Disproportionate abundance between ectomycorrhizal root tips and their associated mycelia

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1 Disproportionate abundance between ectomycorrhizal root tips and their associated mycelia Rasmus Kjøller Department of Microbiology, Biological Institute, University of Copenhagen, Copenhagen, Denmark Correspondence: Rasmus Kjller, Department of Microbiology, Biological Institute, University of Copenhagen, Øster Farimagsgade 2D, 1353 Copenhagen K, Denmark. Tel.: ; fax: ; Received 25 January 2006; revised 27 March 2006; accepted 27 March First published online 22 June DOI: /j x Editor: Karl Ritz Keywords ectomycorrhiza; external mycelia; rhizomorphs; internal transcribed spacer region; Fagus sylvatica ; Xerocomus pruinatus. Abstract Extensive knowledge of various ectomycorrhizal fungal communities has been obtained over the past 10 years based on molecular identification of the fungi colonizing fine roots. In contrast, only limited information exists about the species composition of ectomycorrhizal hyphae in soil. This study compared the ectomycorrhizal external mycelial community with the adjacent root-tip community in a Danish beech forest. Sand-filled in-growth mesh bags were used to trap external mycelia by incubating the mesh bags in the soil for 70 days. The adjacent ectomycorrhizal root-tip communities were recorded at the times of insertion and retrieval of the mesh bags. Ectomycorrhizal fungi were identified by sequencing the internal transcribed spacer region. In total, 20, 31 and 24 ectomycorrhizal species were recorded from the two root-tip harvests and from the mesh bags, respectively. Boletoid species were significantly more frequent as mycelia than as root tips, while russuloid and Cortinarius species appeared to be less dominant as mycelia than as root tips. Tomentella species were equally frequent as root tips and as mycelia. These discrepancies between the root-tip and the mycelial view of the ectomycorrhizal fungal community are discussed within the framework of ectomycorrrhizal exploration types. Introduction Ectomycorrhizal fungi are important root symbionts of tree species in temporal and boreal regions (Smith & Read, 1997). Structurally and functionally, ectomycorrhizal fungi can be partitioned into four parts: the colonized root tip, the external mycelium ramifying into the soil from the colonized tips, sporocarps (with some exceptions), and a sporebank (or other resistant propagules) in the soil. The colonized root tips and the external mycelium are the fundamental structures for the functional symbiosis, as sites for the uptake and exchange of nutrients between the partners. The mycorrhizal tips are acknowledged to have nutrient-absorbing capacity (Harley & Smith, 1983; Buée et al., 2005); in addition, the external mycelium is believed to play a crucial role in the nutrient acquisition process by increasing the soil volume from which nutrients can be absorbed and by reaching into volumes of soil not accessible to roots (Finlay & Read, 1986; Finlay et al., 1988; Read, 1992; Read & Perez-Moreno, 2003). Some ectomycorrhizal species have root tips with a hydrophobic surface and are therefore totally dependent on their hydrophilic external mycelia for nutrient absorption (Agerer, 2001). The majority of the carbon transferred to the fungi is also directed towards the external mycelia rather than to the colonized root tips (Colpaert et al., 1992; Wallander et al., 2001; Högberg & Högberg, 2002). The external mycelium may be undifferentiated hyphae or organized into more or less complex rhizomorphs, with the latter optimized for long-distance transport of nutrients (Agerer, 2001). At sites of an appropriate nutrient source, rhizomorphs may branch into absorbing hyphae. The capacity to form rhizomorphs is to some extent determined by the phylogenetic history of the fungi in question; for example, ascomycete ectomycorrhizal fungi do not have this ability, while boletoid ectomycorrhizal fungi form highly differentiated rhizomorphs similar to those of their saprotrophic relatives, for example Serpula species (Read, 1992; Agerer & Iosifidou, 2004). Based on root-tip and sporocarp surveys, ectomycorrhizal fungal species are known to coexist in species-rich communities far more diverse than those of their host trees (Dahlberg, 2001). However, despite acknowledgement of the functional importance of ectomycorrhizal external mycelia, very little is known about the distribution and abundance of such structures in soil. This is because within each sample, either as soil or as extracted hyphae, there will be a mixture

2 Ectomycorrhizal root tips and their associated mycelia 215 of mycelia of several ectomycorrhizal species as well as other fungal species, which makes identification of all the fungi present a challenge. To meet this challenge, cloning and sequencing or Terminal-restriction fragment length polymorphism (T-RFLP) strategies have recently been employed to analyse ectomycorrhizal mycelial communities in soil (Chen & Cairney, 2002; Dickie et al., 2002; Landeweert et al., 2003, 2005; Smit et al., 2003; Koide et al., 2005). These studies have shown that extracting DNA from soil may retrieve more ectomycorrhizal species than sampling adjacent colonized root tips (Smit et al., 2003; Koide et al., 2005; Landeweert et al., 2005), and that ectomycorrhizal mycelial communities are differentiated vertically in the soil profile (Dickie et al., 2002; Landeweert et al., 2003). In an experiment involving more than 300 individual samples, Koide et al. (2005) found differences in frequencies of individual ectomycorrhizal species between the root tips and the adjacent mycelial community. The dominant root-tip species were generally also found as dominant mycelia, while several dominant mycelia were either absent or of low abundance as root tips (Koide et al., 2005). That ectomycorrhizal communities could have disproportionate abundances between mycelia and root tips was predicted by Agerer (2001), who classified ectomycorrhizal fungi into exploration types based on the structure of the external mycelium and how far from the colonized root tip the mycelia protrude into the soil. The two extremes are the contact exploration type with no or very little external mycelia, and the long-distance exploration type with highly differentiated vessel-like rhizomorphs, with intermediate types in between (Agerer, 2001). Following his scheme, the external hyphal community should be skewed towards species with a larger production of external hyphae per colonized root tip. The present study explores this hypothesis and compares the mycelial community in sand-filled in-growth mesh bags (Wallander et al., 2001) with the adjacent ectomycorrhizal root-tip community. The in-growth mesh-bag technique produces fungal mycelia appropriate for molecular analysis and avoids problems concerning amplification of detached mantle material or resistant propagules that may be associated with total soil DNA extraction. The mesh-bag technique has previously been used to estimate the production of external mycelia in various forest types and soil layers as well as the production of mycelia in response to nitrogen and phosphorus gradients (Wallander et al., 2001; Hagerberg et al., 2003; Nilsson & Wallander, 2003; Nilsson et al., 2005). Materials and methods Experimental site The experimental site was located in Lille Bgeskov, a 200 ha forest on the island of Zealand, Denmark ( N, E). The stand was predominately 80-year-old beech (Fagus sylvatica L.), with some naturally regenerated medium-sized beech trees. Otherwise, the understory was dominated by beech seedlings, Fraxinus excelsior L. seedlings, Maianthemum bifolium (L.) F. W. Smith, Carex sp. L., Galium odoratum (L.) Scop., and Lamiastrum galeobdolon (L.) Ehrend. & Polatschek. The landscape was moraine plain and the soil type a mollisol with ph 4.5 (H 2 O) in the sampled horizon. Lille Bgeskov is the Danish CORE field-station site, and additional information is available at Sampling root tips and mycelia Within a m area in Lille Bgeskov, 20 soil cores were taken randomly on 3 September No other ectomycorrhizal hosts were found within or near the plot. Soil cores were taken using a 2.1 cm diameter soil corer to 15 cm depth (52 cm 3 ). The soils to this depth were all organic and no action was taken to separate the litter and humus horizons within. The soil samples were stored on ice until return to the laboratory, and then at 4 1C until processed (see below). In-growth mesh bags (Wallander et al., 2001) were inserted into the holes left after coring. These comprised nylon bags, 10 4 cm, with mesh size 37 mm, filled with 60 g of acidwashed sand (size mm) and sealed with a plastic sealer. The bags were inserted into the soil such that the tops of the bags were visible when removing the litter layer. After 70 days (13 November) the bags were retrieved. First the bags were pulled out and then a 5 cm soil corer was pushed down around the hole to 10 cm depth (162 cm 3 ) to record the ectomycorrhizal root-tip community around the mesh bag at harvest time. Hyphae were extracted from the sand by floating and decanting, and the mycelia were then frozen and freeze-dried. The roots from the two soil sampling times were retrieved from a series of 2, 1 and 0.5 mm sieves. From each soil sample healthy-looking mycorrhizal tips were detached from the root system and sorted into morphotypes based on macroscopic features including colour, size, branching pattern and surface texture. The abundance of each morphotype within each sample was evaluated by eye and grouped into four abundance categories corresponding approximately to 1 3, 3 10, and 4 30 tips, respectively. From each sample, two replicate root tips of each morphotype were selected for molecular identification and stored in 2 CTAB (cetyltrimethyl-ammonium bromide) lysis buffer (Gardes & Bruns, 1996b). The rest of the root tips were freeze-dried and kept in reserve in case the amplifications did not work or the two replicate tips produced contrasting RFLP results (see below). All root samples were processed within 1 week of sampling.

3 216 R. Kjller Molecular identification of ectomycorrhizal tips and mycelia DNA was extracted from root tips by the method described by Gardes and Bruns (1996b). The internal transcribed spacer (ITS) region was amplified with the primer combination ITS1-F and ITS4 (White et al., 1990; Gardes & Bruns, 1993) and restricted with the enzymes Hinf-I and Hha-I (New England Biolabs, Beverly, MA). RFLP patterns were compared within individual RFLP gels, and from each gel all unique RFLP patterns were sequenced. If contrasting RFLP patterns from the two replicate root tips selected from each morphotype within a sample were obtained, new extractions were made from the freeze-dried material to estimate the abundance of each of the RFLP types. If possible, six new root tips were selected for these extra extractions. In total, 313 root tips were subjected to PCR and RFLP analysis. The PCR products were sequenced at MWG-Biotech (Martinsried, Germany). PCR products were sequenced with one of the forward primers ITS1-F or ITS5 (White et al., 1990; Gardes & Bruns, 1993), but if sequencing proved difficult additional sequencing was carried out using ITS4, ITS2 and ITS3 as sequence primers (White et al., 1990). ITS sequences potentially belonging to Pezizales or Sebacina species were extended to include the D1 D2 region of the large ribosomal subunit by additional amplification with primers ITS1-F and TW13 (Taylor & Bruns, 1999) and sequencing with primers TW13 and LR0R (5 0 -ACCCGCTGAACTTAAGC- 3 0 ). Root-tip sequences belonging to Cortinarius, Pezizales and Tomentella species were always sequenced in both directions to obtain the complete ITS sequence. Chromatograms were analysed using SEQUENCHER version 3.1 (Gene Codes Corporation, Ann Arbor, MI) and aligned in the BIOEDIT Sequence Alignment Editor version 6 (Hall, 1999) with sequences of known species (Table 1). For most genera alignment proved an efficient way to identify sequence types, but for sequences belonging to Tomentella and Cortinarius phylogenetic analysis was carried out to help identify sequence types. Phylogenetic analysis was performed using PAUP version 4.0 (Svofford, 1998). In the remainder of the present paper, the term species will be used for the identified ITS-unique sequence types. After freeze-drying, the DNA was extracted from the mycelia from the mesh bags and ITS amplified as described above for mycorrhizal tips. The ITS products from the mycelia were cloned using the TOPO TA Cloning Kit from Invitrogen (Carlsbad, CA). Following plating, transformed clones were selected, ITS-amplified, and sequenced as described above for mycorrhizal tips. Some amplifications were submitted to restriction analysis before sequencing as described above for mycorrhizal tips. In this case, all unique RFLP types from each original mesh bag were sequenced. Additional PCR, cloning and sequencing reactions were carried out until approximately 20 or more successful sequences were obtained from each mesh bag. Independent PCR and cloning reactions from the same original sample also served as a control of the reproducibility of the method. Reference sequences from sporocarp collections To obtain reference sequences for identifications, sporocarps were collected within the plot in 2001 and The plot was visited five and six times in 2001 and 2002, respectively. For the last collection day in 2001, twigs and small branches on the forests floor within the plot were carefully examined for the presence of Tomentella species. No actions were taken to sample hypogeous species. Additional collections were also obtained from various other beech localities in Denmark. Reference sequences from fruit bodies were generated using the same procedure as described above for mycorrhizal tips. Sequences of sporocarps, root tips and mycelia are deposited at the European Molecular Biology Laboratory (EMBL). The sporocarp sequences are available through the user-friendly Nordic ITS ectomycorrhizal (UNITE) database of ectomycorrhizal fungi where additional information can be sought for the individual collections. Sporocarps were deposited in the herbarium at the Copenhagen Natural History Museum (searchable through /svampebase/search.htm). Data analysis The frequency of single species or species groups found as root-tip or as mycelia was compared using a w 2 test with Yates correction. The relative abundance of each species within a root sample was calculated as the percentage of the sum of ranked abundances within a sample. Likewise, the relative abundance of clones belonging to each species was calculated within each mesh bag. Relative abundances between species or species groups were analysed using oneway ANOVA analysis including Tukey s test of differences of means. Species accumulation curves, which relate the number of species detected to the number of samples taken, were calculated for the two root-tip datasets and the mycelia dataset using PC-ORD software (McCune & Mefford, 1999). This software simultaneously calculates first-order and second-order jacknife estimates of true species richness within the area sampled. In addition, species accumulation curves were calculated for the ectomycorrhizal community found within each individual mesh bag. Results Cloning success and species richness Species identified and their associated EMBL accession numbers are shown in Table 1. Out of the total of 496

4 Ectomycorrhizal root tips and their associated mycelia 217 Table 1. EMBL accession numbers for root-tip-, mycelia- and sporocarp-derived sequences Taxa Root tips Mycelia Sporocarps w Amanita rubescens AM AM AJ889922, AJ Cantharelloid sp. 1 AM Cenococcum geophilum AM Clavulina cristata AM AJ889937, AJ889929, AJ Cortinarius anomalus AM AJ889939, AJ889940, AJ Cortinarius cf. decipiens AM AJ889946, AJ Cortinarius lividoochraceus AM AM Genea hispidula z AJ AM AJ969623, AJ Glischoderma sp. z AJ AM Helvella sp. z AJ Inocybe asterospora AM AJ889950, AJ Inocybe glabripes AM AM AJ Inocybe petiginosa AM AM AJ889956, AM Inocybe sp. 1 AM Inocybe sp. 3 AM Inocybe sp. 7 AM Laccaria amethystina AM AM AM113953, AM113954, AM Laccaria sp. 1 AM AM Lactarius camphoratus AM AJ Lactarius subdulcis AM AJ889963, AJ889964, AJ Melanogaster macrosporus AM Peziza sp. z AJ AM Pezizaceae sp. z AJ AM Piloderma sp. 1 AM Piloderma sp. 2 AM Russula fellea AM AM113957, AM Russula mairei AM AM Russula nigricans AM AM AM113960, AM113961, AM Russula ochroleuca AM AM113963, AM Russula vesca AM AM AM Sebacinoid sp. 3 AM Tarzetta sp. z AJ Tomentella badia AM AM Tomentella botryoides AM Tomentella bryophila AM AM AJ Tomentella punicea AM AM Tomentella sp. 4 AM Tomentella sp. 6 AM AM Tomentella sp. 7 AM AM Tomentella sp. 9 AM AM Tomentella sp. 14 AM Tomentella sp. 16 AM Tomentella sp. 20 AM Tomentella terrestris AM Tuber puberulum z AJ AJ969625, AJ Xerocomus badius AM AM AJ889926, AJ Xerocomus pruinatus AM AM AJ889931, AJ889932, AJ Species or genus names were assigned to root-tip- or mycelia-derived sequences by comparison with sporocarp-derived sequences collected from Danish beech forests in connection with the present study. In lieu of matches to own sporocarp-derived material, names were assigned according to sequence homology with sequences from GenBank and/or UNITE. w Only accessions of sporocarp sequences obtained from material collected in connection with the present study are shown. z For additional information about naming of the Pezizalian root-tip sequences, see Tedersoo et al. (2006). successfully sequenced clones, 411 contained sequences belonging to ectomycorrhizal species while the remaining 17% contained nonmycorrhizal sequences (Table 2). Between 19 and 43 clones were analysed from individual mesh bags, and in total 24 ectomycorrhizal species were identified with an average of 3.1 species per individual mesh bag (Table 2). For

5 218 R. Kjller Table 2. Number of clones analysed and number of ectomycorrhizal (EMF) and nonectomycorrhizal fungal species Mesh-bag data Root-tip data Core Total no. clones analysed EMF clones EMF species Non-EMF species EMF species start EMF species end x w Sums z nd 20 z 31 z 48 z Means Sample 15 was lost during handling. w Sample 17 was harvested after 1 month. z Number of unique species. The total number of unique nonectomycorrhizal species was not determined. Total EMF species ectomycorrhizal root tips, 20 species were identified at the beginning of the experiment, while 31 were identified from the samples surrounding the mesh bags at the second harvest. A total of 48 species were identified for the three sample types taken together (Fig. 1). Eight species were uniquely found as mycelia (Fig. 1). Five species were unique to the initial root-tip sampling and 12 to the second root-tip sampling (Fig. 1). One mesh bag (no. 17) was retrieved after 1 month to monitor the colonization progress and to establish the extraction protocol to be used. This mesh bag was colonized by Xerocomus badius, Xerocomus pruinatus and Melanogaster macrosporus, but these data are not used beyond Table 2. A further sample (no. 15) was damaged in the field and lost. The nonmycorrhizal sequences obtained from the mesh bags were of ascomycete, basidiomycete and zygomycete origin, and in addition there were sequences completely without any match in the sequence databases. Often the identities of the non-mycorrhizal sequences were difficult to determine, as the BLAST results would retrieve a list of closely related sequences but assigned, for example, to both Ascomycota and Zygomycota clades. Community structure -- mycelia The most dominant species obtained from the mesh bags was X. pruinatus, amplified from half of the samples (Fig. 1). Two unidentified Tomentella species followed as the second and third most abundant species, and Tomentella was also the dominating fungal clade, present in 14 mesh bags, followed by the boletoid group, present in 12 (Table 3). From the mesh bags, Tomentella (ten species) was the most species-rich clade (Fig. 1), and often two or three Tomentella species cooccurred in a single mesh bag (data not shown). While tomentellas were also common root tips at the late harvest, the frequency of boletoid fungi was significantly lower from root samples at the late harvest than from mesh bags (Table 3). Lactarius and Cortinarius species were commonly found root tips but were absent as mycelia in the mesh bags, but, owing to the low number of observations, this tendency was not significant nor were comparisons of the russuloid or the Cortinariaceae clade between root tip and mesh bags (P ; Table 3). When boletoid species were present in an individual mesh bag, these typically dominated the population of clones obtained (Table 3). In comparison with

6 Ectomycorrhizal root tips and their associated mycelia 219 Cortinarius cf. decipiens Tarzetta sp. Lactarius camphoratusi Inocybe sp. 3 Inocybe sp. 1 Inocybe asterospora Tuber puberulum Tomentella sp. 14 Sebacinoid sp. 3 Piloderma sp. 2 Piloderma sp. 1 Cortinarius cf. lividoochraceus Cenococcum geophilum cantharelloid sp. 1 Russula ochroleuca Russula mairei Tomentella sp. 4 Russula fellea Helvella sp. Clavulina cristata Lactarius subdulcis Cortinarius anomalus Pezizaceae sp. Tomentella sp. 20 Tomentella terrestris Tomentella botryoides Inocybe sp. 7 Inocybe glabripes Laccaria amethystina Amanita rubescens Tomentella punicea Laccaria sp. 1 Peziza sp. Glischoderma sp. Genea hispidula Inocybe petiginosa Tomentella sp. 16 Tomentella sp. 6 Tomentella bryophila Russula vesca Tomentella badia Xerocomus badius Melanogaster macrosporus Russula nigricans Tomentella sp. 9 Tomentella sp. 7 Xerocomus pruinatus mycelia root tips - start root tips - end Cumulative number of samples with species Fig. 1. Number of root samples or mesh bags with specific ectomycorrhizal species. The bar chart is sorted by the abundance of species in the mesh bags. Inocybe glabripes, Xerocomus badius and Tomentella sp. 9 were found as root tips in the plot at other sampling dates than those used in the present experiment. the boletoid species, the mean relative abundances of Tomentella Russula, Pezizales and Inocybe clones were all significantly lower within mesh bags (Tukey s test, P o 0.05). Community structure -- root tips The root-tip community at the second harvest was dominated by Russula species, followed by Tomentella species and thereafter by Pezizales, Inocybe, Cortinarius and Lactarius species (Fig. 1). There were fewer species found at the first harvest than at the late harvest, but all dominant groups were identified from both harvests. There were significantly more Tomentella roots tips at the second harvest than at the first harvest (w 2 = 6.75; Table 3), although this result should be treated cautiously because of the difference in sampling size between the two root-tip harvests. There were no

7 220 R. Kjller Table 3. Frequency of fungal clades in root-tip and mesh-bag samples Occurrence in no. of samples Fungal clade Root tips start Root tips end Mycelia w 2 (root tips end mycelia) w Mean relative abundance of clones within mesh bags Tomentella a (23) z Boletoid b (15) Russula a (5) Pezizales a (3) Inocybe a (3) Laccaria (2) Amanita (1) Cortinarius n/a Cantharelloid n/a Lactarius n/a Sebacina n/a Cenococcum n/a Piloderma n/a Cortinariaceae a (3) Russulaceae a (5) Several different species from the same clade within one mesh bag or root sample is counted as one hit. w w 2 comparisons between the number of times a specific clade was found within mesh bags or root samples at the last harvest. Values above 3.84 are significant at the 5% level. z Least significant difference was used to separate the means of relative abundances of clones within mesh bags of clades with three or more single species observations. Different letters indicate values that are significally different. The number in parentheses is the total number of single species observations. n/a, not applicable. significant differences between mean relative abundances of individual species (data not shown). No attempt was made to count the total number of root tips in the soil cores sampled, but a rough estimate based on the abundance categories suggests that about 600 and 900 root tips were collected from the first and second root-tip harvest, respectively. Species accumulation curves First- and second-order jacknife estimates indicated that on average about 90% of the true species richness was sampled within individual mesh bags (data not shown). The species accumulation curves for the root-tip and mycelia data indicate that all three communities were undersampled (Fig. 2). Root tips taken at the second harvest were sampled 35 Estimated ectomycorrhizal fungal species mycelia root tips - start root tips - end Number of samples taken Fig. 2. Species accumulation plot of the two root-tip datasets and the data from the mesh bags. First-order jacknife estimates for first- and second-harvested root tips and for the mycelia were 34.2, 43.3 and 35.3, respectively. Secondorder jacknife estimates for first- and secondharvested root tips and for the mycelia were 45.8, 48.1 and 41.8, respectively.

8 Ectomycorrhizal root tips and their associated mycelia 221 most effectively, followed by the mesh bags and then by those taken at the first harvest. Reproducibility of the cloning method Overall, the abundant sequence types were always found in the independent PCR and cloning reactions from the same original sample. Some of the low-abundance sequence types could be absent or present between separate cloning reactions (data not shown). Discussion All mesh bags were heavily colonized by fungal hyphae after 70 days of incubation in the soil. When inspected under a dissection microscope, it was obvious that several different types of hyphae and rhizomorphs were present within each mesh bag. The in-growth mesh-bag method has previously proved suitable for measuring the production of ectomycorrhizal mycelia in soil (Wallander et al., 2001; Hagerberg et al., 2003; Nilsson et al., 2005). The data from the present study shed further light on the community of ectomycorrhizal fungi within the individual mesh bags, and allow these data to be compared with the adjacent root-tip community. Furthermore, most of the clones obtained from the mesh bags contained sequences of ectomycorrhizal fungi. As so many different ectomycorrhizal (and non-mycorrhizal) clades were detected from the mesh bags it is highly unlikely that ectomycorrhizal species have been selectively enriched in contrast to nonmycorrhizal fungi by the PCR and cloning reactions. The data obtained therefore agree with previous studies in which other methodologies have been used to show that sand-filled in-growth mesh bags are primarily colonized by ectomycorrhizal fungi (Wallander et al., 2001, 2003; Nilsson & Wallander, 2003). Boletoid species were more frequent in the mesh bags than as ectomycorrhizal root tips, and two of the boletoid species were not found as roots tips at all. The dominant boletoid species, X. pruinatus, was found as ectomycorrhiza in a few samples but was more frequently encountered as mycelia. Some X. pruinatus mycelia were found up to 10 m from the nearest spot where X. pruinatus root tips were detected, even though there were other sampling spots much closer. Ectomycorrhizal communities are known to be highly spatially heterogeneous (Lilleskov et al., 2004), and one cannot be certain that root tips just adjacent to the ones taken would have revealed X. pruinatus. However, a recent sampling at another Danish beech locality supports the view that mycelia of X. pruinatus are much more widely distributed than the associated root tips (J. Ockelmann and R. Kjller, unpublished). Boletoid species are in general known to form abundant and highly differentiated rhizomorphs and have therefore been classified as long-distance exploration types (Agerer, 2001). Xerocomus species have likewise been described as having rhizomorphs with a thick central hypha forming a core where septae are partially or completely dissolved (Agerer & Rambold, ); that is, as having rhizomorphs optimized for long-distance transport of solutes. Xerocomus pruinatus tips observed in the present study were also associated with thick and stout rhizomorphs. Xerocomus pruinatus, X. chrysenteron and other boletoid fungi such as Suillus and Rhizopogon species have all been reported to be capable of forming large genets, some covering over 300 m 2 (Dahlberg & Stenlid, 1994; Bonello et al., 1998; Fiore-Donno & Martin, 2001; Kretzer et al., 2004). The establishment of large genets by such soilinhabiting fungi is probably connected to their ability to form extensive rhizomorph systems. The root-tip community was dominated by Russula and Lactarius species. A high frequency of russuloid species on root tips is common for many ectomycorrhizal communities, both with beech and with other hosts (Gardes & Bruns, 1996a; Cullings et al., 2000; Taylor et al., 2000; Shi et al., 2002; Buée et al., 2005). Of the seven russuloid species colonizing the root tips only two, Russula nigricans and Russula vesca, were identified from the mesh bags, and less frequently than as root tips. Russula and Lactarius species are generally regarded as contact explorers; that is, as scavenging for nutrients in the close vicinity of the colonized root (Agerer, 2001). As for the boletoid group, a relationship between the amount of external mycelia and genet size seems to be present for russuloid fungi. For russuloid fungi, however, the lack of an abundant external phase correlates with the rather small genet sizes observed for this group (Redecker et al., 2001). Within Russula and Lactarius there are obviously intra-specific variations in the frequency of external mycelia and in the capability of producing rhizomorphs (Agerer, ; Agerer & Rambold, ). Russula and Lactarius species have also been detected in root-free soils (Chen & Cairney, 2002; Dickie et al., 2002; Smit et al., 2003; Genney et al., 2006). However, in two previous studies allowing direct comparisons between root-tip and mycelial frequencies, the dominant Lactarius and Russula species were more frequent as root tips than as mycelia, supporting the results of the present study (Koide et al., 2005; Genney et al., 2006). Tomentella mycorrhizas are described with frequent emanating hyphae but infrequent rhizomorphs (Agerer & Rambold, ). This corresponds well to morphotypes observed in the present study, which later were confirmed to be Tomentella species. The dominating species, Tomentella sp. 7, was found in six mesh bags, five of these adjacent to Tomentella sp. 7 root tips sampled at the second harvest. Tomentella sp. 7 therefore seems to produce abundant mycelia, but, in contrast to X. pruinatus, mainly localized in the vicinity of its root tips. This observation is in good agreement with the classification of Tomentella

9 222 R. Kjller ectomycorrhizas as short- to medium-distance exploration types (Agerer, 2001). The root tips of Cortinarius spp., for example Cortinarius anomalus common at the last harvest, were observed during morphotype sorting to be associated with abundant external mycelia and rhizomorphs. This observation agrees well with the descriptions of Cortinarius ectomycorrhizas (Agerer & Rambold, ). Surprisingly, no mycelia of Cortinarius species were found in the mesh bags even though Cortinarius root tips were found in five samples at both the first and second root-tip harvest. This indicates that some ectomycorrhizal species may avoid the pure sand habitat in the mesh bags. Similarly, no Cortinarius mycelia or rhizomorphs were detected in in-growth mesh bags buried in a southern Swedish spruce forest (Wallander et al., 2003), despite the fact that a Cortinatius sp. previously was found to be the most common species on root tips of that same forest (Mahmood et al., 1999). Cortinarius species are generally regarded as protein fungi (Taylor et al., 2000; Lilleskov et al., 2002a, b), that is, capable of using protein and amino acids as N sources, and may direct their mycelia towards organic substrates rather than into the pure sand found in the mesh bags. Indeed, using direct soil DNA extraction, Genney et al. (2006) showed that Cortinarius mycelia preferentially colonized the organic litter and fermentation horizons of a Scottish pine forest. Cloning and community profiling methods such as T- RFLP and DGGE are two alternative strategies for identifying microorganisms from samples containing mixed pools of DNA. Cloning can introduce a bias, as different PCR products may be more or less prone to cloning; for example, smaller-size PCR fragments generally clone more easily than longer fragments. As discussed above, it is not likely that ectomycorrhizal species were enriched as a group in this study, but some species may have cloned more easily than others. Real-time PCR approaches will in the future allow more accurate quantification of individual species within samples (Schubert et al., 2003). An advantage of cloning is that good-quality sequence data are produced, which normally allows sequences to be unambiguously assigned to ectomycorrhizal or non-mycorrhizal clades. Moreover, cloning and sequencing identifies chimeric sequences. These artificial sequences are created when, during PCR, the DNA polymerase jumps between different DNA strands within a mixed pool of DNA (Hugenholtz & Huber, 2003). About 3% of the sequences generated in this study contained chimeric sequences. Chimeric sequences are difficult to detect by BLAST searches alone, as a chimeric sequence may be made up of two closely related sequences; for example, in the present dataset, chimeric sequences made up of X. badius and X. pruinatus or of several Tomentella species were identified. Therefore, it is necessary to align obtained sequences with related sequences from the relevant fungal clades in order to identify chimeric sequences unambiguously. With the use of T-RFLP and DGGE, chimeric sequences may end up being treated as unique taxa in a community analysis. If using these methods it is therefore strongly advisable only to score bands that are known beforehand to belong to ectomycorrhizal fungi (Koide et al., 2005), or alternatively to dismiss bands/peaks only encountered once. Whether the in-growth mesh-bag technique accurately records the real soil abundance of individual ectomycorrhizal species cannot be fully addressed until it is compared with direct soil extraction. Nevertheless, the mesh-bag technique offers several advantages over direct soil extraction. (1) Hyphae from the mesh bags are easily extracted from the sand, and after a simple CTAB chloroform isopropanol DNA extraction method the extracted DNA is an effective template for PCR amplifications. (2) As the inserted mesh bags initially are tissue-free, the possibility of amplifying slough of ectomycorrhizal mantle tissue or fungal-resistant propagules can be excluded. (3) Problems with DNA amplifying from fungal necromass can also be largely overcome by incubating the mesh bags for short periods as in the present study, where mycelia were already present in the single mesh bag incubated for 1 month and otherwise in the remaining mesh bags incubated for months. (4) The mycelia extracted are of a purity that enables them to be used for quantitative measurements of total biomass as well as elemental composition (Wallander et al., 2001, 2003, 2004; Nilsson et al., 2005). (5) Different kinds of baits can be incubated within the mesh bag to explore fungal preferences towards substrates (Hagerberg et al., 2003). Historically, ectomycorrhizal species richness was monitored from the distribution and abundance of ectomycorrhizal sporocarps (Lange, 1978; Arnolds, 1991; Brandrud, 1995), but the application of molecular techniques is revealing different aspects of ectomycorrhizal communities (Gardes & Bruns, 1996a; Dahlberg et al., 1997; Peter et al., 2001; Lilleskov et al., 2002a, b; Tedersoo et al., 2003; Buée et al., 2005). One of the main results of the latter studies when combined with sampling of sporocarps is that aboveground sporocarp abundance is an inappropriate measure of the below-ground root-tip community, and vice versa (Gardes & Bruns, 1996a, b; Dahlberg et al., 1997; Jonsson et al., 1999). Likewise, analysis of root-tip and resistant propagule communities from the same samples demonstrated a minimal overlap between these two views of the fungal community (Taylor & Bruns, 1999). 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