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Vertical distribution of ectomycorrhizal fungal taxa in a podzol soil profile Blackwell Publishing Ltd.

A. Rosling1, R. Landeweert2, B. D. Lindahl1, K.-H. Larsson3, T. W. Kuyper2, A. F. S. Taylor1 and R. D. Finlay1 1

Department of Forest Mycology and Pathology, SLU, Box 7026, 750 07 Uppsala, Sweden; 2Sub-Department of Soil Quality, Wageningen University, Box

8005, NL-6700 EC, Wageningen, The Netherlands; 3Botanical Institute, Göteborg University, Box 461, SE-405 30 Göteborg, Sweden

Summary Author for correspondence: Anna Rosling Tel: +46 18 671864 Fax: +46 18 673599 Email: [email protected] Received: 11 April 2003 Accepted: 9 May 2003 doi: 10.1046/j.1469-8137.2003.00829.x

• Studies of ectomycorrhizal fungal communities in forest soils are usually restricted to the uppermost organic horizons. Boreal forest podzols are highly stratified and little is known about the vertical distribution of ectomycorrhizal communities in the underlying mineral horizons. • Ectomycorrhizal root tips were sampled from seven horizons in three continuous columns of a 52-cm deep podzol profile. Root tips were sorted into morphological groups and the colonising fungi identified by sequencing of the rDNA ITS region. The vertical distribution of mycorrhizal taxa was examined. • A relationship between ectomycorrhizal species composition and soil horizon was found. Tomentellopsis submollis, three Piloderma species and Dermocybe spp. were found predominantly in the upper horizons while Suillus luteus, Lactarius utilis and three undescribed Piloderma species were associated with the mineral horizons. • Two thirds of the root tips were found in the mineral soil and half of the taxa were restricted to the mineral horizons. The results highlight the need to include the mineral soil in order to gain a more accurate representation of the ectomycorrhizal community. Key words: boreal forest, diversity, ectomycorrhiza, Piloderma, podzol, vertical distribution. © New Phytologist (2003) 159: 775–783

Introduction Boreal forests characteristically develop podzol soils. Slow decomposition rates in these ecosystems lead to the development of a surface layer of organic matter, where partial decomposition results in formation of organic acids, which percolate with rainwater through the soil. In the underlying, upper mineral soil, soluble complexes are formed between the organic acids and Fe and Al, creating a weathered, eluvial E horizon. The organic matter-metal complexes percolate further through the profile and precipitate below the E horizon, creating a characteristic rust coloured illuvial B horizon overlying the C horizon parent material. As few burrowing animals thrive in these soils, mixing is limited, leading to the conservation of visible horizons in the soil profile (Lundström et al., 2000; van Breemen et al., 2000). Podzol soils are poor in easily accessible nutrients, and plants and microorganisms compete for the scarce resources (Lindahl

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et al., 2002). Symbiotic ectomycorrhizal fungi colonise the fine roots of boreal forest trees and play an essential role in nutrient uptake (Smith & Read, 1997). Although the highest fine root density in boreal forest soils is found in the organic and upper mineral soil horizons (Persson, 1980; Sylvia & Jarstfer, 1997; Makkonen & Helmisaari, 1998), tree roots can be found at greater depths (Jackson et al., 1996). At all depths, fine roots are colonised by ectomycorrhizal fungi (Egli, 1981), yet most ectomycorrhizal fungal community studies restrict sampling to the upper, organic part of the soil profile (Horton & Bruns, 2001) and thus ignore the ectomycorrhizal root tips in the deeper mineral soil layers. Chemical and mineralogical properties of soils change with depth, creating a number of different habitats for microorganisms, and the ectomycorrhizal fungal community is likely to change throughout the soil profile. Results from studies that have examined the distribution of morphologically defined

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ectomycorrhizal taxa in soil, either directly in soil samples (Egli, 1981; Goodman & Trofymow, 1998; Fransson et al., 2000) or on bait seedlings in organic and mineral substrates (Danielsson & Visser, 1989; Heinonsalo et al., 2001), suggest that there may be large differences in species composition between the organic layer and the mineral soil. Molecular techniques and the use of sequence databases enable identification of ectomycorrhizal taxa with high resolution (Horton & Bruns, 2001). Recently, Dickie et al. (2002), using T-RFLP analysis of DNA extracted from soil mycelium, found differences in ectomycorrhizal species composition between different components of the forest floor (L, F and H layers) and the B horizon of the mineral soil in a North American Pinus resinosa stand. Zhou & Hogetsu (2002) used T-RFLP to map the three-dimensional distribution of ectomycorrhizal root tips in a Japanese Larix kaempferi stand but found no clear vertical distribution patterns. In the present study, root samples were collected from a Swedish podzol and the distribution of ectomycorrhizal taxa on the root tips was investigated in relation to the location of the roots in different soil horizons. Samples were collected down to a depth of 52 cm and included the different components of the organic and mineral soil (the O, E, B and C horizons). Fungal taxa were identified from ectomycorrhizal root tips to genus or species level using a combination of morphological identification and sequencing of the rDNA ITS region. The aim of this study was to investigate whether the ectomycorrhizal community differed in species composition between different mineral and organic horizons in a podzol soil. This information is an important prerequisite when attempting to assign ecological niches to individual ectomycorrhizal species.

Materials and Methods Study site Soil samples were collected in August 1999 from three columns dug in a podzol profile at a mixed coniferous forest site in the north of Sweden (Nyänget, 64°15′ N, 19°45′ E). The soil has developed from basal glacial till. The dominant tree species were 60–80 yr old Norway spruce (Picea abies [L.] Karst.) and Scots pine (Pinus sylvestris L.) with undergrowth consisting mainly of Vaccinium myrtillus L., V. vitis-idaea L. and Deschampsia flexuosa [L.] Trin. (Ilvesniemi et al., 2000). Soil samples Three 20 × 20 cm vertical soil columns were collected from locations 6 m apart. Columns 1 and 3 were situated close to spruce trees and column 2 close to a pine tree. Each column consisted of four distinct soil horizons: an upper organic horizon (O), a strongly weathered eluvial horizon (E), an enriched illuvial horizon (B) and the parent material (C). The soil horizons were distinguished by their colour and found at

the following average depths: O, 0–3 cm; E, 3–18 cm; B, 18– 35 cm, and C, 40–53 cm. The E horizon was further divided into an upper E1 with visible organic matter and a lower E2 horizon with visibly less organic matter. The B horizon was also further divided into a upper strongly illuvial B1 and a lower partially illuvial B2. An intermediate EB layer was distinguished in two of the columns (1 & 2) where there was no sharp transition between the E and the B horizons. The soil samples from the O horizon down to the B2 horizon were contiguous in each column. To ensure pure parental material, the C-horizon samples were taken close to the bottom of the column, resulting in a gap between the B2 and the C samples. The soil was sealed in plastic bags, transported back to the laboratory and stored at +5°C. Soil and stone (> 1 cm) volume from each sample was recorded. Soil samples were coded according to column number followed by the seven horizon codes O, E1, E2, EB, B1, B2 and C. Extraction and morphological identification of root tips From each of the three intact O horizon samples, three subsamples (17–20% of sample volume) were taken using a 3-cm diameter corer. The whole sample volume was examined in mineral soil samples with low root tip density (i.e. all C horizons, E2 and B2 of column 2 and 3 and B1 of column 3). Other mineral soil samples were carefully mixed before subsampling according to differences in root tip density. In EB and B1 in column 2, 50% of the volume was examined and in the remaining samples, 25% of the volume was examined. Each soil sample was soaked in water for 15 min before roots were extracted by wet sieving using a combination of 2, 1 and 0.5 mm sieves. Root tips were collected from the sieves using a dissection microscope and forceps. No distinction was made between root tips of pine and spruce. Living root tips from each sample were organised into morphological groups according to macro- and microscopic criteria (Agerer, 1987–1998). For each sample the number of root tips in each morphological group and the number of nonmycorrhizal root tips were recorded. The first time a morphological group was distinguished, five representative root tips from the group were selected for genetic identification and individually frozen (−70°C) in micro centrifuge tubes. On the subsequent occasions, when the same morphological group was found, one or two representative root tips were selected and frozen. Genetic identification by DNA extraction, amplification and sequencing DNA was extracted from individual root tips (Gardes & Bruns, 1993) excluding the initial freeze-thawing step. Following a modification of the protocol described by Henrion et al. (1994) the ITS region of the rDNA was amplified by PCR (Mullis & Faloona, 1987). The universal primers ITS1 and ITS4 (White et al., 1990) or the fungal

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specific primer ITS1f and the basidiomycete specific primer ITS4b (Gardes & Bruns, 1993) were used, depending on which primer pair produced the best yield and purest PCR products. PCR was performed in 50 µl and the final concentrations of the reaction mix were the following: 0.2 m of all four nucleotides, 0.3 µ of each primer, 3.1 m MgCl2 and 0.026 U µl−1 of DNA polymerase (Expand™ High Fidelity PCR System, Boehringer Mannheim, GmbH, Germany). Optimal DNA template concentrations were established individually for each sample by testing amplification success using dilution series. DNA template was added as 25% of the final reaction volume. The PCR program started with denaturation at 94°C for 3 min, followed by 35 cycles of 94°C for 30 s, 50°C for 45 s and 72°C for 60 s. The quantity and quality of PCR products were examined by gel electrophoresis (Gardes & Bruns, 1993) and visualised using a GelDoc™ 2000 Gel Documentation Systems and Quantity One v.4.1.0 software (Bio-Rad, Laboratories Svl, Segrate, Italy). Double-banded PCR products were separated on 1% agarose gel at 90 V for 3–4 h. Gel plugs were cut out from the bands with a Pasteur pipette and dissolved in 200 µl of deionised water overnight. Separated bands were re-amplified using the same PCR procedure as used in the initial amplification. From 15 root tip samples, where no species identity was obtained using ITS sequencing, additional sequencing the first 400 bases of the large subunit was performed using the primers ITS3 and Lr21 (Hopple & Vilgalys, 1999). The PCR protocol was modified for this reaction by increasing the annealing temperature to 52°C and running only 30 cycles. PCR products were purified using the QIAquick PCR Purification Kit (250) (Qiagen, GmbH, Germany). Each sample was separately sequenced with both the primers that had previously produced the best PCR product. For samples with problematic sequence products, additional sequencing was performed using the internal primers ITS2 and/or ITS3 (White et al., 1990). Sequence reactions were performed in 10 µl reaction volume, using 4 µl TRRM (ABI PRISM™ BigDye™ Terminator Cycle Seq Kit, Applied Biosystems, Foster City, CA, USA) and a final primer concentration of 0.32 µ; purified PCR product made up 25% of the final sequence reaction volume. The sequencing program performed 25 cycles of 96°C for 10 s, 50°C for 5 s and 60°C for 4 min. The sequence products were purified by ethanol precipitation, resuspended in Template Suppression Reagent (TSR) at 96°C for 2 min and analysed using an ABI PRISM™ 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The electrophoretograms of single stranded sequences were visually examined and pair wise aligned using Sequence Navigator™ v.1.0.1. (Applied Biosystems Foster City, CA, USA), in order to obtain one sequence for each sample. Obtained sequence length and region of overlap between single stranded sequences varied between samples depending on the species-specific length of the ITS rDNA region as well


as the quality of the sequence products. In most cases, the single stranded sequences were clear and the end strands were included in addition to the consensus region as representing the root tip sample. To determine the degree of homology within each morphological group, all sequences within the group were aligned using ClustalW at EMBL (Thompson et al., 1994). Sequences within a morphological group frequently clustered into more than one homology group. If the sequence homology between clusters was less than 95% these were considered to be different sequence homology groups. One representative sequence from each sequence homology group was selected. To determine homology between morphological groups, alignment was repeated using all the selected sequences from all morphological groups. The obtained sequence homology groups (minimum 95% homology) were considered to represent individual taxa. The identity of these taxa was investigated by comparing representative sequences with sequences in the GenBank database at NCBI using the BLAST program (Altschul et al., 1997). Names were assigned to each taxa according to the obtained BLAST matches (Table 1). The identification of several Piloderma species was made possible by comparing the obtained sequences to unpublished sequences obtained from sporocarps (K.-L. Larsson, unpublished). Because of unsatisfactory resolution of morphological groups into genetically identified taxa, a number of taxa encompass subgroups. The homology between these subgroups is greater than 90%.

Results Root distribution and mycorrhizal colonisation Root tip density was highest in the organic horizon, with 1800–3300 root tips dm−3, declining to 8.1 ± 3.6% of the O horizon density in the eluvial horizon (E2), and increasing again in the illuvial horizon (B) to 19.6 ± 3.3% of the O horizon value. The lowest density, 3.7 ± 1.1% of the O horizon density was found in the C horizon. However, because of the greater thickness of the mineral horizons, almost two thirds (65%) of the total number of root tips from all three columns were found in the mineral soil. The average numbers of root tips in the E1, B1 and B2 horizons were, respectively, 64%, 53% and 53% of that in the organic horizon (Fig. 1). Root tip numbers were distinctly lower in the E2, EB and C horizons; 19%, 14% and 15% of the number in the O horizon (Fig. 1). The degree of mycorrhizal colonisation varied between 60% and 98% and no clear patterns could be seen with respect to depth or soil column. Identification of mycorrhizal taxa Using morphological characters, 39 morphological groups were distinguished from a total of 8275 examined root tips.

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778 Research Table 1 Genetically identified ectomycorrhizal taxa sampled from a boreal forest podzol. (a) The 19 mycorrhizal taxa distinguished by genetic identification and included in analysis of vertical distribution. (b) Lists the three taxa that were genetically identified but were excluded from further analysis Taxa (a) Piloderma reticulatum Suillus luteus Cortinarius spp.

Russula decolorans Piloderma sp. 2 Piloderma fallax Tylospora spp. Lactarius utilis Piloderma byssinum Tomentellopsis submollis Russula adusta Piloderma sp. 3. Dermocybe spp.

sub

A B C D

A B A B

A B C

Piloderma sp. 1 Tricholoma portentosum Piloderma sp. JS15686 Wilcoxina rehmii Unidentified#15 Unidentified#12 (b) Helotiales spp.

Black roots

Unidentified doubles

A B C D E F A B C

T bp

Acc nr

Best match

956 786 543 637 649 545 661 950 926 723 568 913 680 557 964 977 731 744 591 770 935 790 903 501 495 747

AF476978 AF476994 AF481374 AF481376 AF476972 AF481377 AF476996 AF476971 AF476982 AF481388 AF476969 AF476970 AF476975 AF477003 AF476974 AF476997 AF476986 AF476989 AF481380 AF481379 AF476998 AF477002 AF476984 AF476993 AF477000 AF477004

Unpublished S. luteus, AJ272413 C. traganus, AF037224 C. umbilicatus, U56032 C. traganus, AF335446 C. traganus, AF335446 R. decolorans, AF418637 New Piloderma sp. P. fallax, AY010281 P. fallax, AY010281 T. fibrillosa, AF052563 T. asterophora, AF052560 + AF325323 L. utilis, AJ534936 P. byssinum, AY010279 Tomentellopsis sp., AJ410766 Russula adusta AY067652 + AF218544 New Piloderma sp. Dermocybe sp. AF323113 C. scaurus AF325563 D. crocea U56038 New Piloderma sp. T. portentosum, AB036896 unpublished W. rehmii, AF266708

882 527 432 402 464 344 469 638 397 470

AF476977 AF476973 AF481384 AF481371 AF481372 AF481370 AF476968 AF476967 AF481389 AF476985

Phialophora finlandia, AF486119 Phialophora finlandia, AJ534704 Salal mycorrhial fungi, AF149082 Phialocephala fortinii, AY078134 Axenic ectomycorrhizal AJ430403 Piceomphale bulgarioides, Z81441 Cenococcum geophilum, AY112935 Pseudotomentella tristis, AF274772 Helotiales sp., AJ308340

H bp

%

657 543 569 649 542 650

99 94 95 95 96 100

569 595 546 885 680 541 577 965

99 95 100 99 95 100 100 99

560 590 635

90 96 100

790

97

501

99

447 513 414 402 454 327 468 503 397

99 98 99 100 97 93 99 97 99

Species or genus names were assigned to most taxa according to sequence homology with sequences from GenBank. Taxa within the genus Piloderma were named according to phylogenetic analysis using unpublished sporocarp sequences. The assigned species or genus names are listed in the first column under ‘Taxa’. Taxa that are only identified to the genus level commonly encompass genetic subgroups, these are listed from A–D in the column ‘sub’. The number of base pairs in the sequence representing the taxa is listed in the column ‘T bp’ and the assigned accession number of sequences submitted to GenBank are listed under ‘Acc nr’. The identified name is listed under ‘Best match’. GenBank matches (17th of January, 2003), are found with sequence name and accession number. The column is left blank for taxa that remained unidentified after alignment to GenBank sequences and reads unpublished for Piloderma taxa identified by phylogenetic analysis. The number of base pairs over which the homology is calculated is presented under ‘H bp’ and the degree of homology is presented as percent base pair identity between the best match and the representative sequence of the taxa, under ‘%’.

Of 247 root tips selected for genetic identification, 75% were successfully sequenced. Alignment with GenBank sequences, as well as with unpublished sporocarp sequences, enabled identification of 95% of the sequenced mycorrhizal tips (Table 1). Of the initial 39 morphological groups, no

sequences were produced from six. Of these, three were represented by few mycorrhizal tips (< 8) and were removed from the analyses. The remaining three, Inocybe sp., Hygrophorus olivaceoalbus and an unidentified Tomentelloid taxon were considered well identified by morphological


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Fig. 1 Average total number of root tips in each soil horizon (O, E1, E2, EB, B1, B2 and C) expressed as the percentage of total number of root tips in the organic horizon. Error bars represent SE of the mean (n = 3).

characters. The remaining 33 morphological groups could be rearranged into 22 taxa (Table 1) on the basis of genetic identification. Eleven of these taxa could be identified to species level and nine to genus level. Two taxa remained unidentified. Of these 22 taxa, three were excluded from further analyses of vertical distribution because of weak correlation between morphological group and genetic identity of sequenced root tips (Table 1b). Furthermore, these three taxa also occurred as secondary colonisers (i.e. double bands) of root tips colonised by other taxa. Apart from one unidentified double colonizer, the removed groups consisted of morphological groups characterized by their black mantles, including root tips colonized by Cenococcum geophilum, Pseudotomentella tristis and Helotiales spp. In the end, the vertical distribution of 22 mycorrhizal taxa was analysed. Seven taxa belonging to the genus Piloderma were recognised. Four of the seven taxa, P. reticulatum, P. byssinum, P. fallax and Piloderma sp. JS15686, were identified to species level by sequence alignment with unpublished sporocarp sequences. The other three, Piloderma spp. 1, 2 & 3, were assigned to the genus based on phylogenetic analysis (K.-H. Larsson, unpublished). The genus Piloderma colonised 52% of all root tips in this study. P. reticulatum was by far the most abundant taxon colonising 41% of all sampled root tips. The second most abundant taxon was Suillus luteus. From horizon EB to B2 in column 2 it colonised 76% of all root tips and in horizon E2 to B2 in column 3 it colonised 21%. The taxon Tylospora spp. encompassed two different species, identified as T. fibrillosa and T. asterophora. Using morphotyping it was not possible to discriminate consistently between the mycorrhizas formed by the two species and they were therefore merged into a single taxon. Morphological


separation within the genus Cortinarius was also low. The taxon Dermocybe could however, be distinguished. It was impossible to distinguish different Cortinarius species satisfactorily using morphotyping and these were therefore grouped into the taxon Cortinarius spp., containing at least three species. Dual colonisation of mycorrhizal root tips In 93 root tip samples (38% of all samples) double PCR bands were observed as a result of amplification with ITS1 and ITS4. From most double-banded samples a single PCR product was obtained when amplifying the samples with the basidiomycete specific primer ITS4b, instead of the universal primer ITS4. Successful gel separation of double PCR products from 19 individual roots resulted in two sequences from each sample. Of these, each root tip generally yielded one sequence homologous to the sequence of the visible ectomycorrhizal coloniser. Apart from this sequence, the majority of double bands yielded sequences within the monophyletic group of Helotiales spp. These sequences were also amplified from root tips that were morphologically identified as Piceirhiza bicolorata. Six double band sequences formed a separate taxon of unidentified double colonizers (Table 1b). The frequency of double bands varied among the different ectomycorrhizal taxa. The occurrence of doublebanded PCR products in 18 out of the 19 analysed root tips colonised by S. luteus was particularly striking. Vertical distribution of mycorrhizal taxa The distribution of the 22 distinguished mycorrhizal taxa in the soil profile is presented in Table 2. Four taxa were identified on root tips from horizons throughout the whole profile.

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780 Research Table 2 Vertical distribution of mycorrhizal taxa throughout the podzol profile O

Tylospora spp. Cortinarius spp. P. reticulatum Piloderma sp. JS15686 Inocybe P. byssinum T. submollis P. fallax H. olivaceoalbus R. decolorans Dermocybe spp. Tomentelloid L. utilis Piloderma sp2 Piloderma sp3 Piloderma sp1 S. luteus unID#15 unID#12 Wilcoxina R. adusta T. portentosum

E1

1

2

I I III

I

3

1

E2 2

I I IV *

III

3

1

*

I

IV *

II

I I

I

I *

I

EB

B1

2

3

1

2

1

* I II

I

IV

I I

II

B2 2

3

1

II

I II I

I

C 2

3

I III I

I

1

2

III I I

3 I I

I I I I I

I I

I

IV *

IV *

I

I I I

II I

* I I I I

I

II I I

I I III

II I I

I I III

II I II

I IV I

I

I

I I IV III

The occurrence of taxa in the horizons O, E1, E2, EB, B1, B2 and C is given for the three soil columns, 1, 2 and 3. Relative abundance of each taxon in each horizon is indicated according to the following intervals * = < 1%, I = 1–25%, II = 26 –50%, III = 51–75% and IV = 76 –100%.

Piloderma reticulatum was common throughout the profile of two columns and Piloderma sp. JS15686 was sparsely detected throughout the profile of column 3. The documented broad distribution for Tylospora spp. and Cortinarius spp. may well be an artefact caused by unsatisfactory morphological separation within these taxa. Two taxa, Inocybe and Piloderma byssinum, were restricted to the organic horizon. The remaining 5 taxa found in the organic horizon, Tomentellopsis submollis, Piloderma fallax, Hygrophorus olivaceoalbus, Russula decolorans and Dermocybe spp., were also found to colonize root tips thoughout the E horizons. Of the 22 recognised taxa, 11 were found only in the mineral horizons. Suillus luteus was common throughout the mineral soil in two columns, colonizing root tips from the lower E horizon downwards. The taxon Tomentelloid was rare and was detected only in E horizons of column 3. Lactarius utilis and the three new Piloderma sp. (1, 2 & 3) were found to colonize root tips in the central horizons of the profile. Two unidentified taxa, unID#12 and unID#15 were found only on roots in the B2 horizon. Three of the taxa in mineral soil, Wilcoxina rehmii, Russula adusta and Tricholoma portentosum, were found only in the C horizon. (Table 2). Chemical properties of the mineral soil were analysed but, as no relationship could be found between the species composition and the chemical properties of the mineral horizons in which they occurred, data are not shown.

Discussion Most studies of below ground ectomycorrhizal diversity have focused on the upper organic horizon where root tip density is high. In the present study, although root tip density was highest in the organic horizon, two thirds of all root tips in the 53 cm deep soil columns were recovered from the mineral soil. The high root tip density in the organic horizon found in this study is in line with earlier findings (Jackson et al., 1996). The majority of taxa typically occurred in only part of the soil profile (Table 2). When occurring in several horizons, taxa generally had a continuous distribution over adjacent horizons rather than a discontinuous distribution. The uneven distribution of species throughout the columns could be a result of single mycelial individuals colonising a large number of adjacent root tips (Zhou et al., 2001; Taylor, 2002). Most taxa that were found in the organic layer were also found in the upper eluvial soil horizon (E1). The major separation in species composition was thus found between the organic and eluvial horizons on one hand and the deeper mineral soil on the other (Table 2). The similar species composition of the eluvial soil and the organic horizon could possibly be explained by the relatively large amount of organic material present in the upper mineral horizons, particularly in E1. Dickie et al. (2002) found differentiation in species composition even between different parts of the forest floor (the O


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horizon). By pooling all components of the forest floor (L, F and H), the present study may have missed parts of the vertical variation. However, by including deeper parts of the mineral soil and separating the different mineral soil components this study included variation that was not covered by Dickie et al. (2002). In the present study one half of the ectomycorrhizal taxa were found exclusively in the mineral soil horizons. The dominance of taxa belonging to the genus Piloderma in this study, is consistent with the ecological importance of this genus in many boreal forest ecosystems (Erland & Taylor, 1999). Piloderma fallax, a species commonly identified in other studies (often referred to as P. croceum), was restricted to the organic and eluvial horizons. Goodman & Trofymow (1998) found P. fallax exclusively in the organic horizon. In a study by Heinonsalo et al. (2001), P. fallax was found on bait seedlings planted in humus material (O horizon) but not on those planted in mineral soil (B horizon). Piloderma byssinum was restricted to the O horizon, but it was only found in one column. The most abundant species in this study, Piloderma reticulatum, occurred predominantly in the O and E horizons. The three other Piloderma taxa that were found in this study appear to be undescribed species. They were all restricted to the mineral soil. Piloderma species do not form conspicuous fruiting bodies and the possible restriction of these new species to the deeper mineral soil may explain why they have not been found on roots in earlier investigations. Tomentellopsis submollis appears to be restricted to the upper part of the profile. Pink morphotypes, most likely T. submollis, are regularly identified on roots from the organic horizon of Scots pine in Fennoscandia (Kõljalg et al., 2002). Suillus luteus occurred in two columns in which it was only found in the mineral soil, occurring from the lower eluvial (E2) horizon and downwards. Both Danielsson & Visser (1989) and Heinonsalo et al. (2001) suggest that Suillus species constitute a higher relative proportion of the total mycorrhizal community on bait seedlings planted in mineral soil than on seedlings planted in organic material. The greater abundance of the pine specific Suillus species in the mineral soil could partially be explained by the greater rooting depth of pine compared to spruce (Mikola et al., 1966). Dermocybe spp. were almost exclusively found in the E-horizon whereas other Cortinarius spp. were predominantly found from the E2 horizon downwards. Species within the genera Suillus and Cortinarius have been highlighted among those forming numerous fruit bodies although being weakly represented in communities of fungi colonising root tips in the organic soil horizon (Gardes & Bruns, 1996; Dahlberg et al., 1997). The common practice of excluding the mineral soil from below ground community studies undoubtedly contributes to the discrepancy frequently observed between perceived above- and below-ground mycorrhizal community structures (Horton & Bruns, 2001). In this study three different sequence groups were obtained from roots with black mantles. Sequences homologous to


Cenococcum geophilum amplified only from root tips found in the organic horizons. These data correspond with those of Goodman & Trofymow (1998) and Fransson et al. (2000) who both found that C. geophilum was more common in the organic layer than in the mineral soil. In the present study Phialophora finlandia sequences were obtained from root tips in other horizons. This is in agreement with the findings of Heinonsalo et al. (2001) where a black morphotype was found to be C. geophilum when colonising roots in the organic soil and P. finlandia when colonising roots in the mineral soil. Phialophora forms an ectomycorrhizal morphotype, commonly referred to as Piceirhiza bicolorata, that can be mistaken for a roughly defined Cenococcum morphotype (Vrålstad et al., 2002). Consequently the abundance of P. bicolorata in the field has been suggested to be greatly underestimated (Vrålstad et al., 2002). The high occurrence of amplification of DNA from fungi other than the main visible mycorrhizal coloniser may be partly explained by the occurrence of ascomycetous double colonisers that coexist with the mycorrhizal fungi on the roots. In other cases, the additionally amplified DNA could be ascribed to other ectomycorrhizal fungi that were abundant in the same soil sample. In laboratory microcosms, the secondary colonisation of mycorrhizal root tips already colonised by mycorrhizal fungi has been observed (Wu et al., 1999). During replacement of one fungus by another, root tips were simultaneously colonised by two fungi. Sometimes conflicting results of morphological and genetic identification may thus be a consequence of the dynamic character of the mycorrhizal community. A potential source of error in genetic identification of the fungal partner of mycorrhizal root tips may be caused by the systematic variation in the efficiency of extraction as well as efficiency of DNA amplification as a result of species-specific sequence differences at the primer site (Glen et al., 2001). It is thus possible that secondary colonisers are present without changing the morphology of the mycorrhiza and still amplify more strongly than the primary coloniser. In addition to the present study, a parallel study was conducted (Landeweert et al., 2003) in which amplification and cloning of soil DNA was used to investigate the distribution of extraradical mycelium in a parallel set of soil samples collected simultaneously with the root samples examined in this study. In general, the same species were detected when comparing the ectomycorrhizal species composition on root tips with the species composition of extraradical mycelia. Of 16 ectomycorrhizal species identified in DNA extracts from root free soil, all but one were also found on root tips. In several cases species that were abundant on root tips were not detected in soil extracts (e.g. S. luteus and P. reticulatum), and others that were abundant in soil extracts were rarely detected on roots (e.g. Dermocybe spp. and Piloderma sp. 3). In conclusion, the results of the present study indicate that there may be significant variation in ectomycorrhizal species

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composition between soil horizons of boreal forest podzols. In some of these soils, a high proportion of the total number of root tips is present in the deeper mineral horizons and more importantly some ectomycorrhizal taxa may be restricted to these deeper horizons. At present there is still little available information about the processes determining this distribution but it is clear that further studies of ectomycorrhizal diversity and function should include the roots sampled from deeper mineral horizons.

Acknowledgements We gratefully acknowledge financial support from The Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS) and from The Netherlands Organisation for Scientific Research (NWO).

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