Vertical niche differentiation by hyphae of ectomycorrhizal fungi in soil (2002)

For a long time, the high species diversity of ectomycorrhizal fungi on various spatial scales has posed a challenge to soil ecologists. Especially challenging is the explanation of species diversity at smaller spatial scales. Monocultures of even-aged tree species harbour a large number of ectomycorrhizal fungal species, and even single trees are commonly colonized by a variety of ectomycorrhizal fungi. The high ectomycorrhizal fungal species diversity has been related to functional diversity and the importance of diversity for ecosystem functioning has been discussed (Leake, 2001). Whereas in the past ecological surveys on ectomycorrhizal fungi relied on sporocarp counts, at present most ectomycorrhizal fungal diversity studies rely on ectomycorrhizal root tip identification, by morphological as well as molecular means (Dahlberg, 2001; Horton & Bruns, 2001). In these studies however, the part of the ectomycorrhizal symbiosis that is arguably most important for the nutrition of the host plant, namely the extraradical mycelium (Read, 1991), is not taken into account. Variations in structure, function and activity of the extraradical mycelium determine the potential nutrient mobilization and translocation capacities of different fungal species. Still, in contrast to the observed variation in ectomycorrhizal root tip structure, which could also provide an indirect demonstration of functional differences, the hyphae of the extraradical mycelium are deceptively similar in morphology. Until recently it has therefore been impossible to localize and identify the extraradical mycelium in situ, let alone to distinguish ectomycorrhizal, from saprotrophic, fungal hyphae in soil. As a result, the ecological question of whether there is spatial niche differentiation among the various fungal species, an important mechanism for resource partitioning, has remained unanswered. Following developments in molecular prokaryotic ecology, however, application of molecular techniques has enabled detection of single fungal species in natural substrates and has more recently also enabled description of total fungal communities in soil (e.g. Smit et al., 1999; Borneman & Hartin, 2000; Pennanen et al., 2001; Chen & Cairney, 2002; Klamer et al., 2002). In this issue of New Phytologist, Dickie et al. (pp. 527–535) describe a molecular method for identifying hyphae of (ectomycorrhizal) fungi. They use their data to describe the ectomycorrhizal fungal community in a soil profile, testing the hypothesis that there is vertical niche differentiation. This is one of the first papers where such methods have been applied to detect ectomycorrhizal mycelium in soil and constitutes an important step forward in ectomycorrhizal ecology. The method used by Dickie et al. consists of a total DNA extraction from soil with subsequent PCR amplification of fungal DNA. Assessment and subsequent taxonomic assignment is based on terminal restriction fragment length polymorphism (T-RFLP). They applied their technique in a monospecific, even-aged stand of red pine (Pinus resinosa) and sampled four soil layers (three organic layers – Litter, Fermentation and Humus – and the mineral B-horizon), taking 40 replicate soil cores for each layer. They identified 36 fungal taxa, most of which were (putative) ectomycorrhizal fungi. Cluster analysis and statistical tests of the distribution patterns led them to distinguish six vertical distribution patterns, which were hypothesized to reflect groups of fungal species with different patterns of resource utilization. In their view, ectomycorrhizal fungi show spatial niche differentiation along a vertical gradient, which supports the hypothesis that this provides an important mechanism contributing to ectomycorrhizal fungal species diversity. Molecular identification of fungal hyphae to species level in natural substrates is by no means an easy task. These methods are based on total DNA extractions and the extraction efficiency will be influenced by the cell lysis method used. Likewise, the primer set chosen for amplification of the fungal DNA will determine what DNA is amplified and thus what fungal species can be detected. This implies that the choice of certain methods and primers involves making rational choices about various trade-offs. Let us mention three of them: Primer choice. The ectomycorrhizal symbiosis has arisen repeatedly during evolutionary history (Hibbett et al., 2000) and therefore ectomycorrhizal fungi do not constitute a monophyletic group. Consequently, there is no set of primers that uniquely amplifies ectomycorrhizal fungal DNA. Using general fungal primers, as Dickie et al. did (their primer pair ITS-1F and ITS-4 amplifies DNA from all fungi), DNA of all ectomycorrhizal fungi should be amplified. However, amplification of a large number of saprotrophic fungi, which occur especially in the organic layers, could increase taxonomic ‘noise’. One way to reduce the noise could be the use of basidiomycete-specific primer pairs, as the large majority of ectomycorrhizal fungi belong to the Basidiomycetes, whereas many saprotrophic species belong to the Ascomycetes. With 19 unknown T-RFLP types and a final cluster analysis that included 13 unknowns and 13 ectomycorrhizal fungal taxa, it is possible that their primer pair choice has been too inclusive. T-RFLP. Several methods are available to separate and identify amplified DNA fragments. A major advantage of the T-RFLP method used by Dickie et al. is that it allows simultaneous identification of several fungi in one sample, without prior DNA separation by gel electrophoresis or cloning. Therefore, sampling and processing of a relatively large number of replicates is possible. This important point should be emphasized. This advantage is, however, traded off against taxonomic accuracy. The level of taxonomic resolution of RFLP in general, and hence also of T-RFLP, is much lower than that of sequence analysis. Kårén et al. (1997) noted that members of the genus Cortinarius were sometimes monomorphic in ITS-RFLP types. They reported that one ITS-RFLP type contained five different species, even though two species of that group showed only 94% sequence similarity (36 base pair difference). RFLP methods are also sensitive to infraspecific variation, and assignment of unknown types (about half of the RFLP types were not identified in this study) to higher taxonomic categories is also very problematic. It has to be said, however, that due to our still incomplete sequence databases, even fungal sequence data sometimes remain unresolved. Quantitative data. Ultimately, we would like to correlate data on niche differentiation with data on the relative importance of the various fungi in the different layers. Making use of molecular data, this relationship is not straightforward, as the correlation between detected amounts of fungal DNA and fungal biomass or fungal activity is unknown. In the Dickie et al. study, the total number of fungal occurrences in the various layers suggests that total fungal abundance declines with depth, consistent with data on rarefied species richness. Differential fluorescence suggests that the terminal fragment of Lactarius sp. is more abundant than that of Clavulina cristata, but is that correlated to fungal abundance or activity? It would be tempting to speculate to what extent the method of Dickie et al. allows such semiquantitative conclusions. In any case, when planning to use these methods for (relative) quantitative analysis, careful removal of all root tips and fragments of hyphal mantles from the samples prior to DNA extraction would be a crucial first step. Under the influence of ‘Petri dish’ ecology, mycologists have often come to see mycelia as two-dimensional structures. This study shows clearly that in their natural substrates mycelia expand in the vertical dimension as well. That observation raises some new questions. To what extent is the occurrence of mycelia in different soil layers due to germination and establishment of individuals at different soil depths? Could vertical differentiation be due to subsequent interactions, either with competing fungal species or with fungivorous soil animals, as demonstrated for vertical differentiation of saprotrophic basidiomycetes by Newell (1984)? A related question is to what extent vertical separation per se indicates differential resource utilization? What resources are actually divided among different fungal species? From the data of Dickie et al. it is clear that, while most species have non-random occurrences, only a few species occur in one layer only. Closer inspection of their Fig. 2 suggests that species which were found in two or three layers always occurred in adjacent layers. Does that pattern indicate that many species are still generalists to some extent, and that the niche axis is not very finely divided? Or could it be that the statistical test employed, which assumes independence of the samples of the different horizons, overestimates the degree to which non-random patterns and hence niche differentiation occur? The paper by Dickie et al. is certainly the first publication where the hypothesis of vertical niche differentiation of the ectomycorrhizal mycelium is explicitly tested and constitutes an important step ahead. We know that in ectomycorrhizal fungal community studies sporocarp data do not necessarily match root tip data and, as Horton & Bruns (2001) stated, it seems likely that the new mycelial view of the ectomycorrhizal fungal community will be different from both fruitbody and root tip views. Realising this, we need the eyes of the molecular ecologists to look into the darkness of the soil layers. In these layers we see new and fascinating patterns.