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Quercus ilex L. (Holm oak) is a characteristic evergreen oak species in the Mediterranean basin (Quézel, 1985; Scarascia-Mugnozza et al., 2000). Despite heavy anthropic pressure, old-growth forests still exist in the island of Corsica (Quézel & Médail, 2003). In such stands, overstorey oaks coexist with understorey chaparral shrubs such as Arbutus unedo L. (strawberry tree) and Erica arborea L. (tree heath). In general, oak trees do not exceed 200 yr of age (Panaïotis et al., 1997). In old-growth forests, mortality leads to tree falls that create numerous small-scale canopy gaps.
A wide variety of ectomycorrhizal (ECM) fungi are symbionts of many tree species in temperate climatic zones. More than 5000 species from the Ascomycetes and Basidiomycetes form ectomycorrhizas on secondary tree roots (Trappe, 1962; Smith & Read, 1997). The majority of ECM species have large host spectra (Molina et al., 1992). This allows a diffuse interaction, i.e. the sharing of common fungal associates by plant individuals of identical or different species. Ectomycorrhizas are critical for nutrition of both partners, and plant protection against soil parasites and toxic compounds. The mycorrhizal network can also reduce carbon costs of ectomycorrhiza formation for some plants, as the extraradical mycelium is already established and sustained by other plants (Högberg et al., 1999).
The fungi that form ectomycorrhizas with trees also form arbutoid mycorrhizas on the roots of ericaceous plants from the Arbutoidea suborder (e.g. Arctostaphylos and Arbutus spp.; Molina & Trappe, 1982). In addition to the fungal sheath and hyphal intercellular growth (Hartig net) that are typical of ECM, hyphae penetrate the cell wall and produce intracellular coils in living cells (Smith & Read, 1997). These fungi may mediate interactions between arbutoid plants and ECM trees. For instance, in Californian chaparral Arctostaphylos glandulosa Eastw. may allow the establishment of Pseudotsuga menziesii (Mirb.) Franco seedlings (Horton et al., 1999) by acting as a symbiont reservoir that may contribute to successional transition to forest stages. In the Mediterranean basin Q. ilex naturally establishes in A. unedo-dominated chaparral (Gamisans, 1999). However, sharing of fungal symbionts between Q. ilex and A. unedo has hitherto not been explored. To our knowledge, studies of A. unedo symbionts have mainly focused on mycorrhizal ultrastructure (Fusconi & Bonfante-Fasolo, 1984; Giovannetti & Lioi, 1990; Münzenberger et al., 1992).
Studies of ECM communities are based either on identification of mycorrhizas (the so-called below-ground view), or on monitoring of fruitbody production (above-ground view). Identification of mycorrhizas can be conducted according to root-tip morphotype or using molecular tools, such as restriction fragment length polymorphism (RFLP) or sequencing of the internal transcribed spacer (ITS) region, an efficient way to dissect ECM communities (Gardes & Bruns, 1996; Horton & Bruns, 2001; Tedersoo et al., 2003). Fruitbody surveys reveal the presence of ECM taxa in a fast and inexpensive way (Vogt et al., 1992; Richard et al., 2004). However these studies, mainly carried out on fleshy macromycetes, often underestimate the presence of numerous resupinate taxa (e.g. Thelephoraceae or Sebacinaceae), hypogeous fungi, and taxa lacking an apparent sexual stage (e.g. Cenococcum geophilum Fr.) (Horton & Bruns, 2001).
Little is known about the below-ground community of ECM fungi in broadleaved forests. For instance, most descriptions of ECM communities in Q. ilex forests have been based on fruitbody surveys (Signorello, 1996; Laganàet al., 1999; Richard et al., 2004). The problems with the use of fruitbody sampling are obvious to anyone who has collected fungi for many years. First, fruiting may vary tremendously from year to year. Second, sampling must be intensive because fruit bodies of many species are short-lived. Thus, in addition to the analysis of fruitbody patterns, there is a need to explore the ECM community in the soil from either ectomycorrhizas or mycelia. A study conducted recently by De Román & De Miguel (2002) has revealed the presence of numerous species of Thelephoraceae in managed Q. ilex stands. However, further research using molecular tools is necessary to document the below-ground diversity in Q. ilex forests.
In a previous study (Richard et al., 2004), we analysed the temporal and spatial patterns of fruitbody production in an old Holm oak forest in Corsica during three consecutive fruiting seasons. Fleshy epigeous macromycetes were surveyed in a permanent plot (160 × 40 m) from September 1999 to March 2002. Here we sampled ectomycorrhizas from Q. ilex and arbutoid mycorrhizas from A. unedo shrubs at the same research site in March 2001. On Q. ilex we collected ectomycorrhizas from seedlings, young saplings and old trees. Our objectives were to: (i) document the below-ground ECM richness in an old-growth Mediterranean forest; (ii) investigate two factors potentially shaping this richness, i.e. host age and host species; and (iii) relate the structure of the ECM community, as determined by mycorrhizas, to that obtained from fruitbody surveys. To identify the fungal symbionts on roots, we compared RFLP types from mycorrhizas to those from fruitbodies of known species collected from the same site. Dominant fungal associates of A. unedo were also sequenced to investigate in more detail the composition of the below-ground community. We relied on this typically Mediterranean plant species to ascertain the relative importance of fungal groups not sampled during our fruitbody survey, such as resupinate or hypogeous fungi.
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The present study confirms the remarkable species richness of the fungal community measured by fruitbody surveys of epigeous macromycetes at the same site (Richard et al., 2004). High values of species diversity estimators were obtained, as illustrated by Fisher's alpha (Table 3), an estimator linking the number of taxa to the number of individuals sampled, which is not unduly affected by sample size (Tokeshi, 1993). Assuming that each RFLP type corresponds more or less to one species (see below), a total of 140 species were detected based on analysis of mycorrhizas (one RFLP type per 2.5 tips investigated, excluding C. geophilum), compared with 166 species that have been found using fruitbody surveys (Richard et al., 2004). Based on these two approaches together, there were at least 260 ECM fungal species at the site between 1999 and 2002. This is more than in most previously described late successional stands covering similar areas (Jonsson et al., 1999; Bidartondo et al., 2000, 2001), although similar values were found in old temperate coniferous forests, either by ECM typing (Dahlberg et al., 1997; Luoma et al., 1997) or fruitbody surveys (Villeneuve et al., 1989; O'Dell et al., 1999; Smith et al., 2002).
Our results suggest that ITS–RFLP data are robust for characterizing community diversity, for two reasons. First, from 158 morphologically defined species that were used in the DNA analysis, 144 (91% of total) yielded a single species-specific RFLP type (Table S1 and data not shown). Second, intraspecific variation was a minor problem. Of the 58 species represented by at least two fruitbodies, 56 (96%) yielded a single RFLP type for all fruitbodies with the exceptions of R. fragilis and R. persicina var. rubrata (Table S1 and data not shown). Together our results highlight the fact that ITS–RFLP data are a valuable tool for grouping ECM species, and for identification of the mycorrhizal symbionts with the fruitbody RFLP-matching approach. These results are similar to those reported by Horton (2002), who also investigated the use of ITS–RFLP patterns to assess diversity of ECM fungi collected across a 7 km coniferous forest. In addition, Kårén et al. (1997) already reported that intraspecific variation was not a problem on a local scale. In the two polymorphic species of Russula, the RFLP variation found in the ITS is the result of variation in two of the four endonucleases (data not shown). Currently, we do not know if the variation observed is a reflection of cryptic species.
The below-ground method revealed the same distribution pattern as the above-ground survey with respect to the relative proportion of abundant vs rare taxa. The below-ground community was characterized by a few common types and a large number of rare types (Fig. 2). This pattern was also observed using fruitbodies (Fig. 2; Richard et al., 2004). Below ground, 50% of the RFLP types collected were represented by one mycorrhiza. The two dominant species were C. geophilum (Table 1) and R. decipiens (Fig. 6a). Cenococcum alone contributed to 35% of the ectomycorrhizas. Of the 120 total plants, 117 (98%) were colonized by this fungus (Table 1). Russula decipiens was found on 17 oaks (data not shown). However, several questions remain concerning the below-ground diversity because of the large number of rare types observed at our site. Which proportion of the local community is really sampled? Would comparable patterns be obtained at another time? Are all abundant species included? Our ability to detect community similarity (e.g. Arbutus vs Quercus) based on species abundance is also limited by the inherent distribution of the diversity.
Fungal species richness was comparable above and below ground, but the two levels exhibited little overlap (< 20%; Table 3) in fungal species composition. This result confirms and extends earlier observations on the complementarity of the two levels in obtaining a comprehensive view of community composition (Gardes & Bruns, 1996; Jonsson et al., 2000; Peter et al., 2001). For instance, without the below-ground approach we would have missed C. geophilum, an ascomycetes species that does not produce fruitbodies at all. It was particularly abundant on oak seedlings and saplings (Table 1). Its high dominance and frequency at our site may arise in part from its ability to sustain xeric conditions by formation of sclerotia (Lilleskov et al., 2004). The role of this fungus in ecosystem functioning is also intriguing – could it provide drought protection to plant roots, as suggested by Jany et al. (2003)? Or, alternatively, could C. geophilum be purely opportunistic, with little relevance to tree physiology? The observation that C. geophilum often dominates in ECM communities, for example in Spanish Q. ilex forests (De Román & De Miguel, 2002); in the Californian chaparral (Borchers & Perry, 1990); or in temperate Fagus sylvatica forests (Blaise & Garbaye, 1983), leads us to question the existence of ecotypes or cryptic biological species (Shinohara et al., 1999).
Combining the species composition viewed above and below ground, the following patterns were observed. Apart from Cenococcum, the community appeared to be dominated by members of the genus Russula and, to a lesser extent, by the genus Inocybe as well as members of the Thelephoraceae and Sebacinaceae (Fig. 4; Richard et al., 2004). In Spanish managed Q. ilex forests, thelephoroid morphotypes accounted for a quarter of the root tips investigated by De Román & De Miguel (2002). Russulaceae and Thelephoraceae also dominated the community in two other Californian Mediterranean ecosystems, the chaparral (Horton et al., 1999) and the bishop pine forest (Gardes & Bruns, 1996), whereas Sebacinaceae were among the most frequently encountered taxa in Eucalyptus sclerophyllous forests in Australia (Glen et al., 2002). An intriguing feature is the absence of hypogeous fungi (at least among dominant taxa on A. unedo, Table 2 and S1), which is perhaps caused by environmental conditions. For instance, the lack of species of Tuber may be explained by acidic soil conditions.
Tree diversity has been suggested to favour ECM diversity on a local scale (Nantel & Neumann, 1992; Kernaghan et al., 2003). We tested the hypothesis that the hosts contribute to ECM fungal diversity. Only 12.9% of the taxa were shared (Fig. 3), less than what was found in mixed forest stands by Horton & Bruns (1998), Cullings et al. (2000) and Kennedy et al. (2003), where multihost fungi dominated, accounting for 30 to 90% of the ECM fungal community in all three studies. Unfortunately our sampling is insufficient to provide statistically significant data, because of the high species richness of the community. Most species were too infrequent to draw conclusions about their distribution, a problem that is often limiting in studies of ECM communities (Horton & Bruns, 2001; Taylor, 2002). Nevertheless, even if restricted to a limited number of fungal taxa, sharing of symbionts may have ecological consequences as Q. ilex seedlings successfully establish and survive in A. unedo-dominated chaparral (Gamisans, 1999). This pattern suggests that A. unedo shrubs may provide conducive conditions for Q. ilex seedlings in early stages of forest succession, perhaps by providing a compatible fungal network.
Despite the important width of the age sequence, the ECM community was quite similar at the various developmental stages of Q. ilex investigated. We observed: (i) similar rank–abundance curves (reflecting high taxonomic diversity and a dominance of rare taxa, Table 3); (ii) among the identified taxa, similar dominance of genera such as Russula, Cortinarius and Amanita (Table 4); and (iii) a high abundance of C. geophilum (Table 1). Our findings support the conclusion that established seedlings recruit ECM symbionts in an opportunistic way among mycobionts colonizing the old surrounding trees. Similar observations were made in multi-aged stands dominated by conifers such as Pinus sylvestris (Jonsson et al., 1999) or Tsuga heterophylla (Kranabetter, 1999; Kranabetter & Friesen, 2002).
An intriguing question is whether or not the sharing of ECM partners between seedlings and old trees is under natural selection. Seedlings may take benefit from established ECM fungi that already have large extraradical soil-exploring mycelia built at the oldest trees’ expense (Högberg et al., 1999). In addition, shared symbionts may even transfer carbon from high-canopy trees to understorey seedlings (Simard et al., 1997; Lerat et al., 2002), counterbalancing low light influx. For the related species Quercus rubra, seedling nutrition and mycorrhization (infection level and diversity) are improved in the vicinity of adult conspecifics (Dickie et al., 2002). Symbiont sharing between seedlings and older Q. ilex could thus result in favouring of kin, as most Q. ilex acorns remain around the mother tree due to barochory (Darley-Hill & Carter Johnson, 1981).