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Worldwide, more than 400 species of parasitic angiosperms in 87 genera, are achlorophyllous and myco-heterotrophic (Leake, 1994), that is, they obtain C from a symbiotic fungus (mycobiont) rather than directly from a photosynthetic host (photobiont) as is the case for plants that are root or stem parasites. In many instances a tripartite symbiosis is involved, where the mycobiont receives photosynthate from a photobiont (or photobionts) and the myco-heterotrophic plant (MHP) in turn receives C from the mycobiont, probably in a different form from that transferred from the photobiont. Although the relationship between MHP and mycobiont in these tripartite symbioses generally has been thought of as mycorrhizal, some feel it is more accurate to refer to the MHP as epiparasites on the photobiont (Björkman, 1960; Cullings et al., 1996; Taylor & Bruns, 1997, 1999; Bidartondo et al., 2000; Bidartondo & Bruns, 2001, 2002). The mycobionts in these associations include both ecto- and arbuscular mycorrhizal taxa (Leake, 1994; Imhof, 2001; Bidartondo et al., 2002). However, those dealt with in this paper are all ectomycorrhizal (EcM). Although MHP are most abundant in the tropics (Leake, 1994), those occurring in north temperate and boreal forests have received most study, especially members of the subfamily Monotropoideae (Ericaceae), and orchids in the genera Corallorhiza, Cephalanthera, and Neottia.
There are four main lines of evidence supporting myco-heterotrophy. First is the consistent association between the roots of some achlorophyllous plants and fungi, and lack of direct connection between the roots of the achlorophyllous plants and those of photosynthetic plants (Björkman, 1960; Trappe & Berch, 1985). Second, field isolation experiments demonstrated the importance of maintaining connections between mycobionts and photosynthetic plants. For example, when putative fungal connections to surrounding plants were severed in Monotropa hypopitys, individuals did not develop, or developed very poorly compared to control plants (Björkman, 1960). Third, radioisotope tracer studies in the field showed translocation of 14C and 32P from trees to M. hypopitys and Sarcodes sanguinea (Björkman, 1960; Vreeland et al., 1981), but not (or in significantly lower concentration) to nearby photosynthetic plants. Fourth, radioisotope tracer experiments in the laboratory demonstrated transfer of 14C from mycobiont to MHP (Smith, 1966, 1967) and from photobiont to MHP via mycobiont (McKendrick et al., 2000).
Although these examples provide strong circumstantial evidence for myco-heterotrophy, few studies show a direct physiological interaction between photobiont, mycobiont, and MHP; only the tracer data provide direct evidence that putative MHP receive C from photosynthetic plants via mycorrhizal fungi. Inconclusive observations also exist. Not all attempts to demonstrate fungal connections have been successful and, in some instances, radioisotope tracer studies in the field failed to detect tracer in MHP (Vreeland et al., 1981, Duddridge et al. unpublished data, cited by Smith & Read, 1997).
The logistical difficulties of conducting radioisotope tracer studies in the field make it unlikely that they will be common in the future. Although laboratory-based studies are easier to implement and offer greater experimental control, it is difficult to assess the relevance of their results to natural systems (Read, 2002). However, measurement of the natural abundances of stable isotopes in field-collected samples could provide an important means of corroboration and assist in elucidating the physiological relationships within MHP systems. Most biologically important elements occur as two or more stable isotopes, with one being far more abundant than the other(s). Fractionation of the isotopes by biological and physical processes leads to concentration differences in substances of biological interest, and these differences can provide insights into fluxes among organisms, between organisms and their abiotic environment, and among compartments of the abiotic environment. An important advantage in using natural abundance stable isotope ratios for ecosystem studies is their ability to present a time-integrated picture of functional processes that often are difficult to examine directly (Robinson, 2001).
During the course of a larger comparative study of N and C stable isotope patterns in two old-growth conifer forests, samples of MHP were collected and analysed. This allowed us to assess whether natural abundance stable isotope ratios could be of value in confirming the myco-heterotrophic nature of MHP, and elucidating other aspects of their biology. To our knowledge, only three N stable isotope values for MHP have been reported previously, and those were from studies comparing N-isotope signatures in N-fixing and nonN-fixing plants (Delwiche et al., 1979; Virginia & Delwiche, 1982). Thus, this is the first report of C stable isotope ratios in MHP, and the first stable isotope study focused on their symbiotic association. In addition to the lack of previous data, understanding of the physiology of the mycorrhizas formed by MHP, including their N and C transfer mechanisms, is limited (Leake, 1994; Smith & Read, 1997; Taylor et al., 2002). Combined, these factors make it difficult to predict relationships between the stable isotope ratios of MHP and ecosystem pools such as green plants, fungi, and soils. Thus, we developed three simple models against which to compare our results, based on reported stable isotope patterns among ecosystem pools (Table 1).
Table 1. Three working models for evaluating the relationships among natural abundance stable isotope ratios (δ13C and δ15N) of myco-heterotrophic plants (MHP), ectomycorrhizal plants (EcMP), and ectomycorrhizal fungi (EcMF)
|Reported relationship (Δ ())||Ref.||Predicted relationship||Reported relationship (Δ ())||Ref.||Predicted relationship|
|Ectomycorrhiza||EcMF > EcMP (3–6)||1, 2, 3, 4, 5||MHP > EcMF||EcMP < EcMF (5–15)||1, 2, 4, 5, 6, 7, 8||MHP ≈ EcMP < EcMF|
|Mistletoe||Host > mistletoe (1–3)||9, 10, 11, 12, 13||MHP ≤ EcMF||Host > mistletoe (< 1)||12, 14||MHP ≤ EcMF|
|Food chain||TLA+1 ≥ TLA (< 2)||15, 16, 17, 18||MHP ≥ EcMF||TLA+1 ≥ TLA (3–5)||18, 19, 20||MHP > EcMF|
The EcM model derives from several studies demonstrating that sporocarps, and in some cases mycorrhizal mantle tissue, of EcM fungi are enriched in both 13C and 15N relative to photobionts. Based on this model, we would expect MHP to be depleted in 15N compared to their mycobionts, as are EcM plants, because the flow of N in both cases is from fungus to plant. However, because C flows in opposite directions – EcM plant to mycobiont and mycobiont to MHP – applying the model to the mycobiont-MHP relationship for 13C is problematic. Thus, we will evaluate the EcM model primarily on the basis of the 15N abundance data.
The mistletoe model is based on observations that both 13C and 15N are often depleted in xylem-tapping mistletoes and root parasites compared to levels in their hosts. The observed differences in 13C abundance usually are attributed to differences in water-use efficiency or CO2 utilization during photosynthesis, and the degree of difference has been used to estimate the degree of heterotrophy exhibited by these hemiparasites (Ehleringer et al., 1985; Marshall & Ehleringer, 1990; Schulze et al., 1991; Richter et al., 1995; Bannister & Strong, 2001). Based on these observations, a holoparasitic (fully heterotrophic) plant should exhibit a 13C abundance very close to that of its host. Indeed, the similarity in C stable isotope signatures has been used to identify a holoparasitic mistletoe species (Kraus et al., 1995). The achlorophyllous MHP in our study are presumably holoparasitic and, consequently, we have assumed under the mistletoe model that a MHP would exhibit 13C and 15N abundances similar to those of its mycobiont.
The food chain model is based on a considerable body of experimental and empirical data that has led to widespread acceptance of the phrase, ‘you are what you eat, plus a few ’ (DeNiro & Epstein, 1976 and additional references cited in Table 1; but cf; Gannes et al., 1997; Eggers & Jones, 2000). Although complexities such as omnivory can complicate application of this model to food webs, the simplicity of the system with which we are concerned (one or two linear trophic transfers) suggests this should not be an issue here. Thus, under the food chain model, we would expect to see a MHP similar to, or slightly higher in, 13C abundance and higher in 15N abundance than its mycobiont.
Because little is known of the physiology of the mycorrhizas formed by monotropes and achlorophyllous orchids, it is difficult to select one of the models as most probable. Studies of the ultrastructure of mycorrhizas formed by species of Monotropa, Pterospora, and Sarcodes (Lutz & Sjolund, 1973; Duddridge & Read, 1982; Robertson & Robertson, 1982) have shown that they clearly differ from EcM. The presence of characteristic pegs formed by the mycobiont suggests a possible analogy with the haustoria formed by parasitic fungi and plants, including mistletoes. If these structures do function in a similar fashion, it would argue for the mistletoe model being the most relevant of the three models. However, the pegs are formed by the host, whereas haustoria are formed by the parasite. Thus, the resemblance could be superficial, and important functional differences could exist between pegs and haustoria (Smith & Read, 1997).
Regardless of which model is best supported by our data, we hypothesize that the relationship between the stable isotope signatures of MHP and EcM fungi will be much stronger than that between MHP and green plants, reflecting the direct physiological connection between MHP and their EcM mycobionts.
Recent DNA-based identifications of the mycobionts of monotropes and achlorophyllous orchids show that a high degree of host-specificity exists (Cullings et al., 1996; Bidartondo & Bruns, 2001, 2002; Taylor et al., 2002; Young et al., 2002). In addition, we have shown significant differences in 15N abundance among different genera and species of EcM fungi in our study areas. For instance, species of Russula exhibit significantly lower δ15N values than species in Tricholoma and Hydnellum (Trudell et al., 2001 and unpublished data). Thus, in addition to the prediction that the isotope signatures of MHP will show closer relationships to those of EcM fungi than to those of green plants, we hypothesize that the signatures of individual MHP will reflect the signatures of their respective mycobionts.