Introduction

The genus Tomentella (Thelephoraceae, Basidiomycota) forms ectomycorrhizas (ECM) with different plant hosts, and develops a well-defined, frequently found morphotype. Though Tomentella fruit-bodies are inconspicuous and rarely found, these fungi are among the most abundant and diverse mycobionts in ectomycorrhizal fungus communities from arctic regions to the tropics (Dahlberg et al. 1997; Trowbridge and Jumpponen 2004; Haug et al. 2005). Based on basidiocarp studies, Tomentella species are distributed mainly in temperate Eurasia (Köljalg 1996; Köljalg et al. 2000) and North America (Larsen 1974). However, they are reported from other continents and islands, e.g., the tropics of India (Thind and Rattan 1971), Korea (Jung 1994) and the Canary Islands (Larsen 1994), and the occurrence of their ECM in South America (Haug et al. 2005), Africa (Yorou et al. 2007) and Australia (Agerer and Bougher 2001a, b) indicates a worldwide distribution, with likely many as yet undetected species.

Host plants of Tomentella ectomycorrhizas belong to several families; however, the main hosts are gymnosperm (Wurzburger et al. 2001; Haug 2002; Burke et al. 2005; Cline et al. 2005; Douglas et al. 2005; Hibbett et al. 2005) and angiosperm trees (Brand 1991; Pritsch et al. 2000; Walker et al. 2005). Although tomentelloids mostly develop typical ectomycorrhizas with trees (Brand 1991; Taylor and Bruns 1999; Köljalg et al. 2000; Jakucs et al. 2005a, b; Tedersoo et al. 2007a), they may also form endomycorrhizas with herbaceous plants and shrubs belonging to the Orchidaceae, Ericaceae, Monotropaceae, and Pyrolaceae (Bidartondo et al. 2000, 2004; Bidartondo and Bruns 2001; Selosse et al. 2002; McCormick et al. 2004; Jun et al. 2005; Julou et al. 2005; Tedersoo et al. 2007b). Given their typically wide host ranges, Tomentella species may be important members of “common mycelial networks” (CMN, Selosse et al. 2006) by connecting different hosts, e.g., trees with photosynthetic and non-photosynthetic herbaceous plants (Shefferson et al. 2005). In the case of achlorophyllous partners, the monotropoid or orchid plant is totally or partially myco-heterotrophic, invading the ECM symbiosis as a “cheater”, and carbon is transferred from ECM to the epiparasitic plant (Taylor and Bruns 1997; McKendrick et al. 2000; Taylor et al. 2003; Selosse et al. 2006).

Some authors suppose that the ECM of Tomentella are frequent in the mineral horizon of soils poor in organic material (Harrington and Mitchell 2005; Baier et al. 2006). This occurrence is characteristic of the smooth or weakly hairy “contact exploration type” ECM (Agerer 2001) to which the majority of tomentelloids belong. In contrast, in broad-leaved forests a significant portion of tomentelloid ECM occur in the organic horizon (Tedersoo et al. 2003), often attached to plant foliar debris; many species form slightly or highly differentiated rhizomorphs, indicating that these morphotypes belong to the “medium distance exploration type” (Agerer 2001). These ECM types generally occur in the organic layer of soils where they may also exhibit saprotrophic activity. Their mycelial networks may actively digest plant and insect remnants. Other fungal species in the Thelephorales (e.g., Thelephora terrestris Ehrh.) produce exocellular enzymes (cellulases, oxydases, polyphenoloxydases) (Burke and Cairney 2002). However, because it is difficult to obtain in vitro cultures of Tomentella species and their mycelia grow very slowly on agar media, experimental data on digestive enzyme production by Tomentella are lacking.

Although Tomentella ectomycorrhizas are common members of ECM communities, little information exists on the morphology, taxonomy, host specificity, geographic distribution, and ecology of most species. Few Tomentella ectomyorrhiza morphotypes are accurately identified in the literature and molecular databases contain a large number of tomentelloid DNA sequences from environmental samples without indicating their exact specification. More than 70 polymerase chain reaction (PCR)-based studies of ECM community structure report the presence of Tomentella ECM (e.g., Comandini et al. 1998; Buée et al. 2004; Kaldorf et al. 2004; De Román et al. 2005; Murat et al. 2005; O’Brien et al. 2005; Baier et al. 2006), but most of these studies lack morphological analysis of the ECM.

The aim of this paper is to review the present state of our knowledge about the morphological-anatomical characteristics of Tomentella ECM. In order to support extended molecular investigations on geographic distribution, ecological role and diversity of this common and widespread mycorrhizal group, we also present a short summary on microscopic features suitable for selecting tomentelloids during morphotyping of ECM root samples.

The taxonomic position of Tomentella ECM

Tomentella ECM are formed by aphyllophoraceous fungi positioned within the Thelephorales (Agaricomycetes, Basidiomycota) (Donk 1964; Corner 1968). The order was taxonomically revised by Stalpers (1993). The morphology of the often dark-colored basidiocarps is highly diverse, varying from resupinate to effused. A characteristic apomorphy (commonly derived phylogenetic character) of the order is the irregularly shaped, non-amyloid, ornamented, and often dark basidiospore with a large apiculus (Larsson et al. 2004). The presence of thelephoric acid, a blue cytoplasmatic pigment specific for the Thelephorales, is also a common character of the order (Agerer et al. 1995).

Stalpers recognized two families: the Bankeraceae Donk /Syn.: Boletopsidaceae Bond. & Singer ex Jülich/ and the Thelephoraceae Chev. /Syn.: Phylacteriaceae Maire; Tomentellaceae Bref.; Botryohypochnaceae (Parm.) Jülich; Lenztiopsidaceae Jülich/ (Stalpers 1993). According to recent molecular phylogenetic analyses, the Thelephorales form a monophyletic clade within the Agaricomycetes (Hibbett and Thorn 2001; Larsson et al. 2004; Lutzoni et al. 2004; Binder et al. 2005; Hibbett et al. 2007) as a sister group of the Polyporoid clade (Matheny et al. 2006). In the Thelephoraceae, Index Fungorum recognizes 72 valid species of Tomentella, in addition to 43 species of Thelephora, seven of Pseudotomentella, and four of Amaurodon, but more than 100 synonyms are indicated. Recent molecular studies show that the resupinate mycorrhizal genus Tylospora, previously positioned in the Thelephorales (Stalpers 1993), is closely related to Piloderma, Amphinema, and Byssocorticium, belonging to the Athelioid clade (Larsson et al. 2004). Surprisingly, though Tomentellopsis is generally accepted as the member of the thelephoroid clade (Larsson et al. 2004; Agerer 2006), Index Fungorum placed it also in the Atheliaceae.

Within the Thelephorales, there are several well-known ECM-forming genera in the Bankeraceae (Hydnellum, Bankera, Phellodon, Sarcodon) and the Thelephoraceae (Thelephora) with effused, clavarioid, flabelliform, or pileate basidiocarps (Agerer 2006). The ECM of Thelephora species, especially T. terrestris Ehrh., are common in pine forests and nurseries and are intensively studied as they are often used for seedling inoculation in reforestation programs (reviewed by Colpaert 1999). For general morphological characters of ECM within the thelephoroid clade, we refer to the recent summary of Agerer (2006).

The fruit-bodies of the genus Tomentella are resupinate, forming inconspicuous, spiderweb-like layers on the surface of soil, twigs, or other plant debris (Köljalg 1996). Therefore, these species had been considered as rare, saprotrophic, wood-decaying fungi (Larsen 1974). However, in the 1980s Tomentella species were shown to form ECM (Danielson and Pruden 1989). The Thelephoraceae species with resupinate sporocarps are often mentioned as “resupinate thelephoroid” or “tomentelloid” fungi (Köljalg et al. 2000), including also Pseudotomentella, Amaurodon, and even some resupinate Thelephora species (e.g., Thelephora caryophyllea (Schaeff.) Pers.) in addition to Tomentella. The ECM of resupinate ECM taxa is reviewed by Erland and Taylor (1999). On the phylogenetic trees constructed on the basis of ITS-rDNA (internal transcribed spacer region of the ribosomal DNA), Tomentella species are often intermingled within a common clade with Thelephora species of resupinate and erect fruit bodies (Köljalg et al. 2000; Binder et al. 2005), but based on microscopic ECM-characters, Thelephora and Tomentella species can be easily distinguished.

In his review on Tomentella and related genera in temperate Eurasia, Köljalg (1996) described 43 species and presented a phylogenetic tree based on morpho-anatomical fruit-body characteristics. However, his tree contradicts the phylogenetic trees that resulted from DNA analyses (Köljalg et al. 2000; Jakucs et al. 2005b). Within Tomentella ECM, the microscopic characteristics of ECM seem to correspond better to molecular results than to sporocarp morphology, as also observed in other taxa (Agerer 2006).

Our present review is restricted only to the ECM of the genus Tomentella because they are more common, they form a distinguished morphotype group, and differ significantly in microscopic characters from the ECM of the remaining resupinate taxa in the Thelephoraceae.

Morphological-anatomical characterization of Tomentella ECM

In the case of Tomentella ECM, microscopic investigation is particularly useful in community studies because these species can be separated relatively easily by morphotyping, and the majority of them have specific characteristics. Anatomical features of the identified tomentelloid ECM are included in the recent comparative review of Agerer (2006). However, for the approximately 70 known Tomentella species, only seven have been accurately identified and their ECM characterized in detail (De Román et al. 2005). In addition to these, several precise descriptions and image documentations of unidentified ECM appeared in Descriptions of Ectomycorrhizae and the Colour Atlas of Ectomycorrhizae. These ECM lack DNA-based identification but belong certainly to Tomentella, based on microscopic similarities. Table 1 summarizes the hitherto published, detailed morphological-anatomical descriptions of seven identified and 18 unidentified but likely Tomentella ECM. Several other tomentelloid ECM morphotypes have been described or documented in the literature, but we consider those descriptions to lack sufficient detail so we restrict our review only to detailed descriptions with complete image documentation.

Table 1 Identified and unidentified Tomentella ECM characterized morphologically in detail
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According to Köljalg et al. (2000), tomentelloid ECM mycobionts encompass species characterized by melanized hyphae and contain one or more of the following three criteria: presence of clamp connections, presence of cystidia, or of a greenish-blue color reaction in KOH indicating the presence of thelephoric acid. However, positive KOH reaction, observed in mantle cells (Fig. 1d) or the presence of cystidia (Fig. 1o) is quite infrequent within Tomentella. ECM of the genus are generally characterized by brown or brown-black mantles, or the color may be yellow, pale ochre, or whitish (Fig. 1a, b), depending on the concentration of cell wall pigments. In most cases, emanating hyphae of the mantle and hyphae of the rhizomorphs are clamped (Fig. 1c, 2e). In contrast to Thelephora, Tomentellopsis, and Pseudotomentella mycorrhizas, which have plectenchymatous mantles, most Tomentella ECM show a mainly angular, pseudoparenchymatous outer layer in their mantle. In some species, the angular cells are organized in a star-like pattern (Fig. 1f), or in others the cells are epidermoid (Fig. 1d, h). Cell wall thickness and the size and shape of cells are highly variable both among and within the species. Plectenchymatous ECM mantles are considered phylogenetically ancient compared to pseudoparenchymatous mantles (Agerer 1995). The presence of both types within the family may lead to the conclusion that the recent representatives of Thelephoraceae have a long evolutionary history (or accelerated evolution) and the pseudoparenchymatous mantle of the typical Tomentella ECM is the apomorphic state within this group.

Fig. 1
figure 1

Typical morphological-anatomical characteristics of tomentelloid ectomycorrhizas. a Monopodial-pyramidal ECM system with yellow-ochre mantle (Tomentella pilosa); b ECM tip with woolly emanating hyphae and dark-brown mantle (T. stuposa); c typical brown, clamped tomentelloid emanating hyphae*; d pseudoparenchymatous-epidermoid mantle with thelephoric-acid-containing cells, stained dark-blue in KOH*; e brown, pseudo-parenchymatous-angular mantle*; f groups of globular cells on the surface of mantle*; g pseudoparenchymatous mantle with star-like pattern*; h yellow, pseudoparenchamatous mantle composed of angular-triangular cells*; i pseudoparenchymatous-epidermoid mantle*; j clamped “fibulocystidium-type”cystidia with large basal cells (T. galzinii); k unclamped, awl-shaped cystidia*; l long, curved, unclamped, thick-walled cystidia*; m spiny cystidia with broad basal parts*; n undifferentiated rhizomorph with clamped hyphae*; o typical tomentelloid rhizomorph with a rind formed by thin, densely entwined, multi-branched, clamped marginal hyphae and cystidia containing black pigment in apical part (T. subtestacea). Asterisk Identified as Tomentella ECM by sequence analysis. a, b Stereomicroscopy, bar = 0.1 mm; c–o Nomarski-DIC, bar = 10 μm

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Fig. 2
figure 2

Emanating elements of Tomentella ECM. a–c Clamped cystidia (‘fibulocystidia’) of T. galzinii emerging from the mantle covered by amorphous gelatinous matrix; d cystidia of T. subtestacea originating from a surface network formed by horn-shaped cells (arrow); e ramification of clamped hypha of T. pilosa. SEM, bars = 10 μm. Photo: K. Bóka (with permission)

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The mantle surface may be covered by a gelatinous matrix in some Tomentella species (Fig. 2b, c) or may bear a hyphal net or a network of angular-triangular, horn-shaped cells on the surface of the mantle (Fig. 2d). In some species (e.g. T. stuposa), groups of globular cells are formed on the mantle (Fig 1e, f). “Quercirhiza squamosa” and some other morphotypes have scales of dead cell remnants on the mantle. Many species also have rhizomorphs or cystidia (Agerer 1995; Jakucs et al. 2005b). The structure of rhizomorphs of some Tomentella species is undifferentiated (like those of Thelephora), but some Tomentella ECM have differentiated rhizomorphs (Fig 1n, o). As rhizomorphs are functional organs involved in water and mineral transport, their morphology and structure indicates different transport mechanisms and exploration types (Agerer 2001 ). The typical Tomentella rhizomorph is characterized by bilateral, nodal ramifications and a rind formed by thin, clamped, densely entwined, multi-branched marginal hyphae as seen in T. subtestacea, T. pilosa, and T. ferruginea (Fig. 1o). The structural heterogeneity of rhizomorphs and the presence of highly specialized rhizomorph types also presume a long evolution history of the genus. Most Tomentella ECM have characteristic cystidia. A special, clamped cystidium-type (“fibulocystidium”) is regarded as an apomorphic character in the group formed by Tomentella galzinii, T. pilosa, and T. subtestacea (Köljalg et al. 2001) (Fig. 1j, 2a–e). The apical part of cystidia of T. subtestacea contains blackish-blue drops of the pigment thelephoric acid (Fig. 1o). In other (unidentified) species, unclamped, awl-shaped cystidia can be observed (Fig. 1k–m).

Specific features of Tomentella ECM

Table 2 contains a comparison of morphological-anatomical characteristics suitable for differentiation within the recently described Tomentella ECM.

Table 2 Differential characters of identified and unidentified Tomentella ECM
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To aid morphotyping of unknown ECM in root samples, we summarize the most important distinctive characteristics of Tomentella ECM. The presence of more than three of the following common features in any combination typically indicates a Tomentella ectomycorrhiza.

  1. 1.

    Color of the ECM black-brown, brown

  2. 2.

    Hyphae clamped

  3. 3.

    Structure of the outer mantle layer angular

  4. 4.

    Cells of mantle organized in star-like pattern

  5. 5.

    Surface network on mantle composed of hyphae or angular-triangular, horn-shaped cells

  6. 6.

    Groups of globular cells on mantle surface

  7. 7.

    Rhizomorphs with bilateral, nodal ramifications and a rind formed by thin, clamped, densely entwined, multi-branched marginal hyphae

  8. 8.

    Clamped cystidia

Because in this group molecular results highly correlate with mycorrhizal characters, further detailed descriptions of Tomentella ECM would contribute to solving taxonomic and phylogenetic problems in the Thelephoraceae. Accurate identification and precise microscopic investigations of Tomentella ECM would allow construction of a key to identify species of the genus for future diversity studies. Although morphological and anatomical analyses of ECM is time-consuming and requires experience (Agerer 1991), combining microscopy with DNA-based analyses is the most effective way to obtain valuable information on ECM community structure. Extending microscopic investigations of Tomentella ECM from new localities would highly support ecological studies on their role in different geographic regions and plant communities.