Mycorrhiza (2013) 23:11–19 DOI 10.1007/s00572-012-0445-z

ORIGINAL PAPER

Ectomycorrhizal fungi of exotic pine plantations in relation to native host trees in Iran: evidence of host range expansion by local symbionts to distantly related host taxa Mohammad Bahram & Urmas Kõljalg & Petr Kohout & Shahab Mirshahvaladi & Leho Tedersoo

Received: 19 March 2012 / Accepted: 2 May 2012 / Published online: 17 May 2012 # Springer-Verlag 2012

Abstract Introduction of exotic plants change soil microbial communities which may have detrimental ecological consequences for ecosystems. In this study, we examined the community structure and species richness of ectomycorrhizal (EcM) fungi associated with exotic pine plantations in relation to adjacent native ectomycorrhizal trees in Iran to elucidate the symbiont exchange between distantly related hosts, i.e. Fagales (Fagaceae and Betulaceae) and Pinaceae. The combination of morphological and molecular identification approaches revealed that 84.6 % of species with more than one occurrence (at least once on pines) were shared with native trees and only 5.9 % were found exclusively on pine root tips. The community diversity of ectomycorrhizal

fungi in the pine plantations adjacent to native EcM trees was comparable to their adjacent native trees, but the isolated plantations hosted relatively a species-poor community. Specific mycobionts of conifers were dominant in the isolated plantation while rarely found in the plantations adjacent to native EcM trees. These data demonstrate the importance of habitat isolation and dispersal limitation of EcM fungi in their potential of host range expansion. The great number of shared and possibly compatible symbiotic species between exotic Pinaceae and local Fagales (Fagaceae and Betulaceae) may reflect their evolutionary adaptations and/or ancestral compatibility with one another. Keywords Symbiotic fungi . Hyrcanian forests . Invasive species . Host specificity . Habitat fragmentation . Island biogeography . Dispersal ability . Pinus sylvestris

Electronic supplementary material The online version of this article (doi:10.1007/s00572-012-0445-z) contains supplementary material, which is available to authorized users. M. Bahram (*) : U. Kõljalg : P. Kohout : L. Tedersoo Institute of Ecology and Earth Sciences, Tartu University, 40 Lai, 51005 Tartu, Estonia e-mail: [email protected] U. Kõljalg : L. Tedersoo Natural History Museum, Tartu University, 40 Lai, 51005 Tartu, Estonia P. Kohout Department of Mycorrhizal Symbioses, Institute of Botany, ASCR, 25243 Průhonice, Czech Republic S. Mirshahvaladi Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran

Introduction Exotic trees have been widely used in forestry and plantations because of their short generation, fast growth and highquality wood (Richardson 1998). There are usually fewer specific pathogens for the trees in a new habitat (Mitchell et al. 2006), which adds to the popularity of exotic trees in forestry practices in a wide range of ecosystems. Nevertheless, introduction of exotic plants may have detrimental ecological consequences to ecosystem functioning. For example, they can suppress the growth of native plants through destroying their mutualistic associations (Stinson et al. 2006) or modifying the soil properties (Wardle 2002; van der Putten et al. 2007). The detrimental effects of exotic plants could be more dramatic if they become invasive (Keane and Crawley 2002) and form associations with local

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symbionts, such as pollinators and mycorrhizal fungi (Pringle et al. 2009). One of the main belowground components of many forest ecosystems are ectomycorrhizal (EcM) fungi that play essential roles in nutrient cycling and function of ecosystems. Some EcM host trees (i.e. Eucalyptus and Pinus) have been widely used in plantations throughout the world (Richardson 1998). The introduction of EcM trees could potentially influence EcM fungi of new habitats in several ways. In addition to the effects of introduced trees on litter quality and quantity (Wardle 2002; Wolfe and Klironomos 2005), which may in turn affect EcM fungal communities (Toljander et al. 2006), co-introduction of exotic fungal species (Mikola 1969; Tedersoo et al. 2007) into the communities can also change the local fungal community structure. Introduced trees and EcM fungi may also extend their mutualistic partners in new habitats (Jairus et al. 2011). Previous studies have demonstrated that plants host relatively species-poor mycorrhizal assemblages outside of their native ranges (Tedersoo et al. 2007; Nuñez et al. 2009; Dickie et al. 2010; Walbert et al. 2010) because of dispersal limitation and co-introduction of few EcM fungal species, and thus limited access to compatible mycobionts. Nevertheless, there is evidence that the potential of compatibility of introduced trees with local mycobionts is higher if the new habitat has similar or closely related host taxa as their original habitat (Kohout et al. 2011; Trocha et al. 2012). Pine trees (Pinus spp.) are among the most popular commercial trees in plantations worldwide (Richardson 1998). Pines are obligatory EcM trees, and thus their growth, establishment and survival depend on EcM fungi. They produce acidic litter with low nutrient concentrations and slow decomposition (Berg and McClaugherty 2008), which may result in specialisation of their belowground microbial associations (Wardle 2002). In fact, most of the EcM fungal species with high host specificity associate with pines (Molina et al. 1992). For instance, Rhizopogon and Suillus spp. are the main pine-specific mycobionts, which depend on animal vectors (Ashkannejhad and Horton 2006) and wind for dispersal, respectively. These features indicate that pine species may need suitable mycobionts for establishment in new areas, the lack of which could lead to the failure of pine plantations in exotic ranges (Mikola 1969; Read 1998). Although the Pinaceae family is native to the Northern Hemisphere, expanding up to the southernmost Caucasian forests, it has no native range in Iran (see Akhani et al. 2010). The Hyrcanian (Caspian) forests are located in the south eastern range of the Caucasian-mixed forests. Lack of Pinaceae is a main feature of the Hyrcanian forests, in contrast to most of northern temperate forests. As ornamental trees, various pine species have been planted in Iran for over half a century. Pre-grown pine seedlings are used for plantations (B Kanjani-Shiraz personal communication).

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Some plantations have been located in the Hyrcanian forests, where native forests have been clear-cut and replaced by exotic trees. As such, these forests provide a unique opportunity to better understand the potential of EcM host range extension in new environments. Our study aimed to characterize EcM fungal community associated with exotic pine plantations in relation to adjacent native Fagales (Fagaceae and Betulaceae) trees in the Hyrcanian forests of northern Iran. We compared EcM fungal community and diversity in the plantations adjacent to native EcM vegetations (non-isolated) with the native EcM trees. In addition, we studied pine plantations that were apart from native trees (isolated) to elucidate the potential spread of EcM fungi from native to exotic trees and vice versa (Wolfe and Pringle 2012). Since most EcM fungal species can associate with intermediate to broad range of host trees (Molina et al. 1992), remnant native species after clear-cutting or species dispersing from nearby EcM trees can colonize the new introduced hosts if they are compatible. The possible common evolutionary origin of EcM symbiosis in Fagales (Fagaceae and Betulaceae) and Pinaceae (Brundrett 2002) in addition to the overlap of their native geographical distribution ranges in the Northern Hemisphere may have resulted in evolutionary adaptation and thus compatibility of their mycobionts (Molina et al. 1992; Cairney 2000). Based on this assumption, we predicted a high similarity between EcM fungal community composition associated with pine plantations and native EcM vegetation of northern Iran. Following the theory of island biogeography (MacArthur and Wilson 1967), which suggests that the degree of isolation of an area from species source affects migration and extinction rate of species, we further hypothesized that increasing isolation from native EcM vegetation affects the community and results in lower diversity of EcM fungi in exotic plantations.

Material and methods Sampling area and design In autumn 2009, sampling was performed in three plantations dominated by the introduced Scots pine (Pinus sylvestris L.) in the Hyrcanian forests of northern Iran. This region has a temperate climate with mild winters (mean temperature >0°C) to warm summers and nearly equal amount of precipitation throughout the year. The forests are dominated by the temperate deciduous trees such as Fagus orientalis Lipsky, Carpinus betulus L. and Quercus castaneifolia C.A.Mey. covering the mountain slopes of the southern Caspian sea. The exotic pine plantations have been established on clear-cut areas of native EcM trees more than 25 years ago (Table 1). The exact sources of planted tree

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Table 1 The dominant ectomycorrhizal host species, age, size and geography of the plantations

Native species Sangdeh

Exotic species

Size (ha) Approximate age Geographic coordinates

Fagus orientalis Pinus sylvestris 60 Quercus castaneifolia

Carpinus betulus Alasht Fagus orientalis Carpinus betulus Kelardasht Carpinus betulus Shafarud

–

Tehran Malayer

– –

seedlings in each of the Scots pine plantations are unknown, but seeds—or possibly seedlings—have been mostly imported from Spain, Armenia, Greece and Yugoslavia (B Kanjani-Shiraz personal communication). Several non-EcM woody plants such as Acer velutinum Boiss., Buxus hyrcana Pojark., Ulmus glabra Huds. and Crataegus sp. are sparsely present in the plots. Picea abies (L.) H.Karst. was another exotic EcM tree in some plots in Alasht and Kelardasht, which was avoided during sampling. In each study site, seven plots were established: one in the native vegetation, three in pine plantations adjacent to native trees and three in the ecotone, where the exotic pines and native trees lie adjacent to each other and occasionally intermixed. The pine and native vegetation plots were established ca 500 m. from the ecotone. The distance among plots ranged from 100 to 1,000 m. In the ecotone, root tips of both pine and native EcM trees were sampled. To examine the effect of proximity to native host plants on EcMF communities of introduced trees, two to three plots were established in three disjunct pine plantations in Shafarud, Tehran and Malayer (Table 1). There are no native EcM trees within 50-km distance to the plots in Tehran and Malayer, and semi-arid climate and vegetation dominate these areas. In Shafarud, which locates near the Hyrcanian forests with nearly relatively climate, the lowland forests (possibly dominated by non-EcM trees such as B. hyrcana and Parrotia persica) have been cut about 40 years ago and replaced with commercial trees including pines (Table 1). The plots in this site were >4 km apart from native EcM forests. Seven soil samples (15×15 cm) were taken to 10-cm depth along transects with 20-m intervals in each plot. This distance was chosen to minimize spatial autocorrelation among samples (Lilleskov et al. 2004; Bahram et al. 2011). EcM roots were separated from attached soil and debris in water. Morphotypes were distinguished according to the color, shape of root tips and presence of cystidia under a stereomicroscope. Percentage of colonization was estimated visually based on relative amounts of EcM and non-EcM

Pinus sylvestris Picea abies Pinus sylvestris Picea abies Pinus taeda Pinus nigra Pinus eldarica Pinus eldarica

40

N 36°04′, E 53°09′

50

35

N 36°07′, E 52°54′

30

45–50

N 36°29′, E 51°09′

150

30–40

N 37°33′, E 49°04′

300 90

30 30

N 35°43′, E 51°31′ N 34°18′, E 48°50′

root tips in each soil sample. Morphotypes were stored in CTAB buffer (1 % cetyltrimethylammonium bromide, 100 mM Tris–HCl (pH 8.0), 1.4 M NaCl and 20 mM EDTA) for transportation and molecular analysis. Molecular analysis DNA was extracted from one healthy root tip from each morphotype and sample with a Qiagen DNeasy 96 Plant Kit (Qiagen, Crawley, UK) according to the manufacturer’s instructions. This step was repeated twice if no DNA was yielded. Primers ITSOF-T (5′-cttggtcatttagaggaagtaa-3′) in combination with reverse primers LB-W (5′-cttttcatctttccctcacgg-3′), ITS4 (5′-tcctccgcttattgatatgc-3′) or ITS2 (5′gctgcgttcttcatcgatgc-3′) were used to amplify the ITS region. For low-quality sequences, PCR was re-performed using taxon-specific primers ITS4-Tom (5′-aactcggacgaccagaggca-3′), ITS4-Seb (5′-tcagcgggtartcctactc-3′), ITS4Russ (5′-agcgggtagtctcaccc-3′), ITS4-Cg (5′-cacatggcaarggcaaccg-3′), ITS4-Clavu (5′-ggtagtcccacctgattc-3′) or LR3Pez (5′-cmtcrggatcggtcgatgg-3′; see Tedersoo et al. 2008, 2011; Bahram et al. 2011). Morphotypes yielding no sequence were re-extracted and re-amplified up to two more times to provide identification to as many EcM root tips as possible. PCR reactions were performed following Tedersoo et al. (2006). Primers ITS5 (5′-ggaagtaaaagtcgtaacaagg-3′) and ITS4 were used for sequencing of the fungal ITS. Plant host identification was confirmed for all morphotypes from mixed plots with native and exotic pine trees as well as one randomly selected root tip from each identified species based on the sequence of plastid trnL intron by using primers TrnC (5′-cgaaatcggtagacgctacg-3′) and TrnD (5′ggggatagagggacttgaac-3′). The TrnD primer was used for sequencing of the plant trnL region. PCR products were visualized on 1 % agarose gels under UV-light and purified using of Exo-Sap enzymes (Sigma, St. Louis, MO, USA). To edit, trim and cluster raw sequences, Sequencher 4.9 software (GeneCodes Corp., Ann Arbor, MI, USA) was

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used. Fungal species were separated based on 97 % ITS sequence similarity threshold. Species were identified based on MegaBLAST and BLASTn searches against the International Sequence Databases (INSD) and UNITE (Abarenkov et al. 2010a) using the massblaster function of the PlutoF workbench (Abarenkov et al. 2010b). Representative sequences were submitted to EMBL under sequence accessions HE687028-HE687187. To trace the potential origin of fungal species, we compared our sequence data against ITS sequences from the previous study that addressed native EcM trees in this region (Bahram et al. 2012). Data analysis Species were assigned to lineages according to Tedersoo et al. (2010). Analyses of community were performed at both the species and lineage level. Bray–Curtis and Euclidean distance measures were used respectively to calculate community and geographical distance matrices. Species information from all seven samples in each plot was combined and plot was used as a sampling unit in the analyses of community structure. Permutational multivariate analysis of variance (ADONIS) was used to explore the source of variation in EcM fungal community as implemented in Vegan package of R version 2.9.1 (R Development Core Team 2007). The dominant host species, age of host trees and site identity were included in the model. Non-metric multidimensional scaling (NMDS) was used to visualize community dissimilarity among plots. Mantel test was used to examine the correlation between approximate distance from native EcM vegetation and community variation of the pine plantations. Alternatively, the correlation between host plant community and fungal community variations was tested by Mantel test. Rarefied species accumulation curves and minimum total richness estimates were calculated using the EstimateS software (Colwell 2006). Two-way ANOVAs were used to compare species richness and the percentage of colonization among different vegetation types.

Results Molecular analysis of 863 root tips and 709 morphotypesample combinations yielded 563 ITS sequences that were divided into 160 species (Table 2; S1). On the plot basis, 47.7 and 27.1 % of species only occurred once (singletons) and twice (doubletons), respectively. Based on the massblaster function, 139 species (86.9 %) showed >97 % identity with INSD records (Table S1), including 66 records from the previous study (Bahram et al. 2012). In total, 28 species (17.5 %) showed 100 % identity with INSD records, from which only eight species matched with records from elsewhere (excluding previous records from Bahram et al.):

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Gymnomyces sp1, Suillus sp2, Genea sp1, Tomentella sp22, Tricholoma sp2, Clavulina sp3, Lactarius sp1 and Cortinarius sp8 (Table S1). In the three main study sites, the average observed species richness per plot was 13.3 (2.6 per sample), 14.2 (2.4) and 14.0 (2.6) in pine plantations, the ecotone and native ranges, respectively. Based on molecular host identification, 94 species associated with pine roots. From species with more than one occurrence associating at least once with pine (68 species), 84.6 % (55 species) were shared between pines and the native trees and only 5.9 % (four species) were found exclusively on pine (Table S1). There were no statistically significant differences among average species richness of plots in different vegetation types (P 00.887). Consistent with this, minimum species richness estimates did not show significant difference between ecotone (38.3± 6.88, mean ±95 % CI), the non-isolated pine plantations (34.7±7.14) and native trees (38.0±11.14; Fig. 1). Percentage of colonization of root tips was 55.98±8.69 %, 72.10± 8.99 % and 69.10±13.68 % in the non-isolated pine plantations, ecotone and native ranges, respectively. The community composition of the plantations adjacent to native trees was different from the isolated plantations (Fig. 2). Besides, EcM fungal richness was remarkably higher in the non-isolated plantations compared to the isolated plantations (F1,14 011.40; P00.005). In Tehran and Malayer, only two EcM fungal species were found associated with pine trees (Geopora sp. and Rhizopogon sp.). In Shafarud, minimum species richness estimate was lower than the non-isolated plantations (29±9.86; Fig. 1), and the abundance of Amphinema sp. (characteristic to coniferous soils) and Rhizopogon sp. (a pine-specific species) was remarkably high (Fig. 2; Table 2). In addition, EcM fungal colonization was also relatively low (21.1±9.5 %) in the isolated plantations in Tehran and Malayer. The proportion of EcM fungal colonization was significantly lower in isolated plantations compared to the non-isolated plantations (F1,14 013.18; P00.003). Across all sites (excluding isolated plantations), the tomentella–thelephora (42 spp.), sebacina (22 spp.), inocybe (18 spp.) and tuber (8 spp.) were the dominant lineages in terms of species richness. The community composition of non-isolated plantations was similar to native trees, and the tomentella–thelephora (27 spp.), sebacina (15 spp.), inocybe (14 spp.) and tuber (7 spp.) were the most species-rich dominant lineages. In addition, 30 species (16 species exclusively) were found from the isolated plantations with the pine-specific mycobionts as the most abundant species and tomentella–thelephora (7 spp.) and sebacina (5 spp.) as the most species-rich lineages. Based on multivariate analysis of variance (ADONIS), site identity explained 16.5 % of the community variation (P 00.001), while the effect of host was not significant

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Table 2 Species richness and most abundant species in the pine plantations, ecotone and native ectomycorrhizal vegetations of Iran

Number of samples

Average richness Total species per plot richness (±95 % CI) (±95 % CI)

Dominant species (frequency)

Native EcM vegetations

38

14.0±4.29

38±11.14

Tuber sp8 (4.3 Cenococcum sp. (2.6 Cortinarius sp9 (2.5 Inocybe sp7 (2.5 Tricholoma sp1 (2.5

%) %) %) %) %)

Ecotone

93

14.2±2.75

92±15.14

Plantations adjacent to native EcM trees

81

13.33±2.20

81±14.98

Isolated plantations

49

6.67±3.6

31±10.2

Tricholoma sp1 (6.7 %) Tuber sp6 (5.4 %) Inocybe sp7 (4.2 %) Inocybe sp6 (2.5 %) Cortinarius sp9 (2.5 %) Tuber sp8 (2.5 %) Tricholoma sp1 (8.5 %) Cenococcum sp. (5.2 %) Tuber sp4 (3.4 %) Tuber sp1 (3.4 %) Clavulina sp3 (2.9 %) Russula sp4 (2.9 %) Tomentella sp22 (2.9 %) Tomentella sp30 (2.9 %) Tuber sp8 (2.9 %) Amphinema sp. (17.7 %) Rhizopogon sp. (10.3 %) Geopora sp. (13.2 %) Pezizales sp2 (5.9 %) Gymnomyces sp2 (5.9 %) Inocybe sp17 (5.9 %)

(P00.473). Likewise, there was no significant correlation between host community and EcM fungal community variation based on Mantel test (P00.452). By including the isolated plantations in the community analysis, site identity explained 32.89 % of community variation (P00.001) and host remained non-significant (P00.163; Fig. 2). The geographical distance from native vegetations was significantly correlated with the variation in community structure (rMantel 00.437, P00.001).

might find no suitable mycobionts in new habitats, especially if they are introduced to a different floristic region (Parker 2001; Tedersoo et al. 2007; Nuñez et al. 2009; Dickie et al. 2010). However, in the presence of phylogenetically related

Discussion The results of our study indicate that native EcM associations strongly drive EcM fungal community and species richness of exotic pines when planted adjacent to native EcM trees in Iran. Previous studies (e.g. Díez et al. 2001; Dickie et al. 2010; Jairus et al. 2011) showed that cointroduction is the main strategy by which introduced plants can maintain their mutualistic associates. Introduced plants

Fig. 1 Species richness rarefied accumulation curves and their ±95 % confidence intervals. Symbols represent the pine plantations adjacent to native EcM trees (diamond), isolated pine plantations (circle), ecotone (triangle) and native trees (square)

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Fig. 2 NMDS ordination illustrating the community dissimilarity among plots in the plantations adjacent to native EcM trees: Sangdeh (closed symboles), Alasht (tinted symbols), Kelardasht (open symbols) a excluding isolated plantations and b including isolated plantations (letters) in Shafarud (S); Tehran (T); and Malayer (M).Non-isolated pine plantations, circles; ecotone, triangle; and native trees, square. Ellipses depict 95 % confidence intervals around centroids (symbols and letters) of each category

native EcM hosts, introduced plants can form new associations with local mycobionts (Kohout et al. 2011; Trocha et al. 2012). The present data provide strong evidence of host range extensions for many EcM fungal species between two phylogenetically distantly related EcM plants, i.e. the native Fagales (Fagaceae and Betulaceae) and exotic pine trees. Since Fagaceae, Betulaceae and Pinaceae overlap in native geographical ranges in most temperate areas of the Northern Hemisphere, their EcM symbionts are perhaps evolutionarily adapted and compatible with each other, in contrast to that of, for example, Dipterocarpaceae and Caesalpiniaceae (Chen et al. 2007). Alternatively, this discrepancy may reflect the ancestral compatibility between EcM symbionts of Fagales and Pinaceae, given their potential common

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evolutionary origin of EcM symbiosis (Brundrett 2002). Host range expansion from Fagaceae to Pinaceae has also occurred in the case of Amanita phalloides (Wolfe and Pringle 2012), a widely distributed invasive species in North America. This European species associates mainly with oaks and rarely with pines in its native range, while it occurs rather exclusively with pine trees on the East Coast. Species may extend the range of their mutualistic partners in order to gain more benefit from others (Zhang et al. 2011). Similar to arbuscular mycorrhizal fungi, which have stronger effect on both native and exotic host plants in their local ranges (Klironomos 2003), local mycobionts may be more beneficial for exotic trees compared to co-introduced fungal species because of adaptation to local soil and climatic conditions. The comparable species richness and the large proportion of shared species between native trees and introduced plantations suggest that local EcM fungal species can outcompete and replace introduced species on the root tips (Jairus et al. 2011). Other studies have similarly reported low EcM fungal diversity in exotic pine plantations in the Southern Hemisphere (e.g. Dunstan et al. 1998; Barroetaveña et al. 2007; Tedersoo et al. 2007). In contrast, comparable richness between exotic and native trees has been demonstrated in the case of exotic pine species overlapping in range with native ones (Kohout et al. 2011; O’Hanlon and Harrington 2011, 2012; Trocha et al. 2012). Additionally, the isolated pine plantations in areas without native EcM trees associated with only a few mycobionts such as Rhizopogon sp., Amphinema sp. and Geopora sp., which are lacking on local tree species (Bahram et al. 2012) and potentially introduced. The dispersal limitation of most EcM fungal species (Peay et al. 2007) and detrimental effect of habitat isolation and fragmentation (Peay et al. 2010) may account for different communities and lower richness in the isolated plantations compared to the plantations adjacent to native EcM trees. Alternatively, different habitat conditions such as pollution (Parrent et al. 2006) and lower precipitation (Bahram et al. 2012) in Tehran and Malayer may contribute to their low species richness. This suggests that species found in the isolated plantations are perhaps more tolerant colonizers compared to other co-introduced mycobionts which might have been outcompeted or disappeared due to harsh conditions. Chapela et al. (2001) also found only three species (Rhizopogon sp., Thelephora sp. and Suillus sp.) associated with an isolated pine plantation. The tomentella–thelephora, russula–lactarius and inocybe were the dominant lineages in both pine plantations and native trees, which is consistent with results from Iran (Bahram et al. 2012) and the Northern Hemisphere (Tedersoo and Nara 2010). Despite the presence of a few pine-specific mycobionts (e.g. Rhizopogon and Suillus), they were rarely found in the pine plantations adjacent to native EcM trees. Members of the suillus–rhizopogon

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lineage are the most abundant and common mycobionts of pine seedlings (Iwanski et al. 2006; Rusca et al. 2006; Kohout et al. 2011) and Rhizopogon is the most common introduced genus globally (Vellinga et al. 2009). The suillus–rhizopogon lineage has not been reported previously from Iran (Bahram et al. 2012), and thus it is likely introduced to this region. These fungi have been introduced most likely through sporadic imports of seedlings or soil. Alternatively, spores of this group and other potentially introduced species can disperse from other regions by wind and colonize the plantations (Peay et al. 2007). Although Cenococcum geophilum is the most frequent species in the communities of native trees of Iran (Bahram et al. 2012), it was lacking in the isolated plantations. This species is the most common EcM fungi in many temperate forests (Horton and Bruns 2001) and is present in the native range of P. sylvestris (Pickles et al. 2010; Kohout et al. 2011). As C. geophilum spreads only through vegetative mycelium and soil movement with sclerotia, it may have low ability to colonize distant or isolated plantations. In addition, Amanita muscaria, the most widespread invasive species of EcM fungi in many ecosystems (Vellinga et al. 2009), and Thelephora terrestris, the most abundant species in Scots pine nurseries (Iwanski and Rudawska 2007; Jonsson et al. 1999; Menkis and Vasaitis 2011), were absent in our study. These species have never been recorded in Iran. Low abundance and unsuitable environmental variables can act as an obstacle for the establishment of introduced species (Wardle et al. 2011). This can also be one of the potential reasons for unsuccessful pine naturalisation in an exotic environment (Mikola 1969; Read 1998; Richardson et al. 2000; Nuñez et al. 2009). Site had the strongest effect on EcM fungal community in the region, but surprisingly the effect of host tree was negligible. Because of recruitment of local EcM fungi, the EcM fungal communities in pine plantations were more similar to the fungal communities of nearby native trees than to other pine plantations. Many other studies have established that host is the main determinant of EcM fungal community at both the local scale (Ishida et al. 2007; Tedersoo et al. 2008) and regional scale (Bahram et al. 2012). Thus, our results demonstrate that the native EcM communities directly determine fungal communities in the adjacent introduced pine plantations, and vast majority of mycobionts associated with exotic trees are host generalists. In conclusion, diversity and community structure of EcM fungal community in exotic pine plantations is greatly determined by adjacent native EcM associations in Iran. In contrast, isolated plantations showed a species-poor community of EcM fungi with strong dominance of pine-specific species. This suggests that introduced pines can easily form EcM associations with native host-generalist mycobionts and may lose their host-specific or potentially co-introduced mycobionts that are dominant in isolated plantations. The great

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number of shared EcM fungal species between exotic Pinaceae and native Fagales (Fagaceae and Betulaceae) may reflect the evolutionary adaptations of their EcM interactions and/or ancestral compatibility with one another.

Acknowledgments We thank Amin Fatahi, Hamed Shoubi, Javad Vanaei and Farhad Maleki for their help during field trips to Iran; Margit Nõukas for assisting in the laboratory; and Vilmar Veldre, Sergei Põlme, Steven Wright and Kessy Abarenkov for useful discussions. We would also like to thank Randy Molina (the editor) and two anonymous reviewers for their constructive suggestions and comments on the manuscript. We are grateful to Baba Khanjani Shiraz, Mehdi Kala Daliri, Khosro Sagheb-Talebi and the Office of Forest and Rangelands in Sari (Iran) for providing information on the plantations. This study was funded by the ESF grants 7434, 8235, RVO 67985939, Doctoral Studies and Internationalization Programme DoRa and FIBIR.

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