mixed with such as Pinus nigra. All the sites are colonization were immediately processed for fungal isolation. located on the top of the three mountains, at 755–1047 m a.s.l. Root portions to be used for molecular analysis were frozen In spring 2006, within each site, plots of 20 m diam were in liquid nitrogen and stored at –80 °C. established in open meadow and surrounding forest habitats based on the presence of fully-photosynthetic and partially Fungal isolation mycoheterotrophic orchid species respectively. Eight orchid Five or six roots per plant were surface-sterilized with species were found: six meadow orchids (Anacamptis consecutive washes of 1:5 sodium hypochlorite (30 s) and morio, Himantoglssum adriaticum, bertolonii, Orchis three rinses of sterile water. Ten 3-5 mm long pieces from pauciflora, O. provincialis, and O. tridentata), and two forest each root were cultured on malt extract agar (MEA) and potato orchids (Cephalanthera damasonium, and C. longifolia). Each dextrose agar (PDA) amended with gentamycin (40 mg/l) and/ plot was randomly established around a flowering orchid of or chloramphenicol (50 μg/ml). Petri dishes were incubated at one of the eight species in the investigated sites, resulting room temperature (20–25 °C) in the dark for up to two months in 25 plots. The centre of each plot was mapped using high- to allow the development of slow-growing mycelia. precision GPS (Trimble Navigation, Sunnyvale, CA) and marked with a metal stake. In order to assess environmental DNA extraction, PCR amplification of fungal ITS, and geographical conditions of the study plots, we recorded cloning, and sequencing the: habitat (meadow or forest), % vegetation cover (divided Both total DNA from orchid root samples and DNA from into tree, shrub, herb, and lichen-moss percentages), litter isolated fungi were extracted using the cetyltrimethyl cover % (ground surface covered by plant debris), rock ammonium bromide (CTAB) procedure modified from Doyle

2 IMA FUNGUS Fungal networks and orchid distribution

& Doyle (1990). Fungal ITS regions were PCR-amplified Legendre 2002). The method produces several vectors that ARTICLE using the primer pair ITS1F/ITS4 (Gardes & Bruns 1993) reflect possible spatial structure in the data, and the subset following the methods described in Pecoraro et al. (2017) of vectors eventually used in our analysis was selected by for PCR reaction, thermal cycling, purification and cloning a multivariate extension of the Akaike information criterion of PCR products. Cloned ITS inserts were purified with a (AIC; Dray et al. 2006). We consequently created a set of Plasmid Purification Kit (Qiagen) and sequenced with the environmental and spatial vectors or factors potentially same primer pair used for amplification. Dye sequencing was accounting for variance in the community structure of fungi carried out on ABI 310 DNA Sequencer (Applied Biosystems, and orchids. Given the observational nature of this study, Carlsbad, CA). multivariate patterns in above-ground fungi (sporophores) and orchids can reciprocally be used as predictors of each Sequence analysis other, but results might change depending on the spatial Sequences were edited to remove vector sequences and to structure of each community, which might not be the same ensure correct orientation and assembled using Sequencher at all scales. In one set of analyses we used orchids as 4.1 for MacOsX (Genes Codes, Ann Arbor, MI). Sequence predictors of fungi, and in another we swapped predictors analysis was conducted with BLAST searches against for responses. For fungi producing sporophores above- the National Center for Biotechnology Information (NCBI) ground, we had information on relative abundance, while for sequence database (GenBank; http://www.ncbi.nlm.nih. the orchids there was only presence/absence information. gov/BLAST/index.html) to determine the closest sequence When we used fungi in response to orchids, we Hellinger- matches that enabled taxonomic identification. The fungal transformed raw data prior to apply distance based RDA community associated with the roots of the investigated (dbRDA) to ensure no inflation of the weights accorded to species has been partly described in Pecoraro et al. (2012a, rare species and omit joint absences (Legendre & Gallagher b, 2013, 2015, 2017). Molecular analyses were performed 2001). We used partial-dbRDA to test for any unique effects for 28 additional orchid individuals from two different species of orchids by controlling for covariation between orchids A. morio (15 samples) and O. provincialis (13 samples). on the one hand, and the sum of environmental correlates Fungal DNA sequences amplified from the eight analysed and spatial autocorrelation on the other. To use orchids as species are deposited in GenBank (Accession nos: A. morio a predictor of fungi, we calculated a PCoA of the orchid KX461964-KX461987, C. damasonium KT122776-KT122789, Jaccard matrix and extracted major PCoA axes to measure C. longifolia KT122767-KT122775, H. adriaticum JQ685234- orchid community structure. These axes were then used as JQ685249, O. bertolonii KP868521-KP868544, O. pauciflora predictors (i.e. explanatory axes) of the fungal communities. JQ723318-JQ723347, O. provincialis KX461988- KX462007, Given the number of available plots, we had to limit the and O. tridentata JN683860-JN683859). number of explanatory PCoA axes to a maximum of three to Phylogenetic analysis was conducted with Mega v. 5.0 reduce the issue of over-fitting to acceptable levels (i.e. low (Tamura et al. 2011). Sequences were aligned with Clustal X predictors to observations ratio). The same overall procedure v. 2.0 (Larkin et al. 2007) and neighbour-joining trees against was used to test for the effect of fungi on orchids, and we selected database sequences were constructed using Kimura used a permutational approach to test for the statistical 2-parameter distances, with bootstrapping of 1000 replicates significance of effects (Oksanen et al. 2012). (Felsenstein 1985). Due to the phylogenetic distance between We also performed a similar multivariate analysis on the the fungi identified from the roots of investigated orchids, fungal community molecularly detected in the roots to test distinct phylogenetic analyses were performed. The globose whether different fungal assemblages were associated with and ellipsoid-spored eichleriana and T. tomaculum different orchids. We calculated environmental and spatial were used as outgroups to root the tulasnelloid fungal tree, factors using the same methods applied to the orchid and while the ceratobasidioid fungi tree was rooted with Laccaria above-ground fungal datasets and tested for any effect of bicolor and portentosum. orchid species on the fungi after excluding environmental and spatial factors. All analyses were performed in R 3.1 Statistical analyses (R Core Team 2015). We performed a Principal Component Analysis (PCA) on the correlation matrix of environmental variables (habitat, tree, shrub, herb, and lichen-moss cover %, litter cover %, rock RESULTS cover %, stone cover %, micro-topography, elevation above sea level, slope %, and orientation; Legendre & Legendre Macromycete diversity and community 1998, Gotelli & Ellison 2004). We then used the axes from structure in orchid sites this PCA to define major environmental gradients that In total, 6365 macromycete sporophores were recorded, potentially correlated with species distribution (environmental representing 268 species in 84 genera (Table S1 Supporting correlates). Using PCA axes as proxies for environmental Information); 157 species in 59 genera were harvested in the gradients allowed us to remove the issue of collinearity of meadow plots, and 153 species in 62 genera in the forest predictors (Gotelli & Ellison 2004) in further multivariate plots. Forty-two taxa were found in both habitats (Table S1 analysis of fungi and orchid community structure. Spatial Supporting Information). information (UTME coordinates) was used to apply PCA to Several species-rich saprobic genera, such as neighbour joining matrices, and so described possible spatial and , were observed. Three of nine identified dependence of plots at multiple scales (PCNM; Borcard & Marasmius species (M. epiphylloides, M. epiphyllus, and

VOLUME 9 · NO. 1 3 Pecoraro et al.

M. quercophilus) yielded 437, 688, and 545 basidiomes respectively, 99 JF912461 from Anacamptis laxiflora 83 JF912475 from Ophrys fuciflora whereas Mycena, represented by 35 taxa, including M. clavicularis Orchis tridentata MC2e* 73 DQ182419 from Cephalanthera longifolia (600 basidiomata), showed the highest species richness of the whole 100 AY634163-from community (Table S1 Supporting Information). 99 DQ182418 from Cephalanthera longifolia Orchis tridentata MR2a*

The mycorrhizal fungal community was represented by 63 100 Orchis tridentata MR3a* Orchis tridentata MR1b* ARTICLE species in 12 genera of basidiomycetes, out of 1127 basidiomes 71 Orchis tridentata MR3e* collected. Amongst these, species accounted for 27.5 % of 98 EF536969 from Dactilorhiza hatagirea DQ102402 from Fragaria sp. the basidiomes and 20.6 % of the taxonomic diversity (13 species), 93 DQ279045 sp. 51 AM040889 from Liparis loeselii while (10 identified species), was the second species-richest EU273525 Ceratobasidium cornigerum

88 AF354093 Ceratobasidium sp. group. dominated basidiome production amongst the 69 DQ279046 Ceratobasidium sp. ectomycorrhizal fungi, with 153 basidiomes recorded, and the second 92 JF912474 from Ophrys fuciflora 99 JF912478 from Ophrys fuciflora highest was I. splendens with 108 basidiomes collected in the forest 99 JF912477 from Ophrys fuciflora 95 JF912473 from Ophrys fuciflora plots of ‘Monte Penna’ (Table S1 Supporting Information). Of all taxa AF200514 Ceratobasidium bicorne 100 AF200519 Ceratobasidium bicorne identified, 42 species were recorded in two out of three of the studied Cephalanthera damasonium MC3c* areas, while only 20 were collected in all three investigated Nature 100 AB303044 from Chamaegastrodia sikokiana AB303051 from Chamaegastrodia sikokiana Reserves. Eight of these 20 taxa belong to Mycena, with M. abramsii, 79 AJ549123 from Dactilorhiza incarnata

97 AF472285 from Psychilis monensis M. aetites, M. flavoalba, M. pura, M. pura f. alba, M. rosea, and M. vitilis JF912482 from Orchis purpurea collected both in meadow and forest habitats, whereas M. leptocephala 99 JF912483 from Orchis purpurea 57 AJ427399 Ceratobasidium albasitensis was only found in meadow plots. From the remaining taxa recorded DQ279056 Ceratobasidium sp. 100 EU152858 Ceratobasidium sp. in all the three studied areas, tenera, Crinipellis scabellus, AJ427404 Ceratobasidium ramicola clypeolaria, and Panaeolus acuminatus were only present in DQ279057 Ceratobasidium sp. 100 EU218892 Thanatephorus ochraceus meadow plots; eburneus, Lepista nuda, and Tricholoma DQ834419 Thanatephorus sp. from vanilla 74 DQ093646 from Pinus sylvestris album were exclusively associated with the forest habitat; and 100 JF912488 from Serapias vomeracea 100 79 DQ279013 Ceratobasidium sp. costata, C. semiglobata, H. sinapizans, and T. atrosquamosum were AJ301902 Ceratobasidium cornigerum found in both meadow and forest plots. DQ279015 Ceratobasidium sp. AF153778 Thanatephorus cucumeris DQ279014 Ceratobasidium sp. 50 AF354059 Thanatephorus cucumeris Orchid root fungal diversity and mycorrhizal DQ279061 Ceratobasidium sp. 100 AJ302009 Ceratobasidium cereale specificity 94 DQ279060 Ceratobasidium sp. Microscopical observation showed that cortical cells of Acamptis morio 69 AJ427402 Ceratobasidium anceps AJ427403 Ceratobasidium angustisporum and Orchis provincialis roots were densely colonized by fungal hyphae 99 AF472302 from Tolumnia variegata forming typical orchid mycorrhizal intracellular coils (pelotons), thus 74 AF472303 from Oncidium altissimum 99 AY634129 from Platanthera chlorantha 94 100 EU218895 from Platanthera obtusata confirming the high level of fungal colonization previously described in DQ093780 from Picea abies decayed root these orchid species (Pecoraro et al. 2012a, b, 2013, 2015). Rhizoctonia- 99 JF912462 from Anacamptis laxiflora 65 100 EF429314 Ceratorhiza oryzae-sativae like hyphae emanating from pelotons were observed in the majority of 86 DQ279058 Ceratobasidium sp. DQ279059 Ceratobasidium sp. root samples. 98 AJ427401 Ceratobasidium papillatum AF472301 from Campylocentrum filiforme Endophytic fungi exhibiting Rhizoctonia-like morphological features 80 100 AJ318425 from Oncidium "Sweet sugar" were obtained by in vitro isolation from the roots. Molecular identification 74 AJ318423 from Phalenopsis sp. 59 AF472299 from Ionopsis satyrioides of these isolates revealed that they belonged to Ceratobasidiaceae 100 AF472279 from Tolumnia variegata AJ419931 from Pinus sylvestris (Table S3 Supporting Information). A non-ceratobasidioid basidiomycete Orchis tridentata MR5b* was also isolated from A. morio sample MC4, the closest match for the 69 AY833047 from Cephalanthera damasonium DQ102444 from Fragaria sp. 97 sequence amplified from the culture being Schizophyllum commune 97 JF912481 from Orchis purpurea

55 JF912480 from Orchis purpurea (Schizophyllaceae, Table S3 Supporting Information). AY634128 from Epipactis helleborine 63 DQ279065 Ceratobasidium sp. Direct root DNA amplification mostly yielded fungal ITS sequences 100 AY787667 from Fraxinus excelsior showing similarity to Rhizoctonia fungi belonging to Ceratobasidiaceae AY643805 from Acianthus pusillus AY634126 from Dactilorhiza majalis (56 % sequences) and Tulasnellaceae (11 % sequences). A variety of 76 AY634127 from Epipactis palustris JF912460 from Anacamptis laxiflora ascomycetes was also identified in 7 out of 14A. morio, and 4 out of 13 67 JF912476 from Ophrys fuciflora O. provincialis individuals (Table S3 Supporting Information). AY634120 from Epipactis gigantea EU218894 Ceratobasidium sp. PCR products were obtained with the universal fungal primer pair 99 AY634119 from Epipactis gigantea JF912464 from Anacamptis laxiflora ITS1F/ITS4 from 88 out of 99 analysed samples from the eight orchid JF912459 from Anacamptis laxiflora species. More than half of the samples (52 %) yielded Rhizoctonia 53 JF912484 from Serapias vomeracea MR4d* sequences with an identity in Ceratobasidiaceae (26 %), Tulasnellaceae 92 JF912485 from Serapias vomeracea 75 JF912489 from Serapias vomeracea (22.5 %), and Sebacinaceae (3.5 %), whereas 33 % of orchids were only Anacamptis morio MR9c* Orchis provincialis MR14c* colonized by ascomycetous fungi, notably Exophiala sp., Leohumicola sp., 72 JF912458 from Anacamptis laxiflora JF912487 from Serapias vomeracea

51 JF912486 from Serapias vomeracea

94 Anacamptis morio MC1e* Anacamptis morio MC2b* Neighbour-joining phylogenetic tree showing the relationship between the 100 Fig. 1. 99 Anacamptis morio MC3a* Ceratobasidiaceae sequences obtained from the analysed orchid species (*) and 99 Anacamptis morio MR2b* Orchis tridentata MR5a* selected database relatives. Kimura 2-parameter distances were used. Bootstrap DQ149869 Laccaria bicolor 100 AF349686 Tricholoma portentosum values are based on percentages of 1000 replicates. The tree was rooted with 0.05 Laccaria bicolor and Tricholoma portentosum as outgroups.

4 IMA FUNGUS Fungal networks and orchid distribution

58 DQ925564 from Leptodontidium sp., Tetracladium sp., and Fusarium sp., and 68 DQ925552 from Cypripedium macranthos ARTICLE 68 DQ925554 from Cypripedium calceolus 9 % by non-Rhizoctonia-like basidiomycetous endophytes, DQ925555 from Cypripedium parviflorum 100 including Cryptococcus carnescens, , 99 DQ925557 from Cypripedium parviflorum JF926492 from Orchis purpurea and Tomentella species. In 13 orchid samples, both 98 64 JF926491 from Orchis purpurea 100 JF926489 from Orchis purpurea Rhizoctonia and non-Rhizoctonia fungal sequences were JF926486 from Orchis purpurea amplified (Table S2 Supporting Information). JF926487 from Orchis purpurea 82 JF926484 from Orchis purpurea Phylogenetic analysis of the Rhizoctonia-like associates JF926488 from Orchis purpurea 100 JF926485 from Orchis purpurea revealed relationships within Ceratobasidiaceae and JF926490 from Orchis purpurea Tulasnellaceae (Figs 1–2). Sequences amplified from DQ925572 from Cypripedium montanum Himantoglossum adriaticum CG2a* roots and from isolated fungi could be aligned with 100 100 Himantoglossum adriaticum CG7b* Himantoglossum adriaticum CG7e* GenBank sequences from fungal strains and sporophores, 83 Himantoglossum adriaticum CG5a* as well as from several plants, including orchid and non- 79 Himantoglossum adriaticum CG1b* Himantoglossum adriaticum CG1a* orchid species. In the neighbour-joining tree from the 77 EU583691 from Orchis x bergonii Ceratobasidiaceae dataset (Fig. 1), sequences from the Himantoglossum adriaticum MC2a* 70 Himantoglossum adriaticum MC3b* 64 66 orchids segregated into five main clusters. In particular, Himantoglossum adriaticum MC3e* DQ925576 from Cypripedium montanum most sequences from O. tridentata co-segregate in a DQ925599 from Cypripedium arietinum cluster including GenBank fungal sequences from different DQ925600 from Cypripedium arietinum DQ925600 from Cypripedium arietinum fully photosynthetic meadow orchids (e.g. A. laxiflora JF926481 from Ophrys fuciflora and O. fuciflora), and partially mycoheterotrophic orchids 100 7b* Ophrys bertolonii 7a* growing in open forest habitats (including C. longifolia Ophrys bertolonii 6b* Ophrys bertolonii 6a* and Epipactis helleborine). An O. tridentata sequence Ophrys bertolonii 7e* from MR5 (clone b) segregated into a distinct cluster with Orchis provincialis MR18b* Orchis provincialis MR21b* ones from orchids of the same (O. purpurea) and different Orchis provincialis MR18d* genera (Acianthus pusillus, C. damasonium, and E. 72 Orchis provincialis MR22b* 74 Orchis provincialis MR21a* 50 helleborine), as well as sequences from non-orchid plant Orchis provincialis MR22a* Orchis provincialis MR22c* species (Fragaria sp. and Fraxinus excelsior) and from a 99 DQ925633 from Cypripedium guttatum 100 Ceratobasidium isolate (DQ279065). The remaining O. 59 DQ925633 from Cypripedium guttatum DQ925635 from Cypripedium guttatum tridentata sequence (MR5, clone a) clustered with the 96 DQ925624 from Cypripedium montanum majority of A. morio sequences in our study, whereas A. DQ925629 from Cypripedium fasciculatum DQ925615 from morio sequences from MR4d and MR9c fell in one including Orchis pauciflora 4CGe*

98 Orchis pauciflora 4CGc* an O. provincialis sequence from MR14c and GenBank 83 Orchis pauciflora 4CGa* sequences from the green meadow orchids A. laxiflora 50 DQ925597 from Cypripedium reginae 100 DQ925593 from Cypripedium reginae and S. vomeracea. The only Ceratobasidium sequence DQ925595 from Cypripedium reginae we amplified was from Cephalanthera damasonium and 85 JF926472 from Ophrys fuciflora JF926476 from Ophrys fuciflora clustered with ones from the achlorophyllous forest orchid 100 JF926479 from Ophrys fuciflora 73 JF926475 from Ophrys fuciflora Chamaegastrodia sikokiana, the photosynthetic meadow JF926473 from Ophrys fuciflora orchids Dactylorhiza incarnata and O. purpurea, and the JF926478 from Ophrys fuciflora JF926474 from Ophrys fuciflora epiphytic orchid species Psychilis monensis. JF926471 from Bromus erectus DQ925503 from Cypripedium californicum The phylogenetic tree of Tulasnellaceae showed the 65 JF926469 from Anacamptis laxiflora investigated orchids associated with different groups of 98 JF926470 from Anacamptis laxiflora 97 JF926498 from Serapias vomeracea tulasnelloid fungi (Fig. 2). Tulasnellaceae sequences DQ925536 from Cypripedium candidum 100 obtained from H. adriaticum likely represented two or 100 DQ925537 from Cypripedium parviflorum 98 71 AJ313456 from Spathoglottis plicata three distinct taxa. Ones from O. bertolonii segregated into 59 EF433954 from Cypripedium guttatum DQ925658 from Cypripedium japonicum two clusters, the smaller including GenBank sequences 89 DQ925644 from Cypripedium japonicum amplified from Cypripedium arietinum and O. fuciflora, 100 AY634130 from Dactylorhiza majalis 100 DQ925664 from Cypripedium macranthos var. speciosum and the larger including ones from O. sphegodes. On the DQ925661 from Cypripedium macranthos var. speciosum basis of ITS sequences, fungi detected in O. pauciflora EU218890 sp. AB369932 from Cypripedium macranthos var. rebunense and O. provincialis spanned a smaller range of variation, Ophrys bertolonii 11a* 99 AM697902 from Ophrys sphegodes appearing to belong to a single taxon for each orchid Ophrys bertolonii 12a* species, the two orchid taxa being represented by closely 66 Ophrys bertolonii 2a* Ophrys bertolonii 10e* related fungi. Ophrys bertolonii 3a* 78 Ophrys bertolonii 3b* 60 Ophrys bertolonii 5c* Ophrys bertolonii 10c* AJ549122 from Ophrys sphegodes Fig. 2. Neighbour-joining phylogenetic tree showing the Ophrys bertolonii 9a* relationship between the Tulasnellaceae sequences obtained Ophrys bertolonii 12b* Ophrys bertolonii 3c* from the analysed orchid species (*) and selected database Ophrys bertolonii 2d* relatives. Kimura 2-parameter distances were used. Bootstrap Ophrys bertolonii 2b* AY373292 Tulasnella eichleriana values are based on percentages of 1000 replicates. The tree 100 AY373296 Tulasnella tomaculum was rooted with Tulasnella eichleriana and T. tomaculum as 0.1 outgroups.

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Table 1. Variance decomposition of fungi (sporophores) community Table 2. Variance decomposition of orchids community structure. structure. The effect of orchids was quantified and tested by The effect of fungi (sporophores) was quantified and tested by controlling for the covariation (“Conditioned”) due to measured controlling for the covariation (“Conditioned”) due to measured environmental variables (environment) and geographical position environmental variables (environment) and geographical position of plots (space). of plots (space). Source Variance (%) P-value Source Variance (%) P-value ARTICLE Conditioned (environment + space) 49 Conditioned (environment + space) 70 Orchid effect 7 >0.05 Fungi effect 7 <0.05 Residual 44 Residual 22

Statistical results were strikingly more structured in terms of environmental and We found variation in tree, shrub, and litter cover was spatial variation, which accounted for 70 % of total variance negatively correlated with variables such as slope, (Table 2). However, while fungi accounted just for 7 % of herbaceous, lichen/moss, rock and stone cover. This orchid variation, this effect was still significant after removing accounted for 57 % of the environmental variation between environmental and spatial variation. Unconstrained PCoA plots (Fig. S1a Supporting Information, PC1) and is due to ordination of fungi (Fig. 3) and orchids (Fig. 4) illustrates including meadows and woodlands in the sampling design. some key aspects of these results. The fungal community Second was an elevation gradient (PC2, 18 %), with meadow was less structured than the orchid community, the first two and woodland plots at various altitudes. These first two PCA PCoA axes of the fungal communities accounting for 16 % axes were therefore used to define: (1) a vegetation index and 13 % of total variance respectively; something to be (PC1) that distinguished meadows from woodlands (S1b partly expected given the high dimensionality (i.e. very high Supporting Information) but also accounted for variation in species richness relative to the number of sampled plots) of the measured variables within meadows and woodlands; and the fungal community. Instead, in the case of orchids, they (2) an elevation gradient index (PC2). respectively accounted for 41 % and 24 % of total variance. Spatial, environmental, and orchid factors could account Also, the two communities were structured in a different way: for 56 % of the total variance in macrofungi (Table 1). The sum in the case of orchids the most important gradient (PCoA 1, of environmental and spatial variation represented the largest 41 %) was environmental and basically due to the difference fraction (49 %) while the unique effect of orchids (7 %) was not between meadow and woodland plots (Fig. 4) by construction, statistically significant at p 0.05 after removing environmental while the second gradient (PCoA 2, 24 %) separated one and spatial variation. A similar scenario was observed when site (‘Monte Penna’) from the other two (‘Monte Cetona’ we reversed predictors and response variables, but orchids and ‘Cornate di Gerfalco’). Instead, in the case of fungi the 1.5 1.5

PennaPenna woodlandwoodland Penna woodland a) b) 1.0 1.0

Cetona woodland woodland Cetona Cetona woodland meadow

0.5 GerfalcoCetona 0.5 woodlandwoodland Cetona meadow Gerfalco meadowCetona Gerfalco woodland Gerfalco Penna woodland Gerfalco GerfalcoCetona Cetona meadow meadowmeadow meadow

0.0 Cetona 0.0 meadow

Penna

PCoA 2 (13%) meadow PCoA 2 (13%)

-0.5 Penna -0.5 meadow

Penna Penna meadowmeadow -1.0 -1.0 Penna meadow Penna meadow

PennaPenna meadowmeadow -1.5 -1.5

-1.0 -0.5 0.0 0.5 1.0 1.5 -1.0 -0.5 0.0 0.5 1.0 1.5

PCoA 1 (16%) PCoA 1 (16%) Fig. 3. PCoA ordination of the fungal community (sporophores) after Hellinger transformation. The two figure panels show exactly the same data but with different labels. Left, plots labelled by site and clustered habitat (meadow vs woodland); Right, plots labelled by habitat and clustered by site (Penna, Gerfalco and Cetona).

6 IMA FUNGUS Fungal networks and orchid distribution

Penna meadow ARTICLE Penna meadow a) b) 1.0 1.0 Penna meadow Penna meadow Penna 0.5 0.5

GerfalcoPenna woodland woodlandPenna woodland

0.0 meadow Cetona 0.0 woodland Cetona meadow CetonaGerfalco meadowmeadow

PCoA 2 (24%) PCoA 2 (24%) Cetona

-0.5 -0.5 Gerfalco

Cetona meadow -1.0 -1.0 Cetona meadow Gerfalco meadow -1.5 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 Fig. 4. PCoA ordination of the jaccard distance matrix of the orchid community. The two figure panels show exactly the same data but with different labels. Left, plots labelledPCoA by site1 (41%) and clustered habitat (meadow vs woodland); Right, plots labelledPCoA by habitat1 (41%) and clustered by site (Penna, Gerfalco and Cetona).

first gradient was in terms of site separation and the second taxa developing basidiomes in the sampled plots, were 13 gradient in the habitat separation, but the clustering pattern Inocybe species, the species-richest and most recorded was much more confused and in any case the variance of the mycorrhizal . Inocybe species are already known to first two axes was similar and low. associate with several orchid species, and have been reported We also performed a similar multivariate analysis on the to support seed germination and seedling development in fungal assemblages molecularly detected in the roots of the C. longifolia and Epipactis atrorubens (Bidartondo & Read different species of orchids. We calculated environmental and 2008) as well as to colonize C. damasonium, E. atrorubens, spatial factors using the same methods applied to the orchid and E. dunensis adult plant roots in Germany (Bidartondo and above ground fungal datasets, by keeping a record of the et al. 2004, Bidartondo & Read 2008). The two analysed plot from which each sequenced orchid root was collected. Cephalanthera species have been previously found to Environmental and spatial factors together accounted for a very associate with several other fungi detected in our sporophore small (<1 %) fraction of root fungal community variation. After surveys, such as species of , , and removing this effect, orchid species identity significantly affected Russula simultaneously ectomycorrhizal with neighbouring the root fungal composition, accounting for about 15 % of total trees (Bidartondo et al. 2004, Bidartondo & Read 2008, community variance in the root. Environmental data, spatial Liebel et al. 2010). position of plots and orchid species identity could account for In spite of the diversity of macromycetes uncovered 31 % of total variance in the fungal assemblage detected in in the investigated sites, no direct evidence of mycorrhizal roots: 19 % was due to the sum of environmental plus spatial associations between above ground fungal species and effects, and 13 % to the effect of orchid species identity. This last analysed orchids was found using molecular below ground fraction was statistically significant at p <0.05. A PCoA ordination methods. None of the 268 macrofungal taxa, growing on the clarifies the relationship between orchid species (Fig. 5), withH. various substrates in the vicinity of the orchids, was found adriaticum, O. bertolonii, and O. pauciflora forming one cluster, to colonize the roots of the 99 orchid samples molecularly and all the other species another. analysed. The eight analysed orchid species were found to associate with a specific range of fungi selected from a DISCUSSION strikingly large variety of potential partners. We propose an intriguing scenario where orchid plants are engaged in a Mediterranean orchid diversity and distribu- complex network of relationships with co-occurring fungi, that tion can be influenced by macromycetes com- is not limited to the restricted number of fungal taxa actually munities associating with their roots. While we have no direct evidence The investigated orchid species shared their meadow and that orchids interact with fungi that were not identified from forest habitats with a wide range of basidiomycetous and orchid roots, the results of our statistical analysis support this ascomycetous fungi, with 268 macrofungal species identified possibility. Statistical analyses indicated a correlation between based on sporophore characters. Several macrofungal macrofungal diversity and orchid community variation, species colonizing the studied plots are known as orchid regardless of the effect of the environmental and spatial mycorrhizal fungi. Among the 63 mycorrhizal basidiomycetous factors of the investigated sites. Surprisingly, macrofungi not

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0.5 O_bertolonii O_provincialis

H_adriaticum O_tridentata

0.0 C_damasonium A_morio

C_longifolia -0.5

O_pauciflora PCoA 2 (21% of the 13% accounted by orchids) -1.0 -1.5 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5

PCoA 1 (53% of the 13% accounted by orchids) Fig. 5. PCoA ordination of the fungal community molecularly detected in orchid roots. Dots are individual root samples while the ellipsoid represent the 95 % confidence limits around each orchid species centroid.

directly detected in the roots explained about 7 % of total orchid seed germination by producing ethylene that is known orchid community variation. This might be explained by to increase germination in vitro (Rasmussen 1995). Finally, in mycorrhizal symbionts varying through stages in the orchid a recently recognized plant-fungus symbiosis between jarrah life-cycle, from seed germination to adulthood (Bidartondo & (Eucalyptus marginata) and the basidiomycete Austroboletus Read 2008, Jacquemyn et al. 2011, Těšitelová et al. 2015), occidentalis, the plant accesses fungus derived enhanced or as well by the different seasons over the collecting period nutrients from the rhizosphere, in the absence of fungal root (Oja et al. 2015, Ercole et al. 2015). The fungal species pool colonization (Kariman et al. 2014). It is tempting to speculate in the soil at a site may therefore play a fundamental role whether fungal mycelia that do not penetrate orchid roots in determining orchid diversity and distribution, providing may have beneficial effects for orchid growth and nutrition, a source of mycorrhizal partners that form associations as recent studies emphasize the role of fungi in soil biogenic with the host plants over time. Second, although factors weathering processes, such as mineral dissolution, increasing governing species composition of fungal communities are nutrient availability for plants in several ecosystems (Finlay et still poorly understood, it is recognized that combinations al. 2009). of fungal species can be either positively or negatively associated (Jumpponen et al. 2004), perhaps indicating Mycorrhizal associations fungus-fungus reciprocal influence that can lead to a certain Molecular results showed that a wide range of basidiomycetes community structure. So, the occurrence of particular fungi, and ascomycetes associated with the analysed orchid such as competitors for the same carbon source or fungal species, rhizoctonioid fungi dominating the orchid root parasites, may influence the presence and abundance of fungal community, a result consistent with microscopical orchid mycobionts in natural environments, thus indirectly observations on both root sections and isolated mycelia. affecting orchid distribution (Rasmussen et al. 2015). An Among rhizoctonioid partners, the orchids primarily inability of some orchid mycorrhizal fungi to compete with associated with members of Ceratobasidiaceae, and to a other fungal species under certain field conditions may be a lesser extent Tulasnellaceae, while Sebacina sequences crucial limiting factor for the establishment of orchid-fungus were only amplified from a few Cephalanthera samples. associations in nature (McCormick et al. 2016, Rock-Blake et Fungi belonging to Ceratobasidiaceae, Tulasnellaceae, and al. 2017). Third, soil fungi that do not usually associate with Sebacinales have been shown to represent the majority orchids may accidentally have a direct influence on orchid of both terrestrial and epiphytic green orchids mycobionts growth and distribution. For instance, they may stimulate (Taylor et al. 2002, McCormick et al. 2004, McCormick

8 IMA FUNGUS Fungal networks and orchid distribution

& Jacquemyn 2014) and their partnership in orchid plant roots. Molecular analysis of the orchid root endophytes ARTICLE mycorrhizas is considered ancestral (Rasmussen 2002, reveals low mycorrhizal specificity and segregation of Dearnaley 2007). Phylogenetic analysis showed that the associated fungal communities in the studied species, which studied orchids associated with several related taxa within may contribute to Mediterranean orchids coexistence in Ceratobasidiaceae and Tulasnellaceae, thus indicating low natural environments. mycorrhizal specificity. Different Rhizoctonia endophytes Further analyses of the whole fungal communities colonized roots of the orchid species both within and between characterizing orchid growing sites in different environments, sites. Nonetheless, co-occurring orchid species showed a based on novel high-throughput sequencing methods, may preference for different Rhizoctonia partners. Segregation be expected to provide important information on the complex of associated fungal communities may contribute to the networks of interactions between orchids and fungi. coexistence of Mediterranean orchid species by reducing competition. Based on the analysis of mycorrhizal fungi in 56 individuals from nine different orchid species co-occurring in ACKNOWLEDGEMENTS 25 × 25 m Mediterranean grassland plots in southern Italy, Jacquemyn et al. (2014) demonstrated that coexisting orchid The experimental work was performed at University of Siena taxa were associated with distinct mycorrhizal communities, as part of the PhD project of L.P., who also acknowledges CAS and suggested niche partitioning as a crucial mechanism 153211KYSB20160029 for supporting his research at Chinese affecting the coexistence. Similarly, Waterman et al. 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(2015) Two widespread 635. green Neottia species (Orchidaceae) show mycorrhizal McCormick MK, Taylor DL, Juhaszova K, Burnett RKJR, Whigham preference for Sebacinales in various habitats and ontogenetic DF, O’Neill JP (2012) Limitations on orchid recruitment: not a stages. Molecular Ecology 24: 1122–1134. simple picture. Molecular Ecology 21: 1511–1523. Vogt KA, Bloomfield J, Ammirati JF, Ammirati SR (1992) Sporocarp McCormick MK, Taylor DL, Whigham DF, Burnett RKJR (2016) production by basidiomycetes, with emphasis on forest Germination patterns in three terrestrial orchids relate to ecosystems. In: The Fungal Community: its organization and role abundance of mycorrhizal fungi. Journal of Ecology 104: 744– in the ecosystem (Carroll GC, Wicklow DT, eds): 563–581. New 754. York: Marcel Dekker. Nurfadilah S, Swarts ND, Dixon KW, Lambers H, Merritt DJ (2013) Waterman RJ, Bidartondo MI, Stofberg J, Combs JK, Gebauer G, Variation in nutrient-acquisition patterns by mycorrhizal fungi et al. 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10 IMA FUNGUS Fungal networks and orchid distribution

BLAST search closest matches of fungal ITS-DNA Supporting Information Table S3. ARTICLE sequences amplified from A. morio and O. provincialis roots Additional Supporting Information may be found in the online version collected in “Monte Cetona” (samples MC1-MC4) and “Monte of this article: Rotondo” (samples MR1-MR24). Sample GenBank accession codes, accession codes for the closest GenBank matches, sequence Table S1. Detailed description of biotic and abiotic factors identity, and overlap of each match are reported. characterizing each investigated plot (p), including list of orchid and macrofungal species, environmental and geographical factors. Fig. S1. Principal Component Analysis (PCA) of environmental Number of sporocarps for each macrofungal taxon is reported. variables (habitat, tree, shrub, herb, and lichen-moss cover %, litter cover %, rock cover %, stone cover %, micro-topography, elevation Table S2. Overview of all root-associated fungi molecularly detected above sea level, slope %, and orientation). in the eight analysed orchid species.

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Fig. S1. Principal Component Analysis (PCA) of environmental variables (habitat, tree, shrub, herb, and lichen-moss cover %, litter cover %, rock cover %, stone cover %, micro-topography, elevation above sea level, slope %, and orientation). Supplementary Table 1 Table S1. Detailed description of biotic and abiotic factors characterizing each investigated plot (p), including list of orchid and macrofungal species, environmental and geographical factors. Number of sporocarps for each macrofungal taxon is reported. Monte Penna Monte Cetona Cornate di Gerfalco Meadow Forest Meadow Forest Meadow Forest FG Species p1 p2 p3 p4 p5 p6 pN pA pB pC pD p1 p2 p3 p4 p5 pA pB pC p1 p2 p3 pN pA pB Sh arvensis Schaeff. 4 4 6 5 1 Sh Agaricus campester L. 13 1 1 3 Sh Agaricus campester L. var. squamulosus (Rea) Pilát 1 1 4 1 Sh Agaricus macrosporus (F.H. Møller & Jul. Schäff.) Pilát 1 Sh Agaricus xanthoderma Genev. var. griseus (A. Pearson) Bon & Cappelli 2 Sh Agrocybe pediades (Fr.) Fayod 1 4 2 1 Sh(Sw) Agrocybe praecox (Pers.) Fayod 1 3 2 Sh Agrocybe pusiola (Fr.) R. Heim 3 Sh Agrocybe vervacti (Fr.) Singer 1 Em Amanita fulva (Schaeff.) Fr. 1 Sw Annulohypoxylon multiforme (Fr.) Y.M. Ju, J.D. Rogers & H.M. Hsieh 1 Sw Auricularia auricula-judae (Fr.) Quél. 1 Sw Auriscalpium vulgare Gray 2 Sw Baeospora myosura (Fr.) Singer 3 33 17 21 1 Sw Bisporella citrina (Batsch) Korf & S.E. Carp. 10 Sc Bolbitius vitellinus (Pers.) Fr. 1 5 Em Boletus edulis Bull. 1 Em Boletus luridus Schaeff. 1 Em Boletus queletii Schulzer 1 4 Sh aestivalis (Bonord.) Demoulin 1 7 3 Sh Bovista pusilla (Batsch : Pers.) Pers. 5 2 2 2 Sh Bovista sp. 1 Sw Calocera cornea (Batsch) Fr. 50 Em rutilus (Schaeff.) O.K. Mill. 23 2 1 Sh Clitocybe candicans(Pers.) P. Kumm. 1 Sh Clitocybe costata Kühner & Romagn. 21 5 1 1 Sh Clitocybe gibba (Pers.) P. Kumm. 1 1 5 7 Sh Clitocybe nebularis (Batsch) P. Kumm. 2 25 Sl Clitocybe nivea Velen. 1 Sh Clitocybe odora (Bull.) P. Kumm. 2 Sh Clitocybe phaeophthalma (Pers.) Kuyper 14 5 Sh Clitocybe rivulosa (Pers.) P. Kumm. 2 1 Sh Clitocybe trullaeformis (Fr.) Quél. 1 Sh Collybia butyracea (Bull.) P. Kumm. 10 221 3 Sh Collybia dryophila (Bull.) P. Kumm. 2 2 2 3 21 P(Sw) Collybia fusipes (Bull.) Quél. 1 Sh Collybia peronata (Bolton) P. Kumm. 1 Collybia sp. 1 1 Collybia sp. 2 2 Sh Conocybe brunneola Kühner & Watling 1 Sh Conocybe cfr. siliginea (Fr.) Kühner 1 Conocybe cfr. sulcatipes (Peck) Kühner 5 Sh Conocybe cyanopus (G.F. Atk.) Kühner 1 Sh Conocybe moseri Watling 1 1 Sh Conocybe pseudopilosella Kühner & Watling 1 Sc Conocybe rickenii (Jul. Schäff.) Kühner 1 Sh Conocybe rubiginosa Watling 1 5 Sh Conocybe semiglobata Kühner & Watling 5 1 2 2 Sh Conocybe sienophylla (Berk. & Broome) Singer 2 Conocybe sp. 1 1 Conocybe sp. 2 1 Sh Conocybe tenera (Schaeff.) Fayod 3 9 2 2 2 3 4 Sc Coprinus cinereus (Schaeff.) Gray 1 Sh Coprinus impatiens (Fr.) Quél. 6 Sh Coprinus leiocephalus P.D. Orton 1 Sc Coprinus niveus (Pers.) Fr. 1 Sw Coprinus xanthothrix Romagn. 1 Em Cortinarius anomalus (Fr.) Fr. 1 Em Cortinarius cfr. pulchripes J. Favre 6 Em Cortinarius duracinus Fr. 5 Em Cortinarius infractus (Pers.) Fr. 15 Em Cortinarius sp. 1 1 Em Cortinarius sp. 2 1 Em Cortinarius umbrinolens P.D. Orton 4 Em Cortinarius uraceus Fr. 1 Sw Crepidotus cesatii (Rabenh.) Sacc. 22 41 Sw Crepidotus lundellii Pilát 31 10 Sl(Sw) Crinipellis cfr. procera G. Stev. 1 Sl(Sw) Crinipellis scabellus (Alb. & Schwein.) Murril 1 1 3 20 31 7 10 Sw Crucibulum crucibuliforme (Scop.) V.S. White 3 3 Sw Cyathus striatus (Huds. : Pers.) Willd. 1 Sh Cystolepiota cystophora (Malençon) Bon 1 1 Sw Dacrymyces tortus (Willd.) Fr. 20 Sw Dacrymyces variisporus McNabb 50 Sw Diatrype disciformis (Hoffm.) Fr. 1 11 Sh cfr. velenovskyi Noordel. 2 Sh Entoloma juncinum (Kühner & Romagn.) Noordel. 1 Sh(Em?) Entoloma lividoalbum (Kühner & Romagn.) Kubicka 1 Sh(Em?) Entoloma nitidum Quél. 6 Sh Entoloma occultopigmentatum Arnolds & Noordel. 1 Sh Entoloma phaeocyathus Noordel. 3 Sh Entoloma pseudoturci Noordel. 2 Sh(Em?) Entoloma rhodopolium (Fr.) P. Kumm. f. nidorosum (Fr.) Noordel. 2 Sh Entoloma sericellum (Fr.) P. Kumm. 1 Sh Entoloma solstiziale (Fr.) Noordel. 1 Entoloma sp. 1 1 Entoloma sp. 2 1 Entoloma sp. 3 1 Entoloma sp. 4 1 Sh Entoloma tenellum (J. Favre) Noordel. 3 Sh Entoloma triste (Velen.) Noordel. 1 P Eutypa flavovirens (Pers.) Tul. & C. Tul. 1 Sw Exidia glandulosa (Bull.) Fr. 10 Sl Flammulaster carpophilus (Fr.) Earle 4 Sw Galerina marginata (Batsch) Kühner 1 Am Galerina vittaeformis (Fr.) Singer 36 Sh Geastrum fimbriatum (Fr.) 1 Sh Geastrum minimum Schwein. 7 141 1 12 2 11 P(Sw) Gymnopilus junonius (Fr.) P.D. Orton 2 Em (Bull.) Quél. 2 Em Hebeloma longicaudum (Pers.) P. Kumm. 3 1 3 Em Hebeloma ochroalbidum Bohus 1 Em (Fr.) Gillet 2 3 6 1 16 Em Hebeloma sordescens Vesterh. 1 1 Em Hebeloma sp. 1 1 Em Hebeloma sp. 2 1 Em Hebeloma sp. 3 3 P Hexagonia nitida Durieu & Mont. 1 Sh Hygrocybe cfr. acutoconica (Clem.) Singer 1 Sh Hygrocybe conica (Schaeff.) P. Kumm. 1 Sh Hygrocybe spadicea (Scop.) P. Karst. 1 Sh Hygrophoropsis aurantiaca (Wulfen) Maire 3 Em Hygrophorus arbustivus (Fr.) Fr. 2 Em Hygrophorus cfr. fagi Becker & Bon 2 Em Hygrophorus chrysodon (Batsch) Fr. 1 Em Hygrophorus eburneus (Bull.) Fr. 3 3 7 1 1 Em Hygrophorus lindtneri M.M. Moser 1 1 Em Hygrophorus penarius Fr. 1 Em Hygrophorus personii Arnolds 2 Sw Hymenoscyphus calyculus (Sowerby) W. Phillips 7 1 Sw Hymenoscyphus serotinus (Pers.) W. Phillips 2 Sw Hyphoderma capitatum J. Erikss. & Å. Strid 1 Sw Hyphoderma cfr. pallidum (Bres.) Donk 1 1 Em Inocybe flocculosaSacc. 2 Em Inocybe furfurea Kühner 1 Em Inocybe fuscidula Velen. 1 16 1 Em (Fr.) P. Kumm. 15 80 Em Inocybe geophylla (Fr.) P. Kumm. var. lilacina (Peck) Gillet 3 20 Em Inocybe pelargonium Kühner 7 Em Inocybe phaeodisca Kühner 50 Em Inocybe posterula (Britzelm.) Sacc. 1 Em Inocybe pusio P. Karst. 1 1 2 Em Inocybe rimosa (Bull.) P. Kumm. 1 Em Inocybe sp. 1 Em Inocybe splendens R. Heim 4 102 2 Em Inocybe whitei (Berk. & Broome) Sacc. 10 Em Laccaria amethystina Cooke 2 Em Laccaria laccata (Scop.) Cooke 10 2 30 1 Sw Lachnellula sp. 10 Sw Laeticorticium polygonioides (P. Karst.) Donk 50 Sh Lepiota castanea Quél. 2 Sh Lepiota clypeolaria (Bull.) P. Kumm. 2 6 2 1 2 1 Sh Lepiota cristata (Bolton) P. Kumm. 3 1 Sh Lepiota sp. 1 Sh Lepiota subincarnata J.E. Lange 2 Sh Lepiota ventriosospora D.A. Reid 1 Sh Lepista nuda (Bull.) Cooke 7 1 1 6 Sh Lepista rickenii Singer 2 Sh Leucopaxillus sp. 1 Sh cfr. foetidum Bonord. 1 Sh Lycoperdon lividum Pers. 1 Sh Lycoperdon perlatum Pers. : Pers. 6 1 5 7 Lycoperdon sp. 1 Sh cucumis (Pers.) Joss. 1 Sh mastoidea (Fr.) Singer 7 5 5 3 1 1 Sh (Scop.) Singer 2 1 4 2 Sh Macrotyphula juncea (A. & S.) Berthier 300 Sw ramealis (Bull.) Singer 50 Sh(Sl) Marasmius androsaceus (L.) Fr. 1 25 Sl Marasmius bulliardii Quél. 13 103 20 Sl Marasmius epiphylloides (Rea) Sacc. & Trotter 10 45 170 210 2 Sl(Sw) Marasmius epiphyllus (Pers.) Fr. 50 4 600 34 Sl(P?) (Bolton) Fr. 5 12 Sl Marasmius quercophilus Pouzar 85 10 60 20 10 10 350 Sw (Scop.) Fr. 4 10 35 47 Sl Marasmius setosus (Sowerby) Noordel. 1 Sl(Sw) Marasmius torquescens Quél. 10 Sh grammopodia (Bull.) Pat. 5 Sh Melanoleuca kuehneri Bon 1 Sh Melanoleuca melaleuca (Pers.) Murril 2 1 10 Sh Melanoleuca subpulverulenta (Pers.) Métrod 1 Sl Micromphale brassicolens (Romagn.) P.D. Orton 2 1 6 10 1 Sh Mutinus caninus (Huds. : Pers.) Fr. 1 Sh(Sw) Mycena abramsii (Murril) Murril 1 2 5 1 2 1 2 9 1 Sw Mycena adscendens (Lasch) Maas Geest 10 10 Sh Mycena aetites (Fr.) Quél. 1 3 5 3 1 14 1 1 21 3 9 2 Sh Mycena amicta (Fr.) Quél. 1 9 Sw(Sh) Mycena arcangeliana Bres. 23 1 1 15 1 Sh Mycena capillaripes Peck 6 Sh Mycena capillaris (Schumach.) P. Kumm. 200 300 100 Sh Mycena clavicularis (Fr.) Gillet 2 Sw Mycena corynephora Maas Geest. 2 Sh Mycena epipterygia (Scop.) Gray 1 5 Sh(Sw) Mycena filopes (Bull.) P. Kumm. 1 Sl(Sh) Mycena flavoalba (Fr.) Quél. 6 3 2 4 1 2 1 10 4 30 Sw(Sh) Mycena flos-nivium Kühner 30 6 1 Sw Mycena galericulata (Scop.) Gray 2 5 3 Sh Mycena galopus (Pers.) P. Kumm. 3 3 50 Sl Mycena graminicola Robich 1 Sw Mycena inclinata (Fr.) Quél. 2 Sw Mycena juniperina Aronsen 50 30 5 Sh(Sw) (Pers.) Gillet 12 3 8 3 1 1 4 Sw Mycena maculata P. Karst. 3 1 1 Sw Mycena meliigena (Berk. & Cooke) Sacc. 3 10 Sh (Fr.) P. Kumm. 10 Sh(Sw) Mycena mirata (Peck) Sacc. 4 1 Sh Mycena olivaceomarginata (Massee) Massee 1 2 6 10 50 Sh Mycena pelianthina (Fr.) Quél. 1 1 Sw Mycena polygramma (Bull.) Gray 3 Sh Mycena pseudopicta (J.E. Lange) Kühner 1 Sh Mycena pura (Pers.) P. Kumm. 8 12 8 4 17 2 5 7 Sh Mycena pura (Pers.) P. Kumm. f. alba (Gillet) Kühner 1 1 2 Sw Mycena renati Quél. 5 4 Sh Mycena rosea Gramberg 1 1 11 5 3 5 8 Mycena sp. 1 3 Mycena sp. 2 4 Sw Mycena vitilis (Fr.) Quél. 10 14 8 4 3 1 1 4 5 5 1 Sl Mycena xantholeuca Kühner 1 Sw Nemania serpens (Pers.) Gray 2 Omphalina sp. 1 4 5 Sh Panaeolus acuminatus (Schaeff.) Quél. 9 1 2 1 6 Sh(Sc) Panaeolus fimicola (Fr.) Quél. 1 1 1 2 Sc Panaeolus sphinctrinus (Fr.) Quél. 1 8 Em Paxillus involutus (Batsch) Fr. 1 14 Sw Peniophora quercina (Pers.) Cooke 5 Sw Phaeomarasmius erinaceus (Pers.) Schaeff. ex Romagn. 1 P Phellinus torulosus (Pers.) Bourdot & Galzin 2 Sw Phlebia radiata Fr. 1 Sw Polyporus arcularius (Batsch.) Fr. 1 Sl Psathyrella cfr. sacchariolens Enderle 1 Sh Psathyrella fusca (Schumach.) A. Pearson 1 Sh Psathyrella murcida (Fr.) Kits van Wav. 1 Sh Psathyrella obtusata (Pers.) A.H. Sm. 1 Sh(Sw) Psathyrella prona (Fr.) Gillet 1 Sh(Sw) Psathyrella prona (Fr.) Gillet var. utriformis Kits van Wav. 2 Sh Pseudoclitocybe cyathiformis (Bull.) Singer 5 3 Sw(Sh) Psilocybe aeruginosa (M.A. Curtis) Noordel. 2 Sh Psilocybe cfr. pratensis P.D. Orton 1 Sc Psilocybe coprophila(Bull.) Quél. 1 2 Sc Psilocybe semiglobata (Batsch) Noordel. 3 5 7 2 3 1 Sw Psilocybe sublateritia (Fr.) Rode 3 Sh(Sw) applicatus (Batsch) Gray 30 1 Em Russula atropurpurea (Krombh.) Britzelm. 3 Em Russula cavipes Britzelm. 4 Em Russula cfr. sardonia Fr. 1 Em Russula cfr. sublevispora (Romagn.) Romagn. 1 Em Russula cyanoxantha (Schaeff.) Fr. 1 Em Russula decipiens (Singer) Svrçek 3 Em Russula fragilis (Pers.) Fr. 5 8 Em Russula lilacea Quél. 2 2 Em Russula turci Bres. 1 Em Russula virescens (Schaeff.) Fr. 1 Sw Rutstroemia cfr. firma (Pers. : Fr.) P. Karst. 2 Sl Rutstroemia echinophila (Bull.) Höhn. 5 Sw Schizopora paradoxa (Schrad.) Donk 1 Sw Steccherinum cfr. ochraceum (Pers.) Gray 2 Sw Stereum hirsutum (Willd.) Gray 6 Sw Stereum ochraceoflavum (Schwein.) Fr. 4 12 Em Suillus collinitus (Fr.) Kuntze 2 1 Em Suillus granulatus (L.) Roussel 114 17 21 1 Sw Trametes versicolor (L.) Lloyd 21 Sw Tremella mesenterica Retz. 10 Sw Trichaptum abietinum (Dicks.) Ryvarden 10 30 Sw Trichaptum fuscoviolaceum (Bull.) Quél. 1 1 Em Tricholoma album (Schaeff.) P. Kumm. 1 25 12 2 Em Tricholoma atrosquamosum (Chevall.) Sacc. 4 17 2 3 Em Tricholoma myomyces (Pers.) J.E. Lange 2 Em Tricholoma orirubens Quél. 2 Em Tricholoma stiparophyllum (S. Lundell) P. Karst. 1 Em Tricholoma sulphureum (Bull.) P. Kumm. 8 Em (Schaeff.) P. Kumm. 2 Sh Tulostoma brumale Pers. : Pers. 1 Sh Tulostoma squamosum J.F. Gmel. : Pers. 1 Sw Volvariella hypopithys (Fr.) Shaffer 1 Sw Vuilleminia comedens (Nees) Maire 4 Sw(P?) Xerula radicata (Rehlan) Dörfelt 1 2 1 Sw Xylaria hypoxylon (L.) Grev. 20 50 40 110 50 Sw Xylaria longipes Nitschke 1 ELEVATION ABOVE SEA LEVEL 936 937 946 948 930 932 949 934 941 942 874 1047 1033 1027 1021 1006 1026 1024 1017 1032 998 969 1012 793 755 ORIENTATION S E_SE N_NE W SW S_SW S N_NW S_SE N NW SW SW W_SW W_SW SW N NW NW S S_SW S E_NE S S SLOPE % 10 20 10 3 30 10 3 4 4 3 3 10 15 20 20 15 5 5 5 20 25 25 10 25 30 ROCK COVER % 3 20 5 7 3 2 5 0 0 0 0 20 25 5 15 20 0 0 0 35 35 15 5 10 5 STONE COVER % 5 10 3 3 2 6 5 0 3 3 2 25 15 15 25 15 0 0 0 10 20 45 15 30 20 LITTER COVER % 0 0 5 0 0 0 0 95 95 95 95 0 0 0 0 0 95 95 95 15 15 20 3 65 80 MICRO-TOPOGRAPHY 1 1 2 2 2 3 3 3 3 3 2 2 2 3 3 3 2 3 3 1 1 2 2 1 2 TOTAL VEGETATION COVER % 95 80 95 90 95 95 90 100 95 100 100 60 70 85 70 70 100 100 100 65 70 70 85 90 100 TREE COVER % 10 10 10 0 0 3 0 90 95 95 100 5 5 5 10 0,1 90 90 75 25 30 40 20 80 90 SHRUB COVER % 8 5 30 20 10 5 0 15 90 15 5 0,1 0,1 1 1 0 10 15 30 10 15 10 3 40 30 HERB COVER % 90 75 65 80 85 90 90 70 0 5 3 50 65 80 60 65 10 5 40 60 60 60 85 15 40 LICHEN-MOSS COVER % 8 8 8 8 1 5 8 5 0 0 0 10 10 10 10 10 1 0 0 8 8 8 8 5 2 m EAST 717624 717636 717619 717560 717519 717598 717590 717589 717862 717967 717573 734929 734950 734926 734917 734928 734428 734418 734403 659256 659440 659610 659373 660303 660468 m NORTH 4735638 4735665 4735718 4735697 4735663 4735629 4735685 4735473 4735879 4735970 4737421 4756615 4756498 4756450 4756417 4756373 4757855 4757860 4757879 4779872 4779729 4779621 4779819 4779166 4779076 ZONE 32T 32T 32T 32T 32T 32T 32T 32T 32T 32T 32T 32T 32T 32T 32T 32T 32T 32T 32T 32T 32T 32T 32T 32T 32T Cephalanthera damasonium (Miller) Druce 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 1 1 0 0 0 0 0 0 Cephalanthera longifolia (Hudson) Fritsch 0 0 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1 Himantoglossum hircinum L. Spreng. subsp. adriaticum (H.Baumann) H. Sund 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 1 1 0 0 0 Ophrys bertolonii Mor. 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 0 0 0 0 0 0 0 0 0 Orchis morio L. 1 1 1 1 1 1 1 0 0 0 0 0 0 1 1 0 0 0 0 0 0 1 0 0 0 Orchis pauciflora Ten. 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 0 0 0 1 1 1 0 0 0 Orchis provincialis Balb. 1 1 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Orchis tridentata Scop. 1 1 1 0 0 1 0 0 0 0 0 1 1 1 0 1 0 0 0 0 0 0 0 0 0 Supplementary Table 2 Table S2. Overview of all root-associated fungi molecularly detected in the eight analysed orchid species. Orchid species Sample Site Plot Microdochium Schizophyllum commune Fungal endophyte (from Paris polyphylla EF495231) Leptodontidium Scolecobasidium Alternaria Tulasnellaceae Cenococcum geophilum Cryptococcus carnescens Sebacinaceae Hymenogastraceae Ceratobasidiaceae Exophiala Tetracladium Tomentella Neonectria radicicola Pezizomycetes Bjerkandera adusta Phlebia acerina Davidiella macrospora Fusarium/Gibberella Phoma Leohumicola Stilbella Hypocreales Cadophora Candida Terfezia Anacamptis morio MR1 Monte Rotondo 5 x x Anacamptis morio MR2 Monte Rotondo 2 x Anacamptis morio MR3 Monte Rotondo 1 x x Anacamptis morio MR4 Monte Rotondo 3 x x Anacamptis morio MR5 Monte Rotondo N x x x Anacamptis morio MR6 Monte Rotondo N x x Anacamptis morio MR7 Monte Rotondo 4 x x Anacamptis morio MR8 Monte Rotondo 4 x Anacamptis morio MR9 Monte Rotondo 4 x Anacamptis morio MR10 Monte Rotondo 4 x Anacamptis morio MC1 Monte Cetona 4 x x Anacamptis morio MC2 Monte Cetona 3 x x Anacamptis morio MC3 Monte Cetona 3 x Anacamptis morio MC4 Monte Cetona 3 x Orchis provincialis MR11 Monte Rotondo 2 x Orchis provincialis MR12 Monte Rotondo 5 x x Orchis provincialis MR13 Monte Rotondo 6 x x Orchis provincialis MR14 Monte Rotondo 1 x Orchis provincialis MR15 Monte Rotondo 1 x Orchis provincialis MR16 Monte Rotondo 1 x Orchis provincialis MR17 Monte Rotondo 1 x Orchis provincialis MR18 Monte Rotondo 1 x x Orchis provincialis MR19 Monte Rotondo 1 x Orchis provincialis MR20 Monte Rotondo 1 x Orchis provincialis MR21 Monte Rotondo 1 x Orchis provincialis MR22 Monte Rotondo 1 x Orchis provincialis MR23 Monte Rotondo 1 x Orchis provincialis MR24 Monte Rotondo 1 x Orchis pauciflora CG1 Cornate di Gerfalco 1 x Orchis pauciflora CG2 Cornate di Gerfalco 2 x x Orchis pauciflora CG3 Cornate di Gerfalco 3 x x x Orchis pauciflora CG4 Cornate di Gerfalco 3 x Orchis pauciflora CG5 Cornate di Gerfalco 3 x x x Orchis pauciflora CG6 Cornate di Gerfalco 3 x x x Orchis pauciflora MC1 Monte Cetona 4 x x x Orchis pauciflora MC2 Monte Cetona 1 x Orchis pauciflora MC3 Monte Cetona 5 x x x Orchis pauciflora MC4 Monte Cetona 5 x x x Orchis pauciflora MC5 Monte Cetona 3 x x Orchis pauciflora MC6 Monte Cetona 2 x x x Orchis tridentata MC1 Monte Cetona 3 x Orchis tridentata MC2 Monte Cetona 3 x x Orchis tridentata MC3 Monte Cetona 5 x x Orchis tridentata MC4 Monte Cetona 2 x

Orchis tridentata MC5 Monte Cetona 2 x x Orchis tridentata MR1 Monte Rotondo 3 x x Orchis tridentata MR2 Monte Rotondo 1 x Orchis tridentata MR3 Monte Rotondo 1 x Orchis tridentata MR4 Monte Rotondo 6 x Orchis tridentata MR5 Monte Rotondo 2 x Ophrys bertolonii 1 Monte Cetona 4 x Ophrys bertolonii 2 Monte Cetona 3 x Ophrys bertolonii 3 Monte Cetona 5 x Ophrys bertolonii 4 Monte Cetona 5 x Ophrys bertolonii 5 Monte Cetona 2 x x x Ophrys bertolonii 6 Monte Cetona 2 x x Ophrys bertolonii 7 Monte Cetona 1 x Ophrys bertolonii 8 Monte Cetona 1 x Ophrys bertolonii 9 Monte Cetona 2 x Ophrys bertolonii 10 Monte Cetona 2 x Ophrys bertolonii 11 Monte Cetona 2 x Ophrys bertolonii 12 Monte Cetona 2 x x Cephalanthera damasonium MC1 Monte Cetona B x x x

Cephalanthera damasonium MC2 Monte Cetona B x Cephalanthera damasonium MC3 Monte Cetona A x Cephalanthera damasonium MC4 Monte Cetona A x x Cephalanthera damasonium MR1 Monte Rotondo D x Cephalanthera damasonium MR2 Monte Rotondo D x x Cephalanthera damasonium MR3 Monte Rotondo D x x Cephalanthera damasonium MR4 Monte Rotondo D x Cephalanthera longifolia MR5 Monte Rotondo D x x Cephalanthera longifolia MR6 Monte Rotondo D x Cephalanthera longifolia MR7 Monte Rotondo C x Cephalanthera longifolia MR8 Monte Rotondo C x Cephalanthera longifolia CG1 Cornate di Gerfalco A x Cephalanthera longifolia CG2 Cornate di Gerfalco B x Cephalanthera longifolia CG3 Cornate di Gerfalco B x Cephalanthera longifolia CG4 Cornate di Gerfalco B x

Himantoglossum adriaticum CG1 Cornate di Gerfalco 3 x x Himantoglossum adriaticum CG2 Cornate di Gerfalco 2 x x Himantoglossum adriaticum CG3 Cornate di Gerfalco 1 x Himantoglossum adriaticum CG4 Cornate di Gerfalco 1 x x Himantoglossum adriaticum CG5 Cornate di Gerfalco 3 x

Himantoglossum adriaticum CG6 Cornate di Gerfalco 2 x Himantoglossum adriaticum CG7 Cornate di Gerfalco 3 x

Himantoglossum adriaticum MC1 Monte Cetona 1 x Himantoglossum adriaticum MC2 Monte Cetona 1 x Himantoglossum adriaticum MC3 Monte Cetona 1 x Supplementary Table 3 Table S3. BLAST search closest matches of fungal ITS-DNA sequences amplified from A. morio and O. provincialis roots collected in “Monte Cetona” (samples MC1-MC4) and “Monte Rotondo” (samples MR1-MR24). Sample GenBank accession codes, accession codes for the closest GenBank matches, sequence identity, and overlap of each match are reported. Orchid species Sample Clone Plot GenBank Best BLAST match(es) Accession Overlap % match code code lenght A. morio MR1 a 5 KX461964 Cadophora luteo-olivacea DQ404349 304 95% c 5 KX461965 Euascomycete (from oat root) AJ246144 944 93% Leptodontidium orchidicola AF486133 919 92% MR2 b 2 KX461966 Ceratobasidium sp. EU218894 378 85% MR3 a 1 KX461967 Uncultured mycorrhizal ascomycete (from Epipactis helleborine ) AY634167 971 98% Leptodontidium orchidicola (from Platanthera hyperborea ) AF486133 933 96% b 1 KX461968 Fusarium oxysporum (from galled roots) AF443071 908 99% c 1 KX461969 Uncultured fungus (from Pterostylis nutans) EF090490 655 94% Leptodontidium orchidicola (from Platanthera hyperborea ) AF486133 650 94% MR4 b 3 KX461970 Fungal endophyte EF495231 1041 99% Exophiala salmonis AF050274 946 95% d 3 KX461971 Ceratobasidium sp. EU218894 1059 97% MR5 a N KX461972 Helotiales (from ectomycorrhizal root tip) EU326174 872 97% Tricladium angulatum AY204610 708 91% d N KX461973 Leohumicola minima AY706329 688 92% e N KX461974 Uncultured ectomycorrhizal fungus (from Betula maximowicziana root tip) AB219857 774 94% Tetracladium furcatum AF411026 747 94% MR6 a N KX461975 Tetracladium furcatum AF411026 354 95% c N KX461976 Uncultured ascomycete (from apple seedling roots) EU003001 859 93% Leptodontidium orchidicola (from Platanthera hyperborea ) AF486133 854 93% MR7 b 4 KX461977 Uncultured ectomycorrhizal fungus (from roots) EU816631 423 90% Tetracladium furcatum AF411026 412 89% d 4 KX461978 Fusarium sp. EU091029 814 97% MR8 Isolated 4 KX461979 Ceratobasidiaceae (from ) AY634127 1092 96% Ceratobasidium sp. EU218894 1083 96% MR9 c 4 KX461980 Ceratobasidium sp. EU218894 1094 96% MR10 Isolated 4 KX461981 Ceratobasidiaceae (from ectomycorrhiza) AY634127 1101 96% Ceratobasidium sp. EU218894 1099 96% MC1 a 4 KX461982 Fungal endophyte EF495231 895 99% e 4 KX461983 Ceratobasidium sp. EU218894 838 96% MC2 a 3 KX461984 Fungal endophyte EF495231 944 99% b 3 KX461985 Ceratobasidium sp. EU218894 679 91% MC3 a 3 KX461986 Ceratobasidium sp. EU218894 419 89% MC4 Isolated 3 KX461987 Schizophyllum commune FJ478109 1146 99% O. provincialis MR11 Isolated 2 KX461988 Ceratobasidiaceae (from ectomycorrhiza) AY634127 1101 96% Ceratobasidium sp. EU218894 1081 96% MR12 a 5 KX461989 Fusarium oxysporum f. sp. vasinfectum AY259214 434 90% d 5 KX461990 Leohumicola minima AY706329 907 98% MR13 a 6 KX461991 Tetracladium furcatum EU883432 994 99% b 6 KX461992 Alternaria longissima (from Linaria hegelmaieri) DQ865104 898 99% MR14 c 1 KX461993 Ceratobasidium sp. EU218894 1064 96% MR15 b 1 KX461994 Thanatephorus ochraceus EU218892 839 89 e 1 KX461995 Ceratobasidiaceae (from Epipactis palustris roots) AY634127 1068 95 MR16 b 1 KX461996 Ceratobasidiaceae (from Dactylorhiza majalis roots) AY634126 1012 95 Ceratobasidium sp. EU218894 1000 95 MR17 Isolated 1 KX461997 Ceratobasidiaceae (from ectomycorrhiza) AY634127 1085 96% MR18 a 1 KX461998 Microdochium sp. AJ279477 841 94% b 1 KX461999 Tulasnellaceae DQ925633 1033 95% MR19 a 1 KX462000 Dark septate endophyte AF168783 1035 97% Leptodontidium orchidicola (from Platanthera hyperborea ) AF486133 1029 97% MR20 a 1 KX462001 Thanatephorus ochraceus EU218892 913 91% MR21 a 1 KX462002 Tulasnellaceae DQ925633 1059 96% MR22 a 1 KX462003 Tulasnellaceae DQ925633 1053 96% c 1 KX462004 Tulasnellaceae (from Cypripedium montanum ) DQ925624 907 93% MR23 a 1 KX462005 Thanatephorus ochraceus EU218892 1016 92% d 1 KX462006 Thanatephorus sp. (from vanilla) DQ834419 747 95% MR24 Isolated 1 KX462007 Ceratobasidium sp. (from Serapias vomeracea ) JF912478 872 93%