Mineral nutrition of most terrestrial plants relies on mycorrhizal fungi that take up both mineral and simple organic compounds from soil solution. While the majority of plant taxa form arbuscular mycorrhizal (AM) symbiosis with members of the phylum Glomeromycota, the ectomycorrhizal (ECM) symbiosis involving various lineages of mostly Basidiomycota and Ascomycota dominates in economically important trees such as Pinaceae, Fagaceae, Dipterocarpacae and Myrtaceae (Brundrett, 2009; Tedersoo et al., 2010a). These ECM fungal lineages have probably evolved their mycorrhizal habit independently and may play a differential role in nutrition and protection of their plant hosts (Lindahl & Taylor, 2004; Bödeker et al., 2009; Tedersoo et al., 2010a). Fungal species belonging to different lineages display substantial differences in ECM morphology – particularly the presence and abundance of extraradical mycelium including rhizomorphs – that are responsible for exploration and transportation of nutrients (Agerer, 2001, 2006). These so-called ECM exploration types are believed to differ in their efficiency of carbon (C) storage, enzymatic activities, nutrient uptake and translocation (Courty et al., 2010; Hobbie & Agerer, 2010; Lilleskov et al., 2011; Pritsch & Garbaye, 2011).
Potential activities of extracellular enzymes involved in the degradation and nutrient release from soil organic matter have been used to address functional diversity among ECM fungi in situ (Courty et al., 2010; Pritsch & Garbaye, 2011). Activity measurements rely on the enzymatic cleavage of specific substrates by functional enzymes present and probably bound at the surface of ECM root tips (Pritsch et al., 2004, 2011). Although ECM root tips support diverse populations of bacteria and microfungi (Frey-Klett et al., 2007; Tedersoo et al., 2009), consistent differences among ECM fungal species and negligible activities in nonmycorrhizal (NM) roots suggest that most of the potential enzymatic activities in root tips are exerted by ECM fungi rather than by other microbes and the host plant itself (Courty et al., 2010, 2011). There are substantial differences in potential enzymatic activities among ECM fungal species, seasons, soil horizons and their interactions (Courty et al., 2005, 2006, 2010; Buée et al., 2007; Jones et al., 2010), supporting the view that ECM fungi display at least some degree of functional complementarity in their benefits to host plants (van der Heijden & Kuyper, 2003; Nara, 2006; Rineau & Courty, 2011).
The relative abundance of stable isotopes in food webs is based on the discrimination against heavier isotopes in several biochemical processes and therefore reflects patterns of water availability, photosynthesis activity and nutrient cycling (Dawson et al., 2002). In ECM symbiosis, nutrient transport from fungi results in depletion of 15N in plants, while ECM fungi become more enriched in the process (Högberg et al., 1999a). Fruit bodies of ECM fungi are therefore typically more enriched in 15N compared with soil and humus saprotrophs (Gebauer & Dietrich, 1993; Taylor et al., 1997). In addition, ECM fungi access recent photosynthesis products rather than C from soil and debris (Högberg et al., 2001; Hobbie & Horton, 2007; Baldrian, 2009), which render them more depleted in 13C compared with saprotrophic fungi. These differences in the natural abundance of stable isotopes enable ECM symbionts to be distinguished from saprotrophs (Gebauer & Taylor, 1999; Hobbie et al., 1999; Kohzu et al., 1999; Mayor et al., 2009) – a phenomenon called the ‘ECM–sap divide’ (cf. Henn & Chapela, 2001). However, stable isotope patterns vary widely among ECM fungal species and differ both spatially and temporally, depending on nutrient availability (Taylor et al., 1997, 2003). While the local 13C abundance in fungal fruit bodies is mostly attributable to host species (Högberg et al., 1999b), 15N abundance depends on exploration depth by the extraradical mycelium (Agerer et al., 2012), N source (Högberg et al., 1999a) and the ratio of chitin to amino acids in fungal tissues (Taylor et al., 1997). Furthermore, fruit bodies of species with greater amounts of extraradical mycelium, including rhizomorphs, are relatively more enriched in 15N, which has been at least partly ascribed to greater internal nitrogen (N) cycling and accumulation of 15N-depleted chitin in the extraradical mycelium (Handley et al., 1996; Zeller et al., 2007; Hobbie & Agerer, 2010). Natural abundance of 15N in fruit bodies and soil mycelial biomass is positively correlated with the ability of the fungal taxa to take up proteins in pure culture experiments (Lilleskov et al., 2002, 2011).
Stable isotope natural abundance has been rarely addressed in root tips because of difficulties in identifying mycobionts and obtaining sufficient material for analysis of root tips; however, these constraints have been overcome along with technological advances in the last decades. Early studies demonstrated that the natural abundance of 15N in tree roots is intermediate between that of foliage and soil (Gebauer & Dietrich, 1993). Owing to a substantial fungal proportion, ECM root tips are more enriched in 15N than NM root tips (Högberg et al., 1996; Zeller et al., 2007). Information about the effect of fungal species and exploration types on stable isotope natural abundance in ECM root tips is still lacking, although 15N natural abundance in fine roots is considered a good indicator of N availability across fertilization gradients and soil horizons (Högberg et al., 1996), and the identity of mycobionts explains a large proportion of root tip respiration and fine root traits in temperate forests (Ostonen et al., 2009; Trocha et al., 2010).
Because of great differences in seasonality, temperature and humidity, soil processes in tropical ecosystems differ substantially from those in temperate and boreal forests (Six et al., 2002; Zhang et al., 2008; Hyodo et al., 2010). While taxonomic information about tropical ECM fungal communities is rapidly accumulating (Tedersoo et al., 2007, 2010b,c; Diedhiou et al., 2010; Peay et al., 2010; Smith et al., 2011), the functional aspects of tropical fungi remain poorly understood. Preliminary research indicates that certain species of tropical ECM fungi are able to access organic N sources that render them functionally similar to fungi in boreal and temperate ecosystems (Brearley et al., 2005). Here we focused on potential enzymatic activities and stable isotope patterns of ECM fungi in an African tropical lowland rainforest. We postulated three main alternative hypotheses: potential enzymatic activities of root tips are stronger in ECM than in NM root tips; potential enzymatic activities and 15N natural abundance in ECM root tips are mostly related to the phylogenetic origin of fungi (lineage); and potential enzymatic activities and 15N natural abundance are correlated and reflect similar aspects of ECM tree nutrition.