n | New ? Phytologist C<97%7%f%A3:ry Myco-heterotroph-fungus marriages - is fidelity over-rated? 'Full appreciation of the evolution ofmyco-heterotrophy Though green coloration is a defining feature of the plant kingdom, there are many nongreen (i.e. achlorophyllous/ nonphotosynthetic) plants which have long sparked the curio- sity of botanists (see Fig. 1). These plants can be divided into two functional groups: those that directly invade other plants to acquire food, such as the mistletoes, and those that do not. Members of the latter group have historically been called 'saprophytes' but are more properly labeled 'myco-heterotrophs', a term which highlights the fact that they acquire all their fixed carbon from mycorrhizal fungi (Leake, 1994; see also Fig. 1). Let's be clear ? we are talking about plants that con- sume fungi. As odd as such a lifestyle may sound, at least 400 myco-heterotrophic species are distributed across nine families of monocots and dicots (Furman & Trappe, 1971; Leake, 1994). The 'crown jewels' of the myco-heterotrophs are the orchids, of which the estimated 30 000 enchanting species encompass nearly 10% of the Angiosperm flora. Of course, the vast majority of orchids are photosynthetic, at least as adults. However, all orchids can be classified as partially myco- heterotrophic because their minute 'dust seeds' lack energetic reserves and must locate a fungus on which to feed during the interval between seed germination and the elaboration of photosynthetic organs months or years later. In addition, multiple independent lineages of terrestrial orchids have given up photosynthesis entirely, becoming 'fully myco- heterotrophic' Members of another widely distributed and well known myco-heterotrophic subfamily, the Monotropoideae (Ericaceae), share many convergent attributes with orchids (Leake etaL, 1994). Myco-heterotrophs interact in a physio- logically intimate fashion with specific fungal partners, providing amusing opportunities for analogies with human relations (Gardes, 2002). Papers in this issue by McCormick etaL (pp. 425-438) and Leake etaL (pp. 405-423) provide import- ant new insights into the marriages' between myco-heterotrophs and their fungal partners. In particular, these papers demon- strate high fidelity of the plants across all life stages, with the glaring exception of one orchid species which switches partners. The infidelity problem Ordinary mycorrhizal interactions involve a reciprocal exchange of photosynthetically fixed plant carbon in return for fungally scavenged soil minerals, and are thus regarded as mutualisms. Most plants display no signs of fidelity to particular fungal partners. For example, ectomycorrhizal Douglas fir has been estimated to associate with at least 2000 fungal species which span tens of families of Ascomycetes and Basidiomycetes (Molina et aL, 1992). By contrast, idiosyncratic and specific fungal associations were described in orchids by the turn of the 20th century (Bernard, 1909), and were shordy thereafter suggested in the Monotropoideae as well (Francke, 1934). Other authors disagreed vociferously with these claims of specificity (Curtis, 1937; Hadley, 1970). In the case of orchids, some of the disagreements can be blamed on the predominance of associations with fungi in the problematic 'taxon' Rhizoctonia. This form-genus encompasses distantly related clades of fungi which rarely fruit in culture, are difficult to identify based on vegetative characteristics, and can interact with plants as mycorrhizae, endophytes or parasites. Recent molecular studies have confirmed mycorrhizal spe- cificity in several fully myco-heterotrophic orchids (reviewed in Taylor etaL, 2002; see also Selosse etaL, 2002b; Taylor etaL, 2003; Taylor etaL, 2004). A parallel series of studies has clarified the fungal associations of most members of the Monotropoideae and documents equal or greater specificity than that found in orchids (Bidartondo & Bruns, 2001, 2002). This specificity is perplexing, since eschewing potential partners must come at a cost. One potential explanation has been presented as follows (Cullings etaL, 1996; Taylor & Bruns, 1997). Fungi form mycorrhizae in order to acquire carbon, and yet myco-heterotrophs remove carbon rather than providing it to their fungal partners. Hence, they can be viewed as parasites upon their ) New Phytologist (2004) 163:217-221 www.newphytologist.org 217 218 Forum Commentary New Phytologist THE PHYTOLOGIST. No. VIL DECEMBER, MDCCCXLI. PRICE 6D. ART. XXXV.? On the parasitic growth of Monotropa Hypopitys. By EDWIN LEES, Esq., F.L.S., &c. ILLUSTRATIONS OF THE MODE OF GROWTH OF MONOTROPA HYrOPlTYS. 1. Base of a mature plant, 14 inches high, and three young unexpanded plants, growing from their radical parasitical knob. 2. Smaller plant in seed 3, 4 & 5. Young plants growing from radical knobs. Fig. 1 The progenitor of this journal, The Phytologist, was an important early forum for discussions and observations on the parasitic nature of Monotropa hypopitys (Lees, 1841). The plant had been assumed to be a typical angiosperm parasite by Linnaeus, but Luxford (1841), Lees (1841), and Rylands (1842a) were perplexed to find no evidence of haustorial attachments to other plants. Their observations, including recognition of fungal mycelium ensheathing its roots (Rylands, 1842b), paved the way for the studies of myco-heterotrophic germination and development of M hypopitys and identification of its fungal partners now reported in this issue. fungi. Parasitism tends to favor specificity because of selection on victims to resist their attackers, and ensuing evolutionary 'arms-races'. These arguments lead to the prediction that fully myco-heterotrophic orchids should be more specific than green orchids. McCormick et oL put this prediction to the test by carefully documenting specificity in three photosynthetic terrestrial orchids using modern molecular-phylogenetic and seed-packet germination field trials and comparing their results to specificity in a previ- ously studied fully myco-heterotrophic orchid. Difficulties in finding suitable partners McCormick etal isolated fungi from individual coils (pelotons) of mycorrhizal fungi teased out of root cells of Goodyera pubescens and Liparis lilifolia. They then amplified and sequenced several diagnostic ribosomal gene regions, including the highly variable ITS. All of the fungi from these orchids belonged to the genus Tulasnella, a member of the Rhizoctonia complex commonly found in orchids worldwide. Remarkably, the 10 isolates from Liparis displayed a maximum of approx. WWW.newphytologist.org ? New Phytologist (2004) 163:217-221 New Phytologist Commentary Forum 219 0.2% sequence divergence, suggesting that L. lilifolia associates with only a single fungal species over a wide geographic area. Isolates from Goodyera were slightly more diverse, but still closely related when compared to the ITS sequence diversity of tomentelloid fungi found associated with the fully myco- fieterotrophic orchid Cephalanthera austinae in a previous study (Taylor & Bruns, 1997). McCormick et al. contribute an additional key piece of information. They found that germinating seeds and protocorms of these two species from packets planted in the field associated with the same narrow clade of fungi as did adult plants. Hence, we see lifelong fidelity in these two photosynthetic orchids, which has also been demonstrated recently in several fully myco-hetero trophic orchids (McKendrick et al., 2000; McKendrick et al., 2002). The results of McCormick et al. are counter to the predictions about fidelity in photosynthetic vs nonphotosynthetic plants and therefore require a re-examination of orchid-fungus marri- ages. However, these species may also depend heavily on fungally supplied carbon ? they produce single leaves (some in winter) and grow on the dusky floors of dense forests. In this context, Otero et al. (2002) have recently reported low ITS sequence diversity among the Ceratobasidium associates of several epiphytic orchids. One would not expect significant myco-heterotrophic carbon gain by adult orchids in the forest canopy, though this possibility deserves examination (Ruinen, 1953). The third species studied by McCormick et al, Tipularia discolor, stands in stark contrast to the first two. Tulasnella isolates, along with other fungi from Tipularia adults, were phylogenetically diverse. Hence, this orchid appears to display relatively low specificity in the adult stage. Does this mean Tipularia establishment is unlikely to be constrained by a lack of suitable partners? Not at all. Perhaps the most exciting result reported by McCormick et al. is that wild Tipularia protocorms associate with a narrow range of fungi that are not Tulasnella species, nor any kind of Rhizoctonia. These fungi could not be isolated, but were characterized using direct molecular approaches. These fungi are relatively distant from any species that have been sequenced and deposited in the public databases, but appear to be allied to the Auricu- lariales. This order of Basidiomycete jelly fungi includes many wood decomposers. Coincidentally, germination of Tipularia seeds seems to occur predominantly, if not exclusively, in decaying wood. Therefore, establishment of this widespread orchid is likely to require both a specific fungal clade and a specific microhabitat. Previous studies have shown that adult Monotropa hypo- pitys in North America have complete fidelity to fungi in the genus Tricholoma (Bidartondo & Bruns, 2002). Tricholoma is ectomycorrhizal, and hyphally links Monotropa to its ultimate carbon source ? autotrophic hosts such as Salix. Leake et al. set out to determine whether the natural distribution of the fungal partners and autotrophic hosts influence the germination and growth of Monotropa seeds. Many thousands of dust seeds contained in hundreds of mesh packets were introduced into two sites with adult Monotropa plants. This method immobilizes the miniscule seeds, permitting their recovery from the soil, while also permitting hyphal entrance and interaction with the seeds. At the first site, packets were planted in two microhabitats: near adult plants and at least 5 m distant from any observed adults. Consider- able germination and seedling growth occurred in plots near adults. Though some germination occurred away from adults, no appreciable growth occurred. At the second site, seed packets were again planted in two microhabitats: under Salix and in interspersed, open grassy areas. Germination and growth were highly variable under Salix, as might be expected if Tricholoma is patchily associated with its autotrophic host, and essentially absent in the grassy areas. In addition to these detailed studies of Monotropa seed germination, molecular analyses of the fungal associates of seedlings and adults were conducted. Leake et al found absolute fidelity to Tricholoma cingulatum in both seedlings and adults growing with Salix, and fidelity to the closely related Tricholoma terreum in adults growing under Pinus. Interestingly, they note that neither of these Tricholoma species is particularly abundant in the Salix and Pinus ecosystems, according to fruiting records. The results presented by Leake et al. provide the strongest evidence to date that the distribution of a single fungus can forcefully constrain the establishment and resulting distribution of an Angiosperm. These findings have obvious and important implications for the conservation and management of threatened myco-heterotrophs. The findings of equally high specificity at the protocorm stage in photosynthetic orchids dramatically widens the conservation implications. Recommendations for coping with infidelity In the decade since the seminal New Phytologist Tansley Review of myco-heterotroph biology by Leake (1994), many vexing problems have been clarified, particularly relating to fungal identities, linkages to autotrophs and seed germination in the field, in prominent papers including the two in this issue. Yet, a number of the key questions posed by Leake (1994), and more recently by Gardes (2002), remain unresolved. The utilization of modern molecular phylogenetic approaches to characterise specificity has provided major advances (Curlings et al, 1996; Taylor & Bruns, 1997; Selosse et al, 2002b; Bidartondo et al, 2003). However, as researchers dig more deeply into specificity, increasingly quantitative methods for comparative analysis will be needed. The tools of phylogenetics and population genetics offer a variety of options by which we may summarize genetic diversity within a set of fungal associates using a single statistic (Taylor et al, 2004), which would be preferable to ad hoc comparisons based on tree topologies alone. These quantitative values can then be compared across species, populations, geographic regions, and so forth. Specificity toward two or more clades ) New Phytologist (2004) 163:217-221 www.newphytologist.org 220 Forum Commentary New Phytologist of fungi could also be summarized using these statistics. The transitions between protocorm and adult stages in Tipularia make it clear that future studies must differentiate life cycle components of specificity. Statistical evolutionary methods will also become increasingly important as we begin to reconstruct the history of mycorrhizal associations in major groups such as the Orchidaceae and Ericaceae. A key question which is just appearing on the research horizon concerns the possible role of switches of fungal partners in the diversification of myco-heterotrophic (and other?) plant lineages. Unfortunately, a full appreciation of the evolution of myco-hetero trophy will remain elusive until the key evolutionary parameter ? fitness ? is measured in both plant and fungus under a variety of conditions. This is perhaps the greatest challenge facing myco-heterotroph research, because of the major obstacles to measuring the fitness of filamentous fungi under natural conditions (Pringle & Taylor, 2002). Other questions that have received considerable attention of late, but are far from resolved, concern the trophic activities of the fungi and associated full or partial myco-heterotrophs. Studies of stable isotopes show matching N and C patterns between particular myco-heterotrophs and their fungi (Trudell et al, 2003), and that photosynthetic orchids of the forest, and even grassland, acquire carbon and nitrogen from their mycorrhizal fungi (Gebauer & Meyer, 2003). However, the quantities and dynamics of carbon gain via fungi remain to be fully characterized in any partial or full myco-heterotroph. To adequately assess the relationship between myco-hetero trophy and specificity, measurements of both carbon dynamics and specificity in a large number of species will be needed to identify trends that stand out against the idiosyncratic evolutionary history of any particular species. Further break- throughs have included the demonstrations that certain Rhizoctonia species belonging to clades within the Sebaci- naceae and Tulasnellaceae form full-fledged, and in some cases abundant, ectomycorrhizae on autotrophic hosts surrounding particular myco-heterotrophs (Selosse et aL, 2002a; Bidartondo et aL, 2003). But the trophic activities of most orchid-associated Rhizoctonia species remain obscure. Are these questions worthy of the considerable research effort they imply? While myco-heterotrophs may not be dominant components of terrestrial ecosystems, they offer important model study systems in at least two respects. First, it is now clear that even 'normal' photosynthetic plants may rob carbon from one another via mycorrhizal fungi (Simard et aL, 1997). Because of the unidirectional net flow of carbon and high specificity in myco-heterotrophs, they provide the most tractable systems with which to study mycorrhizal carbon transfer. Second, much of our understanding of the evolution of parasitism derives from a few stereotypical interactions, such as those between herbivorous insects or pathogenic fungi and their host plants. Myco-heterotrophs turn these interactions on their heads, since it is the plant that preys on the fungi. Ecological and evolutionary patterns in myco-heterotrophs that mirror those in more conventional parasites (e.g. frequent host-switches which are correlated with speciation events) will aid in identifying fundamental attributes of parasitism. Acknowledgements Details of the 'old' Phytologist courtesy of Jonathan Leake; editorial suggestions provided by Ian Herriot. D Lee Taylor University of Alaska, Institute of Arctic Biology, Fairbanks, Alaska, USA (tel +1907 4746982; fax +1907 4746967; email fflt@uaf.edu) References Bernard N. 1909. devolution dans la symbiose. Les orchideeset leur champignons commensaux. Annals of Science Nut Botany')'. 1?196. Bidartondo MI, Bruns TD. 2001. Extreme specificity in epiparasitic Monotropoideae (Ericaceae): Widespread phylogenetic and geographical structure. Molecular Ecology 10: 2285-2295. Bidartondo MI, Bruns TD. 2002. Fine-level mycorrhizal specificity in the Monotropoideae (Ericaceae): specificity for fungal species groups. Molecular Ecology 11: 557-569. Bidartondo MI, Bruns TD, Weiss M, Sergio C, Read DJ. 2003. Specialized cheating of the ectomycorrhizal symbiosis by an epiparasitic liverwort. Proceedings of the Royal Society of London B 270: 835?842. Cullings KW, Szaro TM, Bruns TD. 1996. Evolution of extreme specialization within a lineage of ectomycorrhizal epiparasites. Nature 379: 63-66. Curtis JT. 1937. Non-specificity of orchid mycorrhizal fungi. Proceedings of the Society of Experimental Biology and Medicine 36: 43?44. Francke HL. 1934. Beitrage zur kenntnis der mykorrhiza von Monotropa hypopitys L. Analyse und synthese der symbiose. Flora 129: 1?52. Furman TE, Trappe JM. 1971. Phylogeny and ecolgy of mycotrophic achlorophyllous angiosperms. Quarterly Review of Biology Ad: 219?225. Gardes M. 2002. An orchid-fungus marriage ? physical promiscuity conflict and cheating. New Phytologist 154: 4?7. Gebauer G, Meyer M. 2003. 15N and 13C natural abundance of autotrophic and mycoheterotrophic orchids provides insight into nitrogen and carbon gain from fungal association. New Phytologist 160: 209?223. HadleyG. 1970. Non-specificity of symbiotic infection in orchid mycorrhiza. New Phytologist 69: 1015-1023. Leake J, R. 1994. The biology of myco-heterotrophic (saprophytic) plants. New Phytologist127: 171-216. Leake JR, McKendrick SL, Bidartondo M, Read DJ. 2004. Symbiotic germination and development of the myco-heterotroph Monotropa hypopitys in nature and its requirement for locally distributed Tricholoma spp. New Phytologist 163: 405-423. Lees E. 1841. On the parasitic growth of Monotropa hypopitys. Phytologist 1: 97-101. Luxford G. 1841. Botanical notes. Phytologist \: 43. McCormick MK, Whigham DF, O'Neill JP. 2004. Mycorrhizal diversity in photosynthetic terrestrial orchids. New Phytologist 163: 425^38. McKendrick SL, Leake JR, Taylor DL, Read DJ. 2000. Symbiotic germination and development of myco-heterotrophic plants in nature: Ontogeny of Corallorhiza trifida and characterization of its mycorrhizal fungi. New Phytologist145: 523-537. WWW.newphytologist.org ? New Phytologist (2004) 163: 217-221 New Phytologist Meetings Forum 221 McKendrick SL, Leake JR, Taylor DL, Read DJ. 2002. Symbiotic germination and development of the myco-heterotrophic orchid Neottia nidus-avis in nature and its requirement for locally distributed Sebacina spp. New Phytologist154: 233-247. Molina R, Massicotte H, Trappe JM. 1992. Specificity phenomena in mycorrhizal symbioses: community-ecological consequences and practical implications. In: Allen MF, ed. Mycorrhizal functioning: an integrative plant-fungal process. New York., USA: Chapman & Hall, 357-423. Otero JT, Ackerman JD, Bayman P. 2002. Diversity and host specificity of endophytic Rhizoctonia-l'ikc fungi from tropical orchids. American Journal of Botany %9: 1852-1858. Pringle A, Taylor JW. 2002. The fitness of filamentous fungi. Trends in Microbiology 10: 474-481. Ruinen J. 1953. Epiphytosis. A second review on epiphytism. Annals of Bogorensis 1: 101?157. Rylands TG. 1842a. On the mode of growth of Monotropa hypopitys. _#y#6%iff 1: 329-330. Rylands TG. 1842b. On the nature of the byssoid substance found investing the roots of Monotropa hypopitys. Phytologist 1: 341? 348. Selosse M-A, Bauer R, Moyersoen B. 2002a. Basal hymenomycetes belonging to the Sebacinaceae are ectomycorrhizal on temperate deciduous trees. New Phytologist 155: 183?195. Selosse MAE, Iss MW, JanyJL, Tillier A. 2002b. Communities and populations of sebacinoid basidiomycetes associated with the achlorophyllous orchid Neottia nidus-avis (L.) L.C.M. Rich, and neighbouring tree ectomycorrhizae. Molecular Ecology 11: 1831?1844. Simard SW, Perry DA, Jones MD, Myrold DD, Durall DM, Molina R. 1997. Net transfer of carbon between ectomycorrhizal tree species in the field. Nature (London) 388: 579-582. Taylor DL, Bruns TD. 1997. Independent, specialized invasions of ectomycorrhizal mutualism by two nonphotosynthetic orchids. Proceedings of the National Academy of Sciences, USA 94: 4510-4515. Taylor DL, Bruns TD, Hodges SA. 2004. Evidence for mycorrhizal races in a cheating orchid. Proceedings of the Royal Society of London B 271: 35-43. Taylor DL, Bruns TD, Leake JR, Read DJ. 2002. Mycorrhizal specificity and function in myco-heterotrophic plants. In: van der Heijden MGA, Sanders I, eds. Mycorrhizal ecology. Berlin, Germany: Springer-Verlag, 375-413. Taylor DL, Bruns TD, Szaro TS, Hodges SA. 2003. Divergence in mycorrhizal specialization within Hexalectris spicata (Orchidaceae), a non-photosynthetic desert orchid. American Journal of Botany 1168: 1168-1179. Trudell SA, Rygiewicz PT, Edmonds RL. 2003. Nitrogen and carbon stable isotope abundances support the myco-heterotrophic nature and host-specificity of certain myco-heterotrophic plants. New Phytologist 160:391-401. Key words: Myco-heterotrophs, fungi, mycorrhizas, orchids, achloro- phyllo us/no nphotosyn the tic plants. Meetings The C02 fertilising effect - does it occur in the real world? The International Free Air C02 Enrichment (FACE) Workshop: Short- and long-term effects of elevated atmospheric C02 on managed ecosystems, Ascona, Switzerland, March 2004 It would seem simple. There are only two immediate primary responses of plants exposed to elevated levels of atmospheric C02 concentration above the ambient (which currently averages approx. 375 ppm by volume, 33% up from the preindustrial 280 ppmv). First, in C, species, competition between C02 and 02 at the active site of the photosynthetic enzyme 'rubisco' is shifted in favour of reaction with C02 thereby increasing gross photosynthetic C02 fixation and decreasing C02 loss via photo respiration. Second, in most species, both C, and C^, stomatal aperture narrows thereby reducing stomatal conductance and, com- bined with the photosynthetic response, leads to increased water use efficiency in C-acquisition. That's it. No other primary responses have been identified, although I do wonder about whether there are subtle developmental effects associated with interactions between endogenous ethylene production and action and atmospheric C02 concentration. But nothing in nature is simple and, with there being two known primary responses, the long-term repercussions for ecosystems may be twice as difficult to quantify as the linked issue of impact of the increasing greenhouse gas concentration on global climate for which there is only one primary response ? namely, more of the infrared back radiation emitted from the Earths surface is absorbed in the lower atmosphere. ) New Phytologist (2004) 163:221-225 www.newphytologist.org 222 Forum Meetings New Phytologist Box 1 FACE methodology ? FACE methodology (Lewin etal., 1992; Hendrey eta/., 1999; Miglietta etal., 2001; Okada eta/., 2001) involves a ring of separately controlled C02 release points above the ground in circles from 1 m to 30 m in diameter. ? The point-releases can be computer-controlled to be always upwind of the central experimental zone (or 'sweet-spot'), with the rate of release varied more or less with windspeed. ? There have been 13 large diameter-ring (> 8 m) FACE systems in the world, 10 still operating. ? There are approx. 20 'miniFACE' ring systems 1-2 m in diameter, for which C02 is usually released all around the ring continuously by day. The small ones do not have scope for a wide guard-zone around the sweet-zone and are unsuited to tall vegetation. For both the 'greenhouse' and 'C02 fertilising' effects, debate has persisted over at least half a century as to whether these primary effects are leading, respectively, to global warming, and to increased vegetation productivity and C-stocks in the terrestrial biosphere. In both debates the power of constraints and feedbacks (both negative and positive) developing through time, and operating on various timescales and spatial scales, in the complex, adaptive climatic and ecological systems, have been invoked by some to argue for resilience to change. In both cases the debate continues despite continuing accumu- lation of observational evidence. For the C02 fertilising effect, both new evidence and continuing debate was seen at the recent Free Air C02 Enrichment (FACE) workshop in Switzerland (http://face2004.ethz.ch/index.htm). How resilient are plant processes, crop yields, ecosystems and the terrest- rial C-cycle to modification by elevated atmospheric C02 concentration in the long term? Doubts about long-term field-expression of the C02 fertil- ising effect arise partly because the majority of such research has been in chambers, glasshouses, open-topped chambers, and controlled environments of various types, these often being short-term experiments. However, the longest enrich- ment experiment by far has been in open topped chambers on a salt marsh vegetation on Chesapeake Bay. Bert Drake (Smithsonian Environmental Research Center, Edgewater, MD, USA) reported at the meeting that after 17 yr the elevated C02 concentration had increased the marsh shoot density by > 100% compared with ambient air control chambers. The development of the FACE technology (Box 1) in the mid-1980s by George Hendrey at the Brookhaven National Laboratory (Upton, NY, USA) has provided the opportunity to test responses in the field. FACE versus Chamber- are the minor differences real? Kimball etal. (2002) conducted a quantitative comparison of the conclusions about elevated C02 effects on 11 crops (including grass, cereal, C4 sorghum, tuber and woody crops) from the four FACE experiments then available, compared with results from the large number of prior chamber experi- ments (including open-topped field chambers) over many years. It was comforting to those using both kinds of facility that FACE experiments had, with two exceptions and within the ranges of variability of reported results, confirmed under longer-term field conditions all the prior quantitative chamber- findings on crops grown and measured in elevated C02 concentration compared with ambient C02 concentration (persistently increased light saturated photosynthesis, decreased stomatal conductance, decreased water use, increased shoot biomass growth, increased root growth, decreased specific leaf area, decreased leaf nitrogen concentration, increased soluble carbohydrate content, little effect on phenology, and increased agricultural yield though for small grain cereal yield the increases were at the lower end of the range found in enclosed environments). The two exceptions were for reduction of stomatal conductance and enhancement of root growth relative to shoot growth, both of which were more strongly expressed in the FACE experiments than in the chamber experiments. Lisa Ainsworth reported results of a statistical meta-analysis of results now available from experiments conducted over several years in 12 large-scale FACE facilities on four conti- nents (Long et ah, 2004). This again confirmed, with greater statistical rigour and for a much wider range of species including crops, pasture species and trees, most of the con- clusions of the evaluation by Kimball et al. (2002) for a C02 concentration of 550 ppmv. In addition, she noted that, in the open field, elevated C02 increased apparent quantum yield of light-limited photosynthesis by 13% (a figure close to the theoretical short-term response expectation), that growth under water or N stress exacerbated the response of stomatal conductance to elevated C02 concentration, and agricultural yield increased by 17% (average of C, and C^) a figure similar to the average of 15% (scaled to 550 ppmv C02) reported by Kimball (1986) for prior chamber studies. However, again the responses of rice and wheat yields were found to be lower than in chamber studies. Growth rate of above-ground biomass was also increased on average across all C, and C^ species by 17%. For trees it increased by 28%, though this high result is influenced by the strong positive response of fast growing poplar saplings. Dicots were more responsive than grasses, and legumes more responsive than nonlegume forbs. Interestingly, the decrease in N-content per unit leaf area that has generally been observed in elevated C02 chamber-studies was less pro- nounced in FACE experiments, ?4% on average, a decline WWW.newphytologist.org ? New Phytologist (2004) 163: 221-225 New Phytologist Meetings Forum 223 consistent wholly with the reduction in Rubisco content. To establish whether the apparent, relatively minor, differences in results between the FACE and enclosure experiments are real, coordinated FACE and enclosure experiments are needed as Alistair Rogers observed. Positive and negative feedback A fast-acting negative feedback, which has often been thought might lead to lower fractional response of growth than of photosynthesis rate in the short term (days to weeks), is down- regulation of photosynthesis under continuous exposure to elevated C02 associated with reduced leaf N-content. Ainsworths meta-analysis confirmed that this does usually occur in the field, with the maximum carboxylation capacity (VcmJ decreasing on average by 13% under continuous exposure to elevated C02. Down-regulation of Vc max was more strongly expressed in grasses, shrubs and crops than in legumes and trees. Should this be seen as a mechanism of plants 'resisting' a positive response to elevated C02, i.e. showing resilience to change? Probably not. Stephen Long (University of Illinois, Urbana, IE, USA) presented an elegant exposition of how photosynthetic down-regulation involves an optimisation of the deployment of N from photosynthetic machinery to growth organs such that a balance between C-source and C-sinks is maintained in the plant under elevated C02 concentration ? a response that generally increases the nitrogen use efficiency (Wolfe et aL, 1998). At an ecosystem scale over years to decades, another type of adaptation to continuous elevated C02 concentration could be changes in plant community structure. One might reasonably hypothesise that species that are most responsive in growth to elevated C02 concentration would become more dominant over time thereby leading to a positive feedback. Mike Jones (Trinity College, Dublin, Ireland) described the Megarich study in which monoliths of six grasslands across Europe were exposed to FACE over 3?6 yr. Generally, under competition, occurrence of dicots was enhanced and monocots relatively suppressed by continu- ous elevated CQ2. And there was a significantly increased fraction of legumes in the swards (Teyssonneyre et aL, 2002). While the Megarich study did not include deter- mination of N-fixation, the increased preponderance of legumes in the swards is supportive of the notion that, in the long run, elevated C02 concentration may cause N-fixation to entrain more atmospheric N2 into the ecosystem, leading ultimately to fuller expression of the increased growth and standing biomass potential that the elevated C02 provides (Gifford, 1992). It might take several decades for such a positive feedback to build up in an ecosystem to the extent that it could be measured as increased N-stocks in the field. To date no FACE experiment has been for long enough. If such N-accumulation were eventually to occur much of it would be expected in the soil and, associated with it, more soil C. Does elevated C02 concentration lead to more C accumulation in the soil? Chris van Kessel (University of California, Davis, CA, USA) addressed this question by studying soil C accumulation in the intensely N-fertilised Swiss grassland FACE system. He concluded that over 10 yr elevated C02 concentration had no effect on soil C-stocks, no effect on soil microbial biomass including Rhizobium after an initial surge, and no effect on above ground litter decomposition. From this he posited the 'resilience hypothesis' that initial responses of soil C-cycle and N-cycle processes are short lived and that they relax back to their original stocks and rates. One mechanism for this may be the 'priming' of oxidation of some older more stable forms of soil organic matter by the input of more new easily oxidised organic matter as proposed by Marcel Hoosbeek (Wageningen University, Netherlands; Hoosbeek et aL (2004)). However, the artificial N-input to the Swiss FACE study was extraordinarily high (either 140 or 560 kg ha-1 yr-1 over the 10 yr). From an ancillary study at the same Swiss FACE site towards the end of the treatment decade, Paul Hill (University of Wales, Bangor, UK) observed that the greater potential for sequestration of C below ground was by the swards that had the lower N-supply. This partly agrees with a microcosm study in a controlled environment over 4 yr in which a native C,-grass was grown in a very low-N soil (total initial N of 0.02%) under elevated C02 concentration with only 22-198 kg ha-1 yr-1 N supplied dilutely in the irrigation water. Over 4 yr the soil had gained 15?57% (respectively) more C with elevated C02 concentration than without (Lutze & Gifford, 1998). Thus it is possible that under both extremely high and extremely low N-nutrition, elevated C02 has no effect on soil C concentration while with intermediate N-nutrition elevated C02 increases soil C stocks. If so, that would parallel the tendency for plant N concentration to be unaffected by elevated C02 concentration at extremely low and high N-status, but diminished by elevated C02 concentration in the intermediate range of N-nutrition (Gifford et aL, 2000). Resolution of this issue is one for which long-term investigations are required. The workshop returned again and again to the need for long-term experiments in the field. The profits and pitfalls of FACE Every experimental system in vegetation studies has its advantages and drawbacks. The great advantage of the FACE approach is that it is technically possible ? if funded appropriately ? to apply long-term C02 treatment to patches of existing ecosystem, even tall ones, over the long-term. Also leaf temperature can respond to the reduced transpiration naturally in the open air. George Hendrey urged researchers to be more aware of several inherent limitations with the ) New Phytologist (2004) 163:221-225 www.newphytologist.org 224 Forum Meetings New Phytologist FACE approach. He emphasised particularly the rapid (down to minutes or seconds) and sometimes large fluctuations in concentration of C02 at each point in a FACE-ring owing to the inherent time delays of enrichment associated with sample-line length, with distance from release point to sweet-zone, with wind speed and direction changes, and with the eddy-structure of the atmosphere on the scale of FACE rings. C02 concentration at any one place can undergo large fluctuations within seconds to minutes under FACE, a feature that is not mirrored, in terms of either amplitude or frequency spectrum, in the control treatment. Hendrey's analysis (Hendrey et ah, 1997) of the impacts of such fluctuations combined direct measurements of the fluorescence responses of wheat leaves exposed to such C02 fluctuations, which are embedded in the unweighted mean C02 concentration, concluded that photosynthesis rate can be decreased by 17% or more for the mean concentration reported when that mean is of large C02 fluctuations on the order of half the mean, and the deviations from the mean occur over a minute or longer. This derives from the fluctuating concentration driving the internal leaf concentration into the saturated part of the photosynthetic response curve. The larger the concentrations swing above the saturating con- centration the worse the underestimate becomes of the response at the calculated mean C02 enrichment. A poster by Joe Hokum and Klaus Winter showed experi- mental data supporting Hendrey's conclusion. They showed (Holtum & Winter, 2003) that for two tree species the photosynthetic enhancement by C02 concentration elevated to 600 ppmv was diminished by one third when that concen- tration was an average of subminute fluctuations between 433 and 766 ppmv. They also reported that the 26% growth response of rice seedlings to a stable 600 ppmv C02 was eliminated when that average comprised 30 sec fluctuations having just a 150 ppmv amplitude. Thus extant FACE tech- nology might be systematically understating the effect of globally elevated C02 on ecosystem productivity. However, it is not only FACE facilities that can suffer such fluctua- tions. Open topped chambers and poorly designed or man- aged enrichment systems in C02-enriched growth-chambers can also produce large 'hunting' effects that the investiga- tors may be unaware of. Thus C02 concentration fluctuations in C02 enriched but not ambient treatments may be a more general problem for elevated C02 plant research than even Hendrey and Holtum realised. In chambers, however, it should not be such an insurmountable problem as in FACE. Perhaps a 'second-best' way forward is to routinely characterise the fluctuations and to model the effective concentration that the plants perceive. That would require, however, clear understanding of all the mechanisms involved. There might be other mechanisms. For example, regular fluctuation of C02 concentration on a 10?30 min timescale might resonate with the inherent relaxation time of stomatal opening or closing and sometimes drive the pores fully open or fully closed artificially. A second major potential problem for FACE technology is ethylene contamination of the C02. Carbon dioxide sources vary enormously in their level of trace ethylene. Sup- plier scrubbing methods may be of variable efficacy. In our hands even when the supplier's quality control laboratories indicate virtually undetectable levels, our own routine ethy- lene scrubbing columns (containing proprietary potassium permanganate-based oxidation granules) can change colour at considerably different speeds from batch to batch of C02 gas delivered. Ethylene scrubbing has been a substantial cost for growth chambers studies in my laboratory since identify- ing the problem with our supplies (Morison & Gifford, 1984). For FACE, the huge quantities of gas used might preclude routine on-site scrubbing. Ethylene, being a natu- ral plant hormone, has growth inhibitory and specific develop- mental effects on some, but not all, species in the part per billion range. Apparently this is a problem that no FACE, and not all chamber, investigators have addressed in the past. As with the fluctuating C02 concentration issue, the im- plication is that the methodology may understate the productivity-enhancing effect of elevated C02. However, in some chambers having low air replacement rates, there is the added problem that ethylene naturally produced by the plants themselves can build up to inhibitory levels (Klassen & Bugbee, 2002). As C02 and ethylene interact physiologi- cally (at the higher C02 levels involved in fruit ripening research, at least) this may also produce subtle confounding interactions in some chamber studies too. Perspectives In summary, as with global warming, there are substantial issues yet to be addressed with the C02 fertilising effect, but the evidence for its existence in the real world continues to consolidate. Long-term FACE studies are showing that the C02 fertilising effect on vegetation productivity may not, after all, be an artefact of 'plant physiologists and their greenhouses'. Roger M. Gifford CSIRO Plant Industry GPO Box 1600, Canberra, ACT 2601, Australia (tel +61 26246 5441; fax +61 26246 5000; email roger.gifford@csiro.au) References Gifford RM. 1992. Interaction of carbon dioxide with growth-limiting environmental factors in vegetation productivity: Implications for the global carbon cycle. Advances in Bioclimdtology. 1: 25?58. WWW.newphytologist.org ? New Phytologist (2004) 163: 221-225 New Phytologist Meetings Forum 225 Gifford RM, Barrett DJ, Lutze JL. 2000. The effects of elevated [COJ on the C: N and C: P mass ratios of plant tissues. Plant and Soil 11A: 1-14. Hendrey GR, Ellsworth DS, Lewin KF, Nagy J. 1999. A free-air C02 enrichment system for exposing tall forest vegetation to elevated atmospheric C02. Global Change Biology 5: 293?230. Hendrey GR, Long SP, McKee IF, Baker NR. 1997. Can photosynthesis respond to short term fluctuations in atmospheric carbon dioxide? Photosynthesis Research 51: 170?184. Holtum JAM, Winter K. 2003. Photosynthetic C02 uptake in seedlings of two tropical tree species exposed to oscillating elevated concentrations of COr Planta2l8: 152-158. Hoosbeek MR, Lukac M, van Dam D, Godbold DL, Velthorst EJ, Biondi FA, Peressotti Cotrufo F, de Angelsi P, Scarascia-Mugnozza G. 2004. More new carbon in the mineral soil of a poplar plantation under Free Air Carbon Enrichmment (POPFACE): Cause of increasing priming effect? Global Biogeochemical Cycles 18: GB1040. doi: 10.1029/ 2003GB002127. Kimball BA. 1986. Influence of elevated C02 on crop yield. In: Enoch HZ, Kimball BA, eds. Carbon dioxide enrichment of greenhouse crops, Vol. 2: Physiology, yield and economics. Baton Raton, EL, USA: CRC Press, 105-115. Kimball BA, Kobayashi K, Bindi M. 2002. Responses of agricultural crops to free-air C02 enrichment. Advances in Agronomy 77: 293?368. Klassen SP, Bugbee B. 2002. Sensitivity of wheat and rice to low levels of atmospheric ethylene. Crop Science 42: 746?753. Lewin KF, Hendrey GR, Kolber Z. 1992. Brookhaven National Laboratory free-air carbon dioxide enrichment facility. Critical Reviews in Plant Science 11: 135-142. Long SP, Ainsworth EA, Rogers A, Ort DR. 2004. Rising atmospheric carbon dioxide: Plants FACE the future. Annual Review of Plant Biology 55: (In press.) Lutze JL, Gifford RM. 1998. Carbon accumulation, distribution and water use of Danthina richardsonii swards in response to C02 and nitrogen supply over four years of growth. Global Change Biology 4: 851-861. Miglietta F, Hoosbeek MR, Foot J, Gigon F, Hassinen A, Heijmans M, Peressotti A, Saarinen T, van Breeman N, Wallen B. 2001. Spatial and temporal performance of the MiniFACE (Free Air C02 Enrichment) ? system on bog ecosystems in northern and central Europe. Environmental Monitoring andAssessment 66: 107?127. Morison JIL, Gifford RM. 1984. Ethylene contamination of C02 cylinders: Effects on plant growth in C02 enrichment studies. Plant 7%y?A,6%y75: 275-277. Okada M, Lieffering M, Nakamura H, Yashimoto M, Kim HY, Kobayashi K. 2001. Free-air C02 enrichment (FACE) using pure C02 injection: system description. New Phytologist 150: 251-260. Teyssonneyre F, Picon-cochard C, Falcimagne R, Soussana JF. 2002. Effects of elevated C02 and cutting frequency on plant community structure in a temperate grassland. Global Change Biology'8: 1034-1046. Wolfe DW, Gifford RM, Hilbert D, Luo Y. 1998. Integration of photosynthetic acclimation to C02 at the whole plant level. Global Okw%rAo6%y4: 879-893. Key words: climate change, ecosystem, carbon cycle, crop yield, carbon dioxide, FACE. About New Phytologist New Phytologist is owned by a non-profit-making charitable trust dedicated to the promotion of plant science, facilitating projects from symposia to open access for our Tansley reviews. Complete information is available at www.newphytologist.org Regular papers, Letters, Research reviews, Rapid reports and Methods papers are encouraged. We are committed to rapid processing, from online submission through to publication 'as-ready' via OnlineEarly - average first decisions are just 5-6 weeks. Essential colour costs are free, and we provide 25 offprints as well as a PDF (i.e. an electronic version) for each article. For online summaries and ToC alerts, go to the website and click on 'Journal online'. You can take out a personal subscription to the journal for a fraction of the institutional price. Rates start at ?108 in Europe/$193 in the USA & Canada for the online edition (click on 'Subscribe' at the website) If you have any questions, do get in touch with Central Office (newphytol@lancaster.ac.uk; tel +44 1524 592918) or, for a local contact in North America, the USA Office (newphytol@ornl.gov; tel 865 576 5261) ) New Phytologist (2004) 163: 221-225 www.newphytologist.org