Research perspectives on functional diversity in ectomycorrhizal fungi
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Communities of ectomycorrhizal fungi
We have recently witnessed an increasing number of studies of ectomycorrhizal fungal communities. This interest, in part, stems from the need to understand human impacts on the functioning of natural ecosystems and it has been facilitated by the advent of nucleic acid-based fungal detection methods. We have learned that ectomycorrhizal fungal communities are frequently species-rich, in some cases exceeding 100 taxa in relatively small plots of land (Izzo et al., 2004). Most comprise few, frequently occurring species and many more rare species (Taylor, 2002; Buée et al., 2005; Koide et al., 2005a). Species may spatially partition the forest floor (Dickie et al., 2002; Genney et al., 2006) and interact with each other both positively and negatively (Agerer et al., 2002; Koide et al., 2005b). Moreover, the relationships between the frequency of soil hyphae and the numbers of fruiting structures and colonized roots differ markedly among species (Gardes & Bruns, 1996; Gehring et al., 1998; Koide et al., 2005a).
Taxonomic and functional diversity in ectomycorrhizal fungal communities
From the standpoint of ecosystem function, taxonomic diversity is only relevant insofar as it is reflective of functional diversity. For example, variation in the composition of the ectomycorrhizal fungal community on individual plants influences host growth (Jonsson et al., 2001) probably because the species vary in ability to transport nutrients to the host or in their demand for carbon. This emphasis on function was reflected in the session entitled ‘Functional diversity in mycorrhiza’ at the last International Conference on Mycorrhiza (Granada, July 2006). Because we feel, as did Bengtsson (1998), that there is more utility in understanding the relationship between ecosystem functions and species traits than between ecosystem functions and taxonomic diversity per se (about which there has been much debate), our purpose here is to highlight some methods that can be used to document functional variability among species of ectomycorrhizal fungi, as well as to discuss briefly the utility in doing so. It seems reasonable to concentrate on functions that influence the success of both the fungi and their hosts. Thus, functions relating to the acquisition of water, carbon (C), phosphorus (P) and nitrogen (N), and the exchange of resources between plants and fungi may be among the most relevant.
Field-based methods for documenting variation in function
While there is accumulating evidence that species of ectomycorrhizal fungi differ functionally, most of it results from in vitro studies of a limited number of culturable fungi, or from very simplified experimental systems with young seedlings under controlled conditions. An extrapolation to the function of the fungi in real ecosystems is therefore difficult at present, but some approaches may be of particular help. We highlight them here.
Courty et al. (2005) recently demonstrated a promising field-based method to determine the activities of various enzymes of ectomycorrhizal roots, including phosphatase, glucosaminidase, peptidase, glucosidase, oxidase and others. This approach allows one to characterize differences among roots colonized by different fungal species in their potential ability to access nutrients from various organic substrates. While ectomycorrhizal roots consist of plant tissue, fungal mycelium (ectomycorrhizal and possibly associated saprotrophs) and adhering and encrusted bacteria (Garbaye, 1994), they nevertheless remain ecologically relevant functional units that are frequently enumerated by researchers according to fungal species (morphotypes). Significant variation among species in their enzyme activities (Buée et al., 2005; Courty et al., 2005, 2006) may explain, in part, why species vary in their capacity to absorb and transport N or P to their hosts, or in their demand for host C. Such enzyme assays may be especially relevant for species of ectomycorrhizal fungi that possess the contact type of hyphal exploration strategy (Agerer, 2001). For other species, the hyphae that grow into the soil may be at least as important to nutrient capture as colonized roots. Accounting for the enzyme activities of ectomycorrhizal hyphae in the field is difficult, but one approach is to transplant intact mycorrhizal microcosms from laboratory to field (Nara, 2006). The difficulty in distinguishing between the activities of ectomycorrhizal and saprotrophic fungi may be partly overcome by using sand-filled mesh bags, which allow growth of ectomycorrhizal fungi to the partial exclusion of decomposer fungi (Wallander et al., 2001). Enzyme assays must be interpreted in light of the fact that incubation conditions are generally chosen to insure rapid catalysis and nonlimiting substrate availability. Obviously the conditions in nature may be different. Nevertheless, significant variation among species in potential enzyme activity assessed under standard conditions may be ecologically informative in much the same way that measurements of photosynthesis under standardized conditions have proven to be with respect to plant distributions in nature (Schulze et al., 2005).
Stable isotope probing is another powerful tool that could elucidate variation in hyphal function among ectomycorrhizal fungal species in field settings. In this method, 13C-labeled (Radajewski et al., 2000) or 15N-labeled (Cadisch et al., 2005) substrate is applied to the soil. If an organism has access to the substrate, the heavier isotope will be incorporated into its DNA. Extraction of community DNA from the soil is followed by separation of isotope-enriched DNA from unenriched DNA on the basis of density. The organisms with access to the labeled substrate are then identified by amplification of the denser DNA by PCR and subsequent analysis, such as by T-RFLP or DGGE. The challenges in using this technique are related to sensitivity, as dilution of the heavy isotopic signal by the more common, lighter isotope can occur, and accuracy, as incorporation of the heavy isotope by microbes without access to the labeled substrate may occur following uptake of metabolic intermediates released by the death of microbes that do have access (Radajewski et al., 2000).
Significant functional variability occurs among species of ectomycorrhizal fungi in their ability to utilize complex organic sources of N, particularly protein (Abuzinadah & Read, 1986). The significance of this observation is highlighted by the distributions of species along N availability gradients and changes in communities in response to nutrient additions. For example, the ability to use protein in culture by the various species of ectomycorrhizal fungi found along a N concentration gradient was inversely correlated with the availability of inorganic N at the sites they occupied (Lilleskov et al., 2002). Moreover, sporocarp δ15N was correlated with the ability to use protein (Lilleskov et al., 2002).
The quantity of naturally occurring stable isotopes may also prove to be useful in other contexts. For example, a significant source of functional variability occurs among ectomycorrhizal fungal isolates in their propensity to transfer N to their hosts (Abuzinadah & Read, 1989), and the amount of this transfer may be indicated by natural abundances of 15N in fungal tissue (Hobbie et al., 2005). In general, however, methods based on the quantification of naturally occurring stable isotopes must be used with caution. For example, host specificity may be reflected in the δ15N and δ13C of fungal tissues (Högberg et al., 1999; Kohzu et al., 1999), but those studies also indicate that considerable variation exists among ectomycorrhizal fungal species of a given host in both δ15N and δ13C, which may reflect their use of different sources of C or N in the environment.
Although perhaps less exciting than DNA- or isotope-based approaches, good old-fashioned observation of morphological and anatomical properties among species of ectomycorrhizal fungi is also very important insofar as they influence resource acquisition and transport (exploration types; Agerer, 2001). Villarreal-Ruiz et al. (2006), for example, showed in a Scots pine chronosequence that there was a marked shift from ectomycorrhizal fungal communities with long distance to fringe exploration types as stands aged. Variation among species in the production of large rhizomorphs capable of extracting water from the soil may also determine whether or not ectomycorrhizal fungi influence other microorganisms, such as those responsible for decomposition (Koide & Wu, 2003).
The ability to document functional variability among ectomycorrhizal fungi allows us to address a number of exciting ecological questions, which we discuss here. If we first assume that dispersal is not the primary limitation to the distributions of ectomycorrhizal fungi, then a most intriguing question concerns the relative importance of host plants and the physical environment as determinants of ectomycorrhizal fungal functional diversity. Many studies have shown that ectomycorrhizal fungal communities of particular hosts change with an assortment of environmental variables. For example, the ratio of Basidiomycete to Ascomycete ectomycorrhizal colonization may decrease with drought (Gehring et al., 1998; Swaty et al., 2004). Are such shifts simply caused by individualistic responses of the fungal species to the environment, or can the host additionally select for fungal species based on functions that are most beneficial to it when conditions change, perhaps by disproportionately allocating carbon to the favored species?
Selection of function by host plants may be important in another context. There is limited evidence that host-specific fungal species are more effective in transporting nutrients to their hosts than generalist fungi (Hobbie et al., 2005). Is this sort of functional superiority a prerequisite for the evolution of host specificity? Does functional superiority lead to disproportionate allocation of carbon from host to superior fungi, in turn leading to the evolution of specificity? Do all host-specific fungal species possess particular functions that make them more valuable to the host than those possessed by generalist species?
Whether host plants can select for particular functions as opposed to particular species can be addressed in systems in which individual host plants support fungal communities that are distinct from those on nearby, conspecific hosts (Gehring et al., 1998). In such situations there may be little selection for species of ectomycorrhizal fungi, but is there selection for function? Is the distribution of fungi on hosts random, or is there selection by hosts that results in certain functions being represented on each host irrespective of fungal species? Does this reflect a low degree of functional redundancy among the fungal species on single trees, but a high degree of functional redundancy among trees?
We actually know little about the degree to which the physical environment selects for particular functions of ectomycorrhizal fungi, but this question is easily approached in several existing systems. For example, when disturbances such as clear-cutting or wildfire occur, host plants may be removed wholesale from the ecosystem, and this could place new selection pressures on the mycorrhizal fungi. Will this result in a community of fungi with greater saprotrophic capacity? Many experiments have been conducted to examine the effects of climate change on vegetation. Researchers have also capitalized on natural experiments of climate variation (Swaty et al., 2004). These could provide valuable opportunities to determine the functional responses of ectomycorrhizal fungal communities to environmental change. For example, long-term warming or cooling trends may increase or decrease rates of mineralization. Will this select for fungi with differences in ability to acquire N and P from organic compounds? Will long-term drying or wetting trends lead to selection for or against species that produce water-transporting rhizomorphs?
Another fascinating question concerns the contribution of ectomycorrhizal fungi to overall ecosystem functional diversity. It is clearly possible for communities of ectomycorrhizal fungi and host plants to influence the composition of the other. How much of ecosystem functional diversity as related to nutrient cycling or carbon sequestration, for example, that is currently ascribed to plant diversity is actually the result of ectomycorrhizal fungal diversity?
Within ectomycorrhizal fungal communities, species may interact in both negative and positive ways. On small spatial scales, greater than expected co-occurrence of pairs of ectomycorrhizal fungi may occur (Agerer et al., 2002; Koide et al., 2005b). Does this reflect complementarity of function by the species that allows each of them to posses a higher fitness when growing together than when growing separately? Do negative interactions (less than expected co-occurrence: Agerer et al., 2002; Koide et al., 2005b) occur primarily among functionally similar species?
In addition to the substantial interspecific functional variability, significant within-species functional variability exists in ectomycorrhizal fungi (Cairney, 1999). If a variety of functions are necessary in every ecosystem or on every host, can intraspecific functional variability substitute for interspecific variability? Do we find that communities of low species diversity have higher degrees of intraspecific functional diversity?
Finally, studies of the functions of rare vs frequent and/or abundant species may also prove to be valuable. Do rare species duplicate the functions of frequent species, and will rare species assume the functions of frequent species in the community if for some reason the frequent species becomes locally extinct, thus preserving that function in the community despite community shifts? Or do rare species (individually or collectively) perform functions not performed by frequent species?
Many tools are now available for the study of the functions of ectomycorrhizal fungi, which will allow us to address the mechanistic bases for many fascinating phenomena reported in the past. We hope to witness a growing interest in the functional diversity of this ecologically important guild of fungi.
We thank Håkan Wallander and Iver Jakobsen for organizing the ICOM session entitled ‘Functional diversity in mycorrhiza’, the New Phytologist for inviting us to write this article, and the anonymous reviewers for their stimulating ideas that resulted in considerable improvement of this manuscript.