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Fungal root colonization responses in natural grasslands after long-term exposure to elevated atmospheric CO2 M A T T H I A S C . R I L L I G , * C H R I S T O P H E R B . F I E L D ² and M I C H A E L F . A L L E N * *Center for Conservation Biology, University of California, Riverside, CA 92521-0334, USA, ²Department of Plant Biology, Carnegie Institution of Washington, Stanford CA 94305, USA

Abstract Arbuscular mycorrhizae, ubiquitous mutualistic symbioses between plant roots and fungi in the order Glomales, are believed to be important controllers of plant responses to global change, in particular to elevated atmospheric CO2. In order to test if any effects on the symbiosis can persist after long-term treatment, we examined root colonization by arbuscular mycorrhizal (AM) and other fungi of several plant species from two grassland communities after continuous exposure to elevated atmospheric CO2 for six growing seasons in the ®eld. For plant species from both a sandstone and a serpentine annual grassland there was evidence for changes in fungal root colonization, with changes occurring as a function of plant host species. We documented decreases in percentage nonmycorrhizal fungal root colonization in elevated CO2 for several plant species. Total AM root colonization (%) only increased signi®cantly for one out of the ®ve plant species in each grassland. However, when dividing AM fungal hyphae into two groups of hyphae (®ne endophyte and coarse endophyte), we could document signi®cant responses of AM fungi that were hidden when only total percentage colonization was measured. We also documented changes in elevated CO2 in the percentage of root colonized by both AM hyphal types simultaneously. Our results demonstrate that changes in fungal root colonization can occur after long-term CO2 enrichment, and that the level of resolution of the study of AM fungal responses may have to be increased to uncover signi®cant changes to the CO2 treatment. This study is also one of the ®rst to document compositional changes in the AM fungi colonizing roots of plants grown in elevated CO2. Although it is dif®cult to relate the structural data directly to functional changes, possible implications of the observed changes for plant communities are discussed. Keywords: arbuscular mycorrhiza; elevated CO2, ®ne endophyte, Glomales, grassland, rootcolonizing fungi Received 30 June 1998; resubmitted and accepted 30 September 1998

Introduction Arbuscular mycorrhizal (AM) fungi have been implicated as important controllers of plant and ecosystem responses to elevated atmospheric CO2 (O'Neill et al. 1991; Allen et al. 1995). Resource-balance models (Chapin 1980) predict that as above-ground resource availability increases, below-ground allocation should increase. Correspondence and current address: Matthias C. Rillig Department of Plant Biology, Carnegie Institution of Washington, 260 Panama Street, Stanford, CA 94305, USA, fax + 1/650 325 6857, e-mail [email protected] # 1999

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Increased carbon investment by AM fungi can be bene®cial to host plants by virtue of the role of these fungi in nutrient and water acquisition and protection against fungal root pathogens (Smith & Read 1997). Since the mycobionts are dependent on the host as their only source of carbon (Allen 1991), increased supply of carbon by means of increased photosynthesis and root allocation could be bene®cial to the fungi. Previous research on the effects of elevated atmospheric CO2 on the AM symbiosis has largely been restricted to the study of single species as `model systems' (e.g. O'Neill et al. 1991; Morgan et al. 1994;

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Rogers et al. 1994; Whitbeck 1994; Jongen et al. 1996; Klironomos et al. 1996, 1997; Lovelock et al. 1996; Godbold et al. 1997; 1998b; Lussenhop et al. 1998; Rillig et al. 1998a) and only few experiments have included a comparison of different AM plant hosts that co-occur in the same community (Monz et al. 1994; Sanders 1996; Rillig et al. 1998a). However, none of these latter studies have been performed in the ®eld, but only in monoculture (Monz et al. 1994; Rillig et al. 1998a), or when grown together (Sanders 1996), in experimental microcosm studies. While these microcosm experiments are useful in exploring mechanisms and allow for high experimental control, they cannot necessarily be extrapolated to ®eld conditions. Our understanding of responses of AM to elevated CO2 is also limited by the fact that available data are from short-term experiments. There is little indication that initially observed responses can persist after several years of exposure, or if they disappear through complete homeostatic adjustment. There are also very few data regarding potential changes in the composition of the AM fungal community colonizing roots when plants are grown in elevated CO2. The occurrence in these soils of two easily distinguishable AM fungal groups, ®ne endophyte and coarse endophyte fungi, have allowed us to test for changes in the composition of rootcolonizing AM fungi at this coarse morphological level. Finally, many fungi other than AM colonize plant roots and act (at the very least) as carbon sinks, or even as pathogens. Responses of these fungi to elevated CO2 have been ignored with few exceptions (Klironomos et al. 1996, 1997; Rillig et al. 1998a). It is important that changes in these fungi be monitored, as they may provide important feedbacks to plant growth, and as mycorrhizae `protect' the root from parasitic fungi. In northern California, sandstone and serpentine grasslands occur adjacent to each other, but have strikingly different soil properties (Luo et al. 1996), plant species composition (Baker 1989), above-ground productivity, and responsiveness to CO2 (Field et al. 1996). This provided a convenient opportunity to examine differences in root colonization responses in elevated CO2 in plants from the two communities. The main purpose of this study was to test whether responses of rootcolonizing fungi to elevated CO2 observed in short-term pot experiments can persist after long-term CO2 fumigation in the ®eld.

Materials and methods This research was carried out at the Jasper Ridge Biological Preserve near Stanford, California (37° 24¢ N, 122° 13¢ W, 100 m elevation). The site has a mediterranean-type climate with cool, wet winters and warm, dry summers. At the site two different grassland commu-

nities exist adjacent to each other. The serpentine grassland occurs on serpentine-derived soils, and sandstone grassland on sandstone-derived soils. The CO2 experiment in the ®eld consisted of 30 plots of 0.33 m2 in each of the two grassland communities. Three treatments were started in January 1992, replicated 10 times in both grasslands: no-chamber controls, open-top chambers (cylindrical, 1 m tall) with ambient CO2 and open-top chambers with elevated CO2 (ambient + 350 mL L±1). Comprehensive details on the design of the Jasper Ridge experiment are given in Field et al. (1996). In early April 1997, after six growing seasons of continuous fumigation with elevated CO2, roots were sampled from the chambered plots in both grasslands. Due to the destructive nature of the sampling necessary for this study, repeated measurements throughout the growing season were not possible. However, due to the unusual timing of rainfall and contracted growing season, potentially confounding factors to an assessment of fungal root colonization through only a single harvest measurement were likely at a minimum: At harvest time, there were no signi®cant effects of elevated CO2 on soil moisture content in the two grasslands (S. Hu, pers. comm., 1997). There were also no signi®cant effects of elevated atmospheric CO2 on total above-ground biomass or plant density in the two grasslands (N. Chiariello, in prep.), and plants were in similar phenological stages throughout all plots. There was no signi®cant effect of elevated CO2 on root length in the sandstone, and only a slight increase in the serpentine (M. Rillig, unpubl. obs., 1998). We chose ®ve annual plant host species in each grassland community for the analysis of fungal root colonization. The choice was based on the availability of a suf®cient number of individuals in a suf®cient number of chambers across the two treatments. We also attempted to sample a range of functional types (native, introduced, forbs, grasses). These plant species were Lolium multi¯orum Lam. (introduced grass), Linanthus parvi¯orus (Benth.) E. Greene (native forb), Plantago erecta E. Morris (native forb), Calycadenia multiglandulosa DC. (native forb), and Epilobium brachycarpum C. Presl. (native forb) for the serpentine, and Brachypodium distachyon (L.) Beauv. (introduced grass), Lolium multi¯orum, Euphorbia spathulata Lam. (native forb), Sherardia arvensis L. (introduced forb), and Avena barbata Link (introduced grass) for the sandstone (nomenclature follows Hickman 1993). At least three individuals per species were taken from a chamber, and for Plantago and Epilobium up to six. Roots were carefully excavated to a depth of 10 cm using a knife, and only roots attached to an identi®ed plant were used. All roots of a species were pooled per chamber, the experimental unit. The roots were washed in tap water and immediately stored in 70% ethanol until # 1999

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R ELEVATED CO2 AND ROOT COLONIZING FUNGI Table 1 Results (F and P-values) from univariate ANOVAs on coarse, ®ne and total AM hyphal percentage colonization, arbuscular percentage colonization, percentage colonization with both ®ne and coarse hyphae (both AM), coarse (c) and ®ne endophyte (FE) AM hyphal colonization intensity, and nonmycorrhizal (Non-M) fungal percentage colonization. Stars behind a response variable indicate that arcsine square-root transformed data were used for analysis

Species Response variable F

P

Sandstone Coarse AM Fine AM Total AM Arbuscules* Both AM* Intensity (c) Intensity (FE) Non-M*

8.40 8.93 5.52 10.98 7.02 8.08 4.34 23.12

< 0.0001 < 0.0001 0.0007 < 0.0001 < 0.0001 < 0.0001 0.003 < 0.0001

Serpentine Coarse AM Fine AM Total AM Arbuscules Both AM Intensity (c) Intensity (FE) Non-M*

5.489 35.71 12.77 15.70 19.36 1.06 22.06 1.79

0.0008 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.38 < 0.0001 0.14

analysis. Roots were cleared in 10% KOH (90 °C) for 1 h, acidi®ed in 1% HCl for 15 min, stained in 0.05% Trypan Blue in lactoglycerol (90 (C) for 30 min, and then stored in lactoglycerol (Brundrett 1994). Randomly selected root fragments were mounted on slides, and at each gridline intersect at 250 3 magni®cation, presence and absence of fungal colonization (arbuscular mycorrhizal and other fungal colonization) was noted (McGonigle et al. 1990; Rillig et al. 1998a). AM fungal hyphae were distinguished from nonmycorrhizal fungal hyphae mostly by following a connection with AM structures like arbuscules (the de®ning criterion) or coils and vesicles. Other, secondary factors we took into account were melanization (rare for AM fungal hyphae), cross-wall septation (absent or irregular septation in AM hyphae), `knobbiness' of the hyphal wall and angular projections (AM fungi), and hyphal branching (typically not at a right angle for AM hyphae). We were further able to distinguish two different morphological groups of arbuscular mycorrhizal fungal intraradical hyphae: so-called `®ne endophyte' (FE) hyphae and coarse AM hyphae. FE hyphae are very thin (» 1±2 mm, compared to 3±10 mm for coarse hyphae), stain intensely with Trypan Blue, form fan-shaped hyphal structures and characteristic small vesicles, and are connected to arbuscules (Greenall 1963; Hall 1977; Gianinazzi-Pearson et al. 1981). We measured percentage root colonization for both AM morphogroups separately, and noted total AM percentage colonization, as well as percentage root colonized by both types simultaneously. Furthermore, we measured colonization intensity of # 1999

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F

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CO2 3 Species P

F

P

1.53 0.21 1.23 8.33 1.29 5.63 0.08 2.37

0.22 0.64 0.27 0.005 0.26 0.02 0.77 0.19

2.08 0.59 2.46 2.97 1.76 0.79 0.95 6.07

0.09 0.66 0.054 0.026 0.14 0.53 0.44 0.0004

6.16 3.73 0.61 38.97 0.28 2.40 1.48 2.70

0.016 0.058 0.43 < 0.0001 0.59 0.12 0.23 0.10

1.823 3.67 2.24 2.53 3.33 0.57 6.01 1.37

0.13 0.009 0.07 0.04 0.015 0.68 0.0004 0.25

coarse and FE hyphae separately by scoring the proportion of root cortex colonized by hyphae at every crosshair intersect with AM hyphae present on a scale from 1 (one quarter or less colonized) to 4 (completely occupied) as more fully described in Rillig and Allen (1998). Intensity of colonization per root system was calculated by dividing the sum of the individual intensity scores by the number of cross-hair intersects with FE or coarse AM hyphae present. Thus intensity of colonization and percentage colonization are independent assessments of hyphal colonization; it is possible that hyphal proliferation in roots occurs without signi®cant changes in percentage colonization (Rillig et al. 1998b). For each grassland, we carried out multivariate ANOVAs (MANOVA) including all eight response variables measured for root colonization (total AM, ®ne, coarse, ®ne and coarse both present, arbuscules, Non-M fungi, intensity for coarse and FE hyphae). We chose MANOVA in order to account for possible multivariate responses and to protect against in¯ation of the experiment-wise type I error. Wilks' Lambda was used as the multivariate criterion (SAS Institute 1994). Subsequently, univariate 5 3 2 factorial ANOVAs were used to test for effects of Plant Species, CO2 and Species 3 CO2 (all ®xed effects) on individual response variables within one grassland. Univariate ANOVAs were also used to test for CO2 effects on all the response variables for each of the 10 different host plant species. In order to meet assumptions of normality (Shapiro±Wilks W-test) and homogeneity of variances (Levene's test) for the analyses, some variables

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Results In both grasslands we found evidence for changes in fungal root colonization in response to the CO2 treatment. MANOVAs including the eight measured root colonization response variables yielded highly signi®cant (P < 0.0001) Species, CO2, and Species 3 CO2 effects for both the serpentine and sandstone grassland. Therefore, for both grasslands, the effect of elevated CO2 on the suite of measured fungal root colonization variables depended on plant host species. Univariate ANOVAs (Table 1) revealed which response variables were responsible for the signi®cant multivariate effects. Except for intensity and nonmycorrhizal fungal colonization in the serpentine, the Plant Species main effect was signi®cant for all response variables. There were several signi®cant CO2 3 Plant Species interactions terms (for coarse AM, total AM, arbuscules, Non-M) for fungi from the sandstone, and for ®ne AM, arbuscules, both AM, and FE intensity in the serpentine. For both grasslands, the CO2 main effect for arbuscular colonization was highly signi®cant. Responses in root colonization by different fungal hyphal types to elevated CO2 are shown in Fig. 1 (sandstone) and Fig. 2 (serpentine). Trends for decreases and signi®cant decreases in percentage Non-M fungal colonization in high CO2 were observed for Avena, Sherardia, Linanthus, Plantago and Epilobium. The only plant species that showed a trend for a small increase in percentage Non-M colonization was Euphorbia, and for all other plant species there was no signi®cant difference. Euphorbia was the only plant species in the sandstone for which total AM percentage colonization signi®cantly increased (Fig. 1). This increase in AM colonization appeared to be due to an increase in coarse AM fungal hyphae only, with no signi®cant difference in percentage colonization with FE hyphae. In the serpentine grassland, the only signi®cant change in total percentage AM

Fig. 1 Sandstone: Percentage root colonization with different hyphal types (nonmycorrhizal, total arbuscular mycorrhizal, coarse, and ®ne endophyte AM hyphae) in elevated and ambient CO2 for ®ve plant species. Error bars indicate SE of the mean. Sample sizes (ambient, elevated) were: Brachypodium (8, 6), Lolium (5, 5), Euphorbia (8, 7), Sherardia (7, 9), and Avena (8, 8). Within a response variable, * (P < 0.05), ** (P < 0.01), *** (P < 0.001) denote signi®cant differences (1-way ANOVA), and + indicates a trend (0.05 < P < 0.15). # 1999

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colonization was the increase in Lolium (Fig. 2). Again, only coarse AM hyphae were responsible for this increase. Interestingly, coarse AM hyphal percentage colonization also increased for Linanthus and Epilobium, and FE percentage colonization decreased for Calycadenia, but in each case without concomitant changes in total AM percentage colonization. This apparent con¯ict is resolved when considering the data presented in Figs 3 (sandstone) and 4 (serpentine): we demonstrate here that the colonization `overlap' of the two AM fungal groups, i.e. the percentage of root colonized by both FE and coarse hyphae simultaneously, can be changed by the CO2 treatment. For example, the overlap of FE and coarse hyphae increased signi®cantly for Euphorbia (Fig. 3). In the serpentine, the overlap increased signi®cantly for Linanthus, but decreased for Calycadenia (Fig. 4). Percentage colonization with arbuscules increased signi®cantly for two out of ®ve species in the sandstone and for four of the ®ve species from the serpentine. For the remaining species, no signi®cant difference was found. An increase in overlap of the two AM fungal groups could lead to an increase in the intensity of overall AM fungal colonization. This was dif®cult to measure since the two hyphal types have such different growth patterns and hyphal diameters. However, for each AM hyphal group separately, such intensity estimates were possible, and are presented in Figs 5 and 6 for sandstone and serpentine, respectively. There was a trend for an increase in intensity of colonization with coarse hyphae for Euphorbia and Avena in the sandstone, and a signi®cant increase for Lolium in the serpentine. FE colonization intensity only changed signi®cantly for Plantago (increase) and Calycadenia (decrease), and showed a trend for decrease in Lolium.

Discussion We found signi®cant responses of root-colonizing fungi, arbuscular mycorrhizal and nonmycorrhizal, after longterm CO2 enrichment with intact host plant and fungal communities in the ®eld. These changes were dependent on the host plant species. We also found shifts in the composition of root colonizing AM fungi (FE and coarse

Fig. 2 Serpentine: Percentage root colonization with different hyphal types (nonmycorrhizal, total arbuscular mycorrhizal, coarse, and ®ne endophyte AM hyphae) in elevated and ambient CO2 for ®ve plant species. Error bars indicate standard errors of the mean. Sample sizes (ambient, elevated) were: Lolium (10, 10), Linanthus (9, 6), Plantago (5, 4), Calycadenia (4, 5), and Epilobium (8, 7). Signi®cance levels as in Fig. 1.

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Fig. 3 Sandstone: Percentage root colonized with both AM hyphal types (FE and coarse) simultaneously (solid bars), and percentage root colonized with arbuscules (slashed bars) in elevated and ambient CO2 for ®ve plant species. Error bars indicate SE of the mean (sample sizes and signi®cance levels as in Fig. 1).

Fig. 4 Serpentine: Percentage root colonized with both AM hyphal types (FE and coarse) simultaneously (solid bars), and percentage root colonized with arbuscules (slashed bars) in elevated and ambient CO2 for ®ve plant species. Error bars indicate SE of the mean (sample sizes and signi®cance levels as in Fig. 2).

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Fig. 5 Sandstone: Intensity of infection by coarse (solid bars) and ®ne (slashed bars) endophyte AM hyphae in elevated and ambient CO2 for ®ve plant species. Error bars indicate standard errors of the mean (sample sizes and signi®cance levels as in Fig. 1). # 1999

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Fig. 6 Serpentine: Intensity of infection by coarse (solid bars) and ®ne (slashed bars) endophyte AM hyphae in elevated and ambient CO2 for ®ve plant species. Error bars indicate SE of the mean (sample sizes and signi®cance levels as in Fig. 2).

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hyphae). Although caution is necessary when trying to extrapolate morphological/structural measurements of root symbionts to function, our results indicate that responses of root-colonizing fungi to elevated CO2 can persist after long-term exposure. The host plant dependence of the response of rootinhabiting AM fungi to elevated CO2 is important in two respects. It underlines the necessity to study more than one plant species, as there is no `representative' plant (Rillig et al. 1998a). For example, in the sandstone, only Euphorbia reacted with signi®cant total AM fungal percentage root colonization responses to the CO2 treatment; had only this plant been studied, conclusions about the sandstone grassland would have been quite misleading. Second, although the functional signi®cance of root colonization is not clear in all cases (Smith & Read 1997), it is possible that differential responses of AM and Non-M fungal colonization as a function of host plant have important consequences for plant community composition. It is known that AM fungi can in¯uence plant competition and plant communities (Allen & Allen 1990; Allen 1991; Zobel et al. 1997). The relatively uniform increase in arbuscular colonization for the serpentine grassland species is remarkable. It points to an increase in AM functioning, as these structures are responsible for carbon/nutrient exchange and are presumed to be shortlived (Allen 1991; Smith & Read 1997). We found decreases in Non-M fungal colonization in elevated CO2 for several plant hosts from the two different grasslands. The consequences of these changes in Non-M fungal colonization to plant growth are presently unknown in these grasslands. It is remarkable that a similar pattern arose in a short-term pot study (Rillig et al. 1998a) and in this longer-term ®eld study. The fact that Non-M fungal root colonization generally decreased in elevated CO2 is quite surprising, since carbon supply for rhizosphere organisms should have increased. The decrease can hence only be explained by biotic interactions among root-colonizing organisms. For example, there could have been increased competition for nutrients other than carbon or for root colonization sites among AM fungi and Non-M fungi. Clearly, future research should focus explicitly on this relatively poorly understood group of root-colonizing fungi and their feedback to plant growth in general, particularly in response to global change. We used the measurement of FE and coarse AM fungal hyphae as a coarse indicator of possible shifts in the composition of AM fungi colonizing roots of plants grown in elevated atmospheric CO2. It is currently not possible to measure quantitatively the species composition of AM fungi in roots, although recent advances in molecular methods are encouraging (e.g. Clapp et al. 1995; Harney et al. 1997). In some cases, different fungal

genera may be distinguished by careful examination of intraradical morphology (Abbott 1982) or by labelled antiserum (Weinbaum et al. 1996). Our results on FE and coarse hyphae root colonization indicate strongly that the composition of AM symbionts in roots can change with elevated atmospheric CO2. Given that the two differed functionally from each other in the few examples studied (Powell 1979; Wilson & Trinick 1983; Abbott & Robson 1984), it is possible that the altered ratio of coarse/FE colonization has consequences for plant growth. It is particularly interesting that the FE hyphae were not observed to increase for any of the host plant species in elevated CO2, whereas this was the case for coarse endophytes. It seems that FE fungi are not well-adapted to changed resource conditions in the rhizosphere as brought about by elevated CO2. Another important point arises with respect to the measurement of AM fungal responses to plants grown in elevated CO2. If only total percentage AM fungal colonization had been measured, some very interesting AM fungal responses would have been missed in our study. For example, there was no signi®cant difference in total percentage AM fungal colonization for Linanthus in elevated and atmospheric CO2. However, coarse hyphal colonization increased signi®cantly, leading to an increase in the overlap of the two hyphal types in roots. In this case, a signi®cant response at the total percentage colonization level was hidden in this increase in overlap. Also, for Calycadenia there was no change in total percentage colonization, but a decrease in FE hyphal colonization, leading to a decrease in overlap of the two groups. This suggests that due caution should be exercised when interpreting percentage AM colonization results in elevated CO2 studies.

Acknowledgements The Jasper Ridge CO2 Experiment was supported by grants from the U.S. National Science Foundation and U.S. Department of Energy. We thank Dr Nona Chiariello for help with identifying the plants in the ®eld, and Wendy Weick and Steve Damberger for help with the lab analysis. This study was supported by a U.S. Department of Energy (P.E.R.) grant to M.F.A. The Studienstiftung des deutschen Volkes (Bonn, Germany) supported M.C.R. with a Doctoral Fellowship during this research.

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