The study of myco-heterotrophic plants is very much in its infancy, and research has been largely driven by methodological developments in molecular and plant physiological ecology. And yet, a reluctance to accept established dogmas regarding the ecology and physiology of the mycorrhizal symbiosis relative to myco-heterotrophy has led to answers that have been provocative and unconventional, for example the epiparasitic mode of life of most myco-heterotrophs and their specialization upon particular lineages of mycorrhizal fungi (reviewed by Bidartondo, 2005
), bidirectional carbon flow in a green orchid–mycorrhizal association (Cameron et al., 2008
) and coevolution between plants and mycorrhizal fungi (Merckx and Bidartondo, 2008
). These answers in turn have prompted the emergence of novel perspectives on symbioses (Sachs and Simms, 2006
; Selosse et al., 2006
). These new theoretical frameworks are conceptually sound because myco-heterotrophic plants stand apart from the major models of cheating within mutualisms (yucca–yucca moth, fig–fig wasp, ant–lycaenid butterfly) as the only non-animal system. Furthermore, myco-heterotrophs provide a prime example of the subversion of mutualisms in nature and offer a system with which to examine further how symbioses remain robust and exclude cheaters. It is also a practical system as it sheds light on the importance of all-too-often ‘black-box’ below-ground interactions and provides mechanistic approaches to conservation and management of biodiversity. Indeed, myco-heterotrophy shows that the biology, evolution and conservation of many plants cannot be understood without a direct focus on individual species of fungi that may, for example, determine plant distribution.
Despite considerable progress in our understanding of myco-heterotrophic plants, they continue to present major challenges in scientific investigations. Our current inferences regarding the nutrient acquisition strategies of myco-heterotrophic plants are based on a limited number of case studies with a notable bias towards myco-heterotrophic plants from temperate areas, where ectomycorrhizas are abundant. Yet to be determined are the isotope signatures of myco-heterotrophic plants that depend on the more widespread arbuscular mycorrhizal fungi. Therefore, it remains unknown whether ectomycorrhizal and arbuscular mycorrhizal myco-heterotrophic plants are physiologically convergent; and although isotope signatures give insight into the carbon and nitrogen acquisition strategies of myco-heterotrophs, the physiological mechanism of these nutrient transfers is completely unknown and thus prone to speculation. In addition to the signals involved in triggering myco-heterotrophic plant seed germination and mycorrhization, the identity of the fungal hosts of most myco-heterotrophic and closely related partial myco-heterotrophic plants, particularly during germination, also remains largely unknown. Within a robust phylogenetic context, this information will eventually uncover the evolutionary history of mycorrhizal specialization by myco-heterotrophic plants. Comparing fungal specialization between recent and old myco-heterotrophic lineages will, in turn, reveal the timing of the process.
Another prominent question is whether myco-heterotrophic plants are in fact parasites of their fungal hosts and/or the autotrophic plants that are part of common mycorrhizal networks. In other words: are there measurable costs for mycorrhizas that have been invaded by a myco-heterotrophic plant? This is a methodologically and conceptually challenging question, particularly for tripartite symbioses, and to date there are no experimental data to address this matter. Although it may seem obvious that a myco-heterotrophic plant exploits its host fungus, there may be benefits for the hosts as well. Some myco-heterotrophic plants may stimulate the growth of their fungal partner and thus perhaps compensate for, or exacerbate, carbon loss (Bidartondo, 2005
). It has been proposed that myco-heterotrophic plants specialize on mycorrhizal fungi that are particularly efficient at tapping into carbon sources from autotrophic hosts (Egger and Hibbett, 2004
), so, similar to other cheaters of mutualisms (Bronstein, 2001
), the carbon cost imposed by a myco-heterotrophic plant on a fungus may be negligible.
In terms of the ecological theory of plant community dynamics, myco-heterotrophic plants provide the clearest evidence for the existence of common mycorrhizal networks in nature, a controversial topic in itself. Myco-heterotrophic plants are the only obvious examples for the potentially widespread phenomenon of plant-to-plant net C transfer via shared mycorrhizal fungi. This functional role of mycorrhizal networks still remains one of the most hotly contested topics in mycorrhizal biology, owing to a combination of technical difficulties and challenging implications. Thus far, the many tests for net carbon transfer from arbuscular mycorrhizal or ectomycorrhizal fungi to green plants have either failed or, if successful, been criticized on methodological grounds (Francis and Read, 1984
; Simard et al., 1997
; Fitter et al., 1999
; Lerat et al., 2002
; Pfeffer et al., 2004
). For instance, Pfeffer and co-workers could not detect carbon transfer in vitro
from Glomus intraradices
to transformed arbuscular mycorrhizal carrot roots growing on glucose. This has been recently confirmed in vitro
with G. intraradices
and whole plants of Medicago truncatula
(Voets et al., 2008
). As previously mentioned, the only exceptions have been field studies of green orchids and Pyroleae (subfamily Monotropoideae of the family Ericaceae) that are closely related to fully myco-heterotrophic plants and generally grow in the dark understorey of forest habitats. These understorey plants, although photosynthetic, fulfil a significant proportion of their adult nutritional needs with fungal-derived carbon and nitrogen. Thus, we know there are at least partially myco-heterotrophic plants. Arbuscular mycorrhizas are by far the dominant mycorrhizas on Earth; roughly 70 % of plant families depend on glomeromycete fungi to obtain soil mineral nutrients and these fungi depend entirely on host plants to obtain carbon. However, none of the photosynthetic plants closely related to non-photosynthetic arbuscular mycorrhizal plants has been examined for facultative mycorrhizal cheating, despite the fact that well over 3000 plant species may fall into this category (e.g. Polygalaceae, Gentianaceae, Dioscoreales, Iridaceae). Judging from studies of ectomycorrhizal partially myco-heterotrophic plants, one would conclude that closely related relatives of fully myco-heterotrophic plants are the best initial candidates for testing whether facultative cheating occurs within the arbuscular mycorrhizal symbiosis.
Finally, there are still many gaps in our understanding of the evolution and ecology of myco-heterotrophic plants. For some myco-heterotrophic plant genera, basic information including distribution, life history, pollination biology, dispersal agents, ecology and taxonomic position is not available. This is mainly due to the fact that species belonging to these genera appear to be rare and ephemeral. Fieldwork is therefore an inevitable first step towards a better understanding of these remarkable plants. Because most myco-heterotrophic plants grow in threatened forest habitats and ex-situ conservation is currently not possible, prompt action should be undertaken to study these plants. Collections of myco-heterotrophic plants should consist of alcohol-preserved material for taxonomic identification, silica-gel-dried material of above-ground parts for DNA extraction, and lysis-buffer- or spirit-preserved root material for the molecular identification of fungi. Dried material of above-ground parts of myco-heterotrophic plants and autotrophic reference plants is necessary for the identification of carbon and nitrogen gains through isotope abundance analysis. In addition, photographs, GPS coordinates and field notes can provide critical information on the ecology of many rare species. These data are essential for the design of realistic experiments to address fundamental questions about mycorrhizal cheating both in the field and in the laboratory.