Nitrogen-Wxing Bacteria, Arbuscular Mycorrhizal Fungi, and the Productivity and Structure of Prairie Grassland Communities

Oecologia (2012) 170:1089–1098 DOI 10.1007/s00442-012-2363-3

COMMUNITY ECOLOGY - ORIGINAL RESEARCH

Nitrogen-Wxing bacteria, arbuscular mycorrhizal fungi, and the productivity and structure of prairie grassland communities

Jonathan T. Bauer · Nathan M. Kleczewski · James D. Bever · Keith Clay · Heather L. Reynolds

Received: 26 October 2011 / Accepted: 4 May 2012 / Published online: 10 June 2012 © Springer-Verlag 2012

Abstract Due to their complementary roles in meeting from the presence of the appropriate rhizobia symbionts. plant nutritional needs, arbuscular mycorrhizal fungi Sorghastrum nutans declined 44 % in the presence of W W W (AMF) and nitrogen- xing bacteria (N2- xers) may have N2- xers, with the most likely explanation being increased synergistic eVects on plant communities. Using greenhouse competition from legumes. Panicum monocultures were V W microcosms, we tested the e ects of AMF, N2- xers (sym- more productive with AMF, but showed no response to W biotic: rhizobia, and associative: Azospirillum brasilense), N2- xers, although inference was constrained by low and their potential interactions on the productivity, diver- Azospirillum treatment eVectivity. We did not Wnd interac- W sity, and species composition of diverse tallgrass prairie tions between AMF and N2- xers in communities or Panicum communities and on the productivity of Panicum virgatum monocultures, indicating that short-term eVects of these in monoculture. Our results demonstrate the importance of microbial functional groups are additive. W AMF and N2- xers as drivers of plant community structure and function. In the communities, we found a positive eVect Keywords Symbiosis · Diversity · Panicum virgatum · of AMF on diversity and productivity, but a negative eVect Azospirillum · Rhizobia W W of N2- xers on productivity. Both AMF and N2- xers aVected relative abundances of species. AMF shifted the communities from dominance by Elymus canadensis to Introduction Sorghastrum nutans, and seven other species increased in abundance with AMF, accounting for the increased diver- Soil micro-organisms are increasingly appreciated as impor- W sity. N2- xers led to increases in Astragalus canadensis and tant drivers of the productivity and structure of plant Desmanthus illinoense, two legumes that likely beneWted communities (Bever et al. 2010; Reynolds et al. 2003). Mycorrhizal fungi (AMF) and nitrogen-Wxing bacteria W (N2- xers) are well-known microbial functional groups that Communicated by Catherine Gehring. are key in meeting plant nutritional needs for phosphorus (Smith et al. 2003) and nitrogen (Cleveland et al. 1999), Electronic supplementary material The online version of this V article (doi:10.1007/s00442-012-2363-3) contains supplementary respectively. However, while the independent e ects of AMF W material, which is available to authorized users. and N2- xers on plant communities have been addressed, much less is known about how these two functional groups J. T. Bauer (&) · J. D. Bever · K. Clay · H. L. Reynolds might interact to inXuence the productivity and structure of Department of Biology, Indiana University, 142 Jordan Hall, 1001 East Third Street, Bloomington, IN 47405, USA plant communities. These symbionts may have interactive e-mail: [email protected] eVects on plant communities by mediating plant resource partitioning through their complementary roles in supplying N. M. Kleczewski limiting nutrients, or alternatively by competing for plant Department of Botany and Plant Pathology, Southwest Purdue Agricultural Research Center, photosynthate (Larimer et al. 2010; Reynolds et al. 2003). Purdue University, 4329 North Purdue Road, In experimental manipulations, plant productivity is Vincennes, IN 47591, USA generally higher in communities with AMF than in 123 1090 Oecologia (2012) 170:1089–1098 communities without AMF or where AMF have been sup- the rhizosphere of plants may lead to signiWcant levels of V W pressed (Hoeksema et al. 2010), although this e ect varies N2- xation (Montanez et al. 2009; Wickstrom and Garono with soil fertility (Hoeksema et al. 2010; Johnson 1993). 2007), and studies have also shown increased plant growth This increase in productivity is likely due to increased as a result of growth hormones produced by the bacteria or phosphorus uptake (van der Heijden et al. 1998), although enhanced mineral uptake associated with colonization by AMF may have other beneWts including improved pathogen Azospirillum (Dobbelaere et al. 2003; Lin et al. 1983; Okon resistance (Borowicz 2001). Plant species can vary widely and Labandera-Gonzalez 1994). in their response to AMF, however, and some studies Wnd As a result of their role in providing nutrients that are little change in productivity with AMF suppression as a frequently limiting to plant productivity, AMF and nitro- result of compensation from species less reliant on mycor- gen-Wxing bacteria may have synergistic eVects on plant rhizae (Hartnett and Wilson 1999; O’Connor et al. 2002). hosts by reducing both phosphorus and nitrogen limitation Variation in the mycorrhizal dependence of plants can lead (Larimer et al. 2010; Stanton 2003). However, these symbi- to shifts in plant competitive dominance and subsequently onts may also compete for photosynthate (Orfanoudakis plant community diversity in the presence versus absence et al. 2004), as AMF and rhizobia both place high demands of AMF (Hartnett et al. 1993; Vogelsang et al. 2006). For on the carbon Wxed by plants (Jakobsen and Rosendahl example, in a grassland dominated by non-mycorrhizal C3 1990; Schulze et al. 1999; Warembourg and Roumet 1989). grasses, the presence of AMF increased diversity by pro- A meta-analysis found that plant performance did not moting the subordinate mycorrhizal species (Grime et al. exceed additive eVects when plants were co-infected with 1988; Stein et al. 2009). In contrast, in grasslands domi- both rhizobia and AMF, although this result could be attrib- nated by C4 grasses that are highly responsive to AMF, the utable to variation among studies in level of soil fertility, presence of AMF may decrease diversity by allowing com- plant growth stage, or coevolutionary history of plants and petitive suppression of subordinate species (Hartnett and symbionts (Larimer et al. 2010). In contrast, where studies Wilson 1999; Smith et al. 1998), an eVect observed to be manipulate soil fertility in addition to symbionts, condi- dependent on level of phosphorus (Vogelsang et al. 2006). tions of low soil fertility produce cases of synergistic inter- The presence of symbiotic (e.g., rhizobia) or associative actions between rhizobia and AMF (Jia et al. 2004) and W (e.g., Azospirillum spp.) N2- xers can also increase produc- between Azospirillum and AMF (Mishra et al. 2008; tivity of plants and plant communities through increased Subba Rao et al. 1985). Given the potential for increased availability of nitrogen (Cleveland et al. 1999). Rhizobia pri- resource partitioning in association with microbial symbi- marily associate with legumes, and the presence of rhizobia onts (Reynolds et al. 2003; Temperton et al. 2007), syner- W can increase total community nitrogen by direct supply to gies between plants, N2- xing bacteria, and AMF may be legumes (van der Heijden et al. 2006a) or indirectly via root more likely to manifest at the level of plant communities exudates, common mycorrhizal networks. and litter decom- rather than individual plant hosts. However, to our knowl- W position associated with legumes (He et al. 2004; Johansen edge, synergies between AMF and N2- xing bacteria in and Jensen 1996; Paynel and Cliquet 2003; Thomas and plant communities have not been tested. Asakawa 1993). This may increase total community produc- These synergies are potentially important in a number of tivity by increasing total ecosystem nitrogen (Thomas and applied settings, particularly in low nutrient soils. Panicum Bowman 1998) and through increased nitrogen partitioning virgatum and diverse native grassland communities are between legumes versus plants that rely on pools of soil being considered as potential feedstocks for cellulosic bio- inorganic nitrogen (Reynolds et al. 2003; van der Heijden fuel production (Tilman et al. 2006; Wright and Turhollow et al. 2006a). The presence of rhizobia may also shift com- 2010). Including appropriate microbial symbionts in these munity composition by allowing legumes to compete with systems is likely to be important for sustainably increasing dominant species (van der Heijden et al. 2006a; Wurst and productivity and increasing ecosystem services generated van Beersum 2009), shifting communities from nitrogen to by these crops, such as increased carbon sequestration and phosphorus limitation (Thomas and Bowman 1998), and improved soil quality (Rygiewicz and Andersen 1994; van because of variation in species ability to beneWt from der Heijden et al. 2006b). Mycorrhizae and nitrogen-Wxing increased nitrogen (Marty et al. 2009). bacteria may also improve establishment of plants on Azospirillum spp. and other free-living nitrogen-Wxing degraded sites, such as mine spoils (Roy et al. 2007; bacteria may also be important sources of nitrogen to soil Walker et al. 2004). Plant microbial symbionts are increas- (Eisele et al. 1989; Solheim et al. 1996). Most experimental ingly recognized as important for the successful establish- work has focused on associative nitrogen-Wxing bacteria ment of desirable species in ecological restoration associated with the rhizospheres of agriculturally important (Middleton and Bever 2011; Richter and Stutz 2002). V W plants, especially grasses (Dobbelaere et al. 2003; James Here, we test the e ects of AMF and N2- xers (rhizobia W and Olivares 1998). Associative N2- xers associated with and Azospirillum) on the productivity, diversity, and 123 Oecologia (2012) 170:1089–1098 1091 species abundances of nutrient-poor grassland microcosms. soil included the top 2.1 m of alluvial silt and clay loam A, Using Panicum virgatum as a case study, we also examine B, and C horizons, which had been removed, partially the separate versus interactive eVects of these microbial mixed, and stockpiled, then tilled to 0.3-m depth before functional groups in monoculture as opposed to diverse delivery (Boyles, personal communication). These mixed communities. We tested the following hypotheses: (1) com- layers had a pH (H2O) of 5.8, contained 1.4 % organic mat- W ¡ + plementary roles of AMF and nitrogen- xing bacteria will ter, and tested at 1.2 ppm NO3 –N, 2.7 ppm NH4 –N, and V ¡ have a synergistic positive e ect on total plant productivity 5 ppm PO3 –P (1 N KCL-extractable N, Bray 1-extractable and diversity; synergistic eVects of microbial symbionts P; Michigan State University, Soil and Plant Nutrient Labo- will be stronger in communities than monoculture due to ratory, East Lansing, MI, USA). These low levels of avail- increased resource partitioning; and (2) the presence of able N and P in stockpiled soils, where plant uptake is not a AMF and nitrogen-Wxing bacteria will shift plant commu- large factor, are consistent with low soil fertility (Binkley nity composition in favor of plant species that beneWt most and Vitousek 1989). Using a cement mixer (Gilson mixer from these associations. 59015B; CF Gilco, Grafton, WI, USA), we mixed this soil 4:1 with coarse river sand to further reduce fertility and to lighten texture to promote root recovery at harvest. In order Materials and methods to eliminate background microbes, the soil mixture was pasteurized at 90 °C over two consecutive days for 120 min Study system each day, using an electric soil sterilizer (Model SS-60; Pro-Grow Supply, BrookWeld, WI, USA). Our focus was native tallgrass prairie restoration on land reclaimed after strip mining for coal, a system that we Plant treatments expected to be impoverished in ambient microXora and levels of plant nutrients and thus responsive to manipula- Microcosms were planted either with monocultures of 24 W tions of AMF and N2- xers (Corbett et al. 1996; Saxerud individuals of Panicum virgatum or with mixtures of 2 indi- and Funke 1991). Modern coal strip mining in the US viduals each of the following 12 species: Elymus canadensis, involves removal and stockpiling of surface soil layers, Panicum virgatum, Sorghastrum nutans (Poaceae); typically the A horizon and portions of the B and C hori- Astragalus canadensis, Desmanthus illinoensis, Desmo- zons, for capping the mine pit after coal seams are dium canadense (Fabaceae); Echinacea purpurea, Liatris removed, and the pit is backWlled with mine spoils (Indi- spicata, Symphyotrichum novae-angliae, Ratibida pinnata ana Code 2011). Mixing of richer A horizons with less (Asteraceae); Asclepias tuberosa (Apocynaceae); and fertile B and subsoil horizons dilutes soil fertility, and Monarda Wstulosa (Lamiaceae). Seeds were purchased both mixing and stockpiling disrupts and reduces micro- from Prairie Moon Nursery, Winona, MN, USA. All these bial communities (Saxerud and Funke 1991; Williamson species associate with AMF, though they vary in the bene- and Johnson 1990). This study system is of interest given Wts they gain from this association (Hartnett et al. 1993; that many thousands of acres of lands are still being strip Wilson and Hartnett 1998). The three legumes form associ- mined and reclaimed in the Midwest U.S. and elsewhere, ations with rhizobia, but the importance of associations W and there is increasing interest in restoration with native between our study plant species and free-living N2- xers is grassland for its beneWts to wildlife habitat, recreation, unknown. Plants were initially grown from surface-steril- carbon sequestration, and water puriWcation among other ized seed (2 min soak in 50 % ethanol followed by 1 min in ecosystem services (Halofsky and McCormick 2005; 5 % Clorox bleach and two rinses in sterile water for MRCSP 2006; Scott and Lima 2004). If eVective, restor- 1 min) in 2 £ 2 £ 4 cm cells containing pasteurized Metro ing the soil fertility of these lands via microbial inocula- Mix 360 (Sun Gro Horticulture) potting soil (except for tions would be a more sustainable approach than Panicum, which is described below). Seedlings were application of chemical fertilizer, whose production and grown for 1 month and then transplanted into experimental use entail substantial inputs of fossil fuels and greenhouse microcosms. Tools were sterilized between replicates by gas emissions, nutrient leaching and associated degrada- washing in soap and water, rinsing, and immersing in 95 % tion of water quality, and inhibition of the soil microbial ethanol. To further minimize cross-contamination, we community (Broussard and Turner 2009; Robertson et al. planted mesocosms in order of no-microbe controls to sin- 2000; Tilman et al. 2002). gle-microbe treatments to AMF £ N2-Wxer treatments. Tallgrass prairie microcosms were established in 11.3-L Plants were regularly spaced in the microcosms, and in the nursery pots (Hummert International, Earth City, MO, case of the 12-species mixtures, a randomly generated USA) Wlled with Cory and Iva series soil obtained from the arrangement of species was consistently used across micro- Lewis Mine (Solar Sources, Vigo County, IN, USA). This bial treatments. 123 1092 Oecologia (2012) 170:1089–1098

Microbial treatments sterile buVer solution was added to each hole. Then, seedling plugs were planted into the holes and the pots were W AMF and N2- xers (rhizobia and Azospirillum) treatments watered thoroughly. were imposed in a 2 £ 2 factorial design (sterile control, We included a Panicum endophyte inoculation treatment W W W AMF, N2 xers, AMF £ N2 xers). The AMF treatment with six endophytic fungi that we had previously identi ed consisted of a mixture of fungal cultures isolated from Indi- as having beneWcial eVects on the growth of Panicum seed- ana and Illinois prairie soils and cultured with Sorghum lings (Kleczewski et al. 2012). BrieXy, Panicum seeds were bicolor, and included Acaulospora spinosa, Entrophospora cleared of preexisting endophytes by heat treating in 50 °C infrequens, Scutellospora fulgida, Glomus claroideum, water for 30 min. Seeds were germinated in autoclaved G. lamellosum, and G. mosseae. The soil and coarsely sand and then inoculated with endophytes (Kleczewski chopped roots from these cultures were used as inocula for et al. 2012). After 14 days, seedlings were transferred to this experiment. Uninoculated controls received an equiva- Metro Mix immediately prior to their incorporation into the lent volume of sterilized soil of the same type used for the experimental microcosms. Panicum seedlings were smaller AMF cultures. AMF were identiWed by species-deWning and less vigorous than those of the other study plant spe- characteristics of their asexually produced spores. AMF are cies, which did not receive hot water treatments and were generally thought to have low speciWcity of association, but sown directly into Metro Mix. In tests for presence of endo- growth of an individual plant species may depend upon the phytic fungi in Panicum leaves after several months of particular fungal partner. We inoculated with a diverse growth, we were not able to consistently reculture inocu- community as previous work has demonstrated that a lated fungi from Panicum. Furthermore, we did not detect diverse community provides the beneWt of the best fungus signiWcant eVects of endophyte treatments in our initial sta- (Vogelsang et al. 2006). A microbial wash is often included tistical analyses of harvested plant biomass. Thus, the endo- in uninoculated treatments to control for other micro-organ- phyte treatment was removed from the Wnal analysis and isms introduced in the AMF cultures, but we did not will not be discussed further. include this background wash to reduce the potential for For both Panicum monocultures and prairie communi- W contamination with N2- xing bacteria. A meta-analysis of ties, each microbial treatment was replicated 6 times. How- AMF studies found that the absence of this microbial wash ever, due to the lack of an endophyte eVect, our replication provides a conservative test of the beneWts of AMF and rec- was eVectively doubled so that each treatment in both com- ommends this approach for factorial manipulation of AMF munities and monocultures was replicated 12 times. W and rhizobia (Hoeksema et al. 2010). The N2- xer treatment included cysts of wild-type Azospirillum brasilense, Data collection V ATCC 29145, suspended in 10 mM KPO4 bu er (pH 6.8; 1 £ 107 cfu/mL) and rhizobia as cultures attached to granu- Plants were allowed to grow in microcosms for 16 weeks, lar peat (»1 £ 108 rhizobia per g). Azospirillum cysts were from June 6 to September 28, 2009. At this point, we induced on minimal salts media (MSM; as described by clipped all aboveground biomass plus any root crowns. Neyra et al. 1997) in the laboratory of Carl Bauer at Indiana Plant tissue was then dried at 60 °C to a constant mass and University. Rhizobia strains were obtained from the Rhizo- weighed. Belowground biomass was sampled from each bium Research Laboratory at the University of Minnesota microcosm by taking three 5.1-cm diameter cores the full where they were originally isolated from tallgrass prairie in depth of the microcosm. The three cores from each micro- Minnesota. A strain of Rhizobium giardinii and Mesorhizo- cosm were bulked, sealed in plastic bags, and held at 4 °C bium huakuii were used as the inoculants for Desmanthus until processing. Bulked soil was subsampled to test for the W and Astragalus; respectively. The strain used as inocula for abundance of associative N2- xers in the soil by plating a Desmodium was not identiWed to species, but had previ- dilution series of 1 g soil in 10 mL water, then further dilut- ously been identiWed as “inoculant quality” for Desmodium ing 10, 100, and 1,000£. Dilutions were plated on N-free (Beyhaut et al. 2006; Tlusty et al. 2004, 2005). Pots were media containing Congo Red, which binds to the cell walls Wlled two-thirds full with sterile background soil, then of Azospirillum and changes color in the presence of nitro- 80 mL of AMF inocula or a control of sterile soil of the gen-Wxing bacteria (Caceres 1982). Colony morphology same soil type was added. Five grams of rhizobia inocula was also compared to reference strains for identiWcation as for each of the three legume species (15 g total) or 15 g of a Azospirillum. Roots were washed and nodules present on sterile granular peat control were then added to each pot the roots were counted to assess rhizobia inoculation. Roots which was topped with a layer of sterile soil. Pots were were then clipped into 1-cm segments and a representative watered lightly, and then a template was used to create reg- subsample from three replicates of each treatment was ularly spaced holes for plugs of the appropriate plant spe- weighed and placed in tissue cassettes (HistoPrep OmniS- cies. Before plants were added, 1 mL of Azospirillum or ette; Fisher ScientiWc Healthcare, Pittsburgh, PA, USA). 123 Oecologia (2012) 170:1089–1098 1093

Following standard protocols (McGonigle et al. 1990), 1.32 § 0.3 nodules; ANOVA p = 0.03). We did not Wnd roots were cleared with KOH and stained with trypan blue evidence that AMF aVected nodulation or that rhizobia V W for analysis of percentage fungal colonization (number of a ected AMF colonization (non-signi cant AMF £ N2- hyphae, arbuscules, vesicles or coils found at 30–40 root Wxer interactions). There was extensive colonization of intersections per root subsample). The remaining roots most pots by Azospirillum by the end of the experiment so were then dried at 60 °C and weighed. Dry weights of root that counts of plated colonies did not diVer signiWcantly W subsamples were estimated using a wet to dry weight con- between the N2- xers treatment and controls (p = 0.20). We version factor obtained from the main root mass. therefore make the conservative assumption that any eVects W of the N2- xer treatment on plant responses in this experi- Data analysis ment were due to rhizobia, and thus limited to the 12-species communities where legumes were also present. Data were analyzed using Proc GLM in SAS (v.9.3.1, 2010; SAS Institute, Cary, NC, USA). For each 12-species Productivity microcosm, total aboveground productivity was determined W W V by adding the biomass of all species and the Shannon AMF and N2- xer treatments signi cantly a ected above- Diversity Index (HЈ) was calculated using the biomass of ground productivity in communities, with productivity each species. We tested for microbial treatment eVects on increasing in response to AMF (Fig. 1a) and decreasing in W each of these response variables and root biomass, percent response to N2- xers relative to controls (Fig. 1b). There V W colonization of roots by AMF, and root nodulation using were no interactive e ects of AMF and N2- xers on com- two-way ANOVA. munity productivity. Panicum virgatum monocultures pro- Shifts in plant community composition in the 12-species duced more above-ground biomass with AMF, but, microcosms were analyzed with non-metric multidimen- consistent with the poor Azospirillum treatment eVectivity, V W sional scaling (NMS) using a Bray Curtis distance measure we did not detect an e ect of N2- xers or an AMF £ N2- and starting coordinates from a Bray Curtis Ordination. The Wxers interaction on above-ground biomass (Fig. 1c; resulting axis scores were used as a response variable in a Table 1). Above-ground productivity in Panicum monocul- V W MANOVA to test for e ects of AMF, N2- xers, and inter- tures increased by 71 % in response to inoculations with actions on community composition. Changes in the abun- AMF, in contrast to the 12 % increase observed in commu- dance of each species were further tested using a nities. We did not detect any treatment eVects on root bio- W W MANOVA with AMF, N2- xers, and AMF £ N2- xers mass in either Panicum monocultures or in communities interactions as Wxed eVects and the biomass of each species (Table 1). as response variables. Community composition and diversity

Results Inoculation with AMF had a positive eVect on the diversity (HЈ) of community microcosms, whereas inoculation with V W W V Ј Treatment e ectivity N2- xers did not signi cantly a ect H (Fig. 2). For the NMS ordination, a three-axis solution was signiWcantly Percent colonization of roots by AMF was signiWcantly diVerent from random data (p = 0.005) and accounted for higher in the microcosms inoculated with AMF (44 § 3%, 96.3 % of the variation in our data. The ordination showed mean § SE) than in controls (7 § 1%, p < 0.0001). Counts diVerences in composition of community microcosms due W of nodules on harvested roots found that microcosms inocu- to AMF and N2- xer treatments, with axes 1 and 2 explain- W lated with N2- xers had more nodules per root sub-sample ing 84 and 8.9 % of the variation, respectively (Fig. 3; axis W W than controls (N2- xers: 4.25 § 1.2 nodules; control: 3 explained 3.6 % of variation). The signi cance of these

Fig. 1 The eVect of a AMF and W (a) (b) (c) b N2- xers on total above- ground biomass in community microcosms and the eVect of c AMF on above-ground productivity in Panicum monocultures

123 1094 Oecologia (2012) 170:1089–1098

V W Table 1 Results of ANOVA for main eVects and interactions of AMF Di erent plant species responses to AMF versus N2- xer W and N2- xers on above-ground biomass in 12 species community treatments help explain changes in NMS scores (Fig. 4). microcosms, above-ground biomass in Panicum monocultures, root Ј Accordingly, a MANOVA of individual species’ perfor- biomass and on diversity (H ) in 12 spp. microcosms W V mance found signi cant main e ects of AMF (F12,33 = 7.83, Productivity df F P W p < 0.0001) and N2- xers (F12,33 = 5.17, p < 0.0001) treat- W 12 spp. communities ments, but no signi cant interaction (F12,33 = 1.14, p = 0.36, ESM Resource 1). Most strikingly, Elymus and AMF 1,44 6.33 0.0156 Sorghastrum switched dominance based on AMF treatment N -Wxers 1,44 6.70 0.0130 2 (Fig. 4a). Elymus was the most important species in micro- AMF £ N -Wxers 1,44 0.05 0.8204 2 cosms without AMF, whereas Sorghastrum dominated Panicum monocultures microcosms with AMF. Additionally, Desmodium AMF 1,44 35.88 <0.0001 increased dramatically with AMF along with positive N -Wxers 1,44 2.10 0.1565 2 responses of Symphyotrichum, Liatris, Ratibida, Echina- AMF £ N -Wxers 1,44 1.70 0.2012 2 cea, and Monarda (Fig. 4a). In contrast, Elymus and Root biomass Sorghastrum were codominant in microcosms without AMF 1,44 1.33 0.2550 N -Wxers, whereas Elymus alone was dominant in micro- W 2 N2- xers 1,44 0.10 0.7547 W X W V cosms with N2- xers, re ecting a signi cant negative e ect £ W AMF N2- xers 1,44 0.00 0.9630 of N -Wxers on Sorghastrum abundance (Fig. 4b). Liatris, Ј 2 Diversity—H Desmodium, and Symphyotrichum also responded nega- AMF 1,44 15.99 0.0002 W V W tively to N2- xers, although this e ect was signi cant only W N2- xers 1,44 0.95 0.3348 for Liatris (Fig. 4b). The legumes Desmanthus and Astragalus AMF £ N -Wxers 1,44 1.39 0.2456 W 2 responded positively to N2- xers, as did Ratibida (Asteraceae) (Fig. 4b).

Discussion

We hypothesized that our low fertility microcosms, including co-occurring tallgrass prairie plants and microbial symbionts, would be an ideal context for synergistic eVects of W AMF and N2- xers on plant productivity and community structure. We expected greater interactive eVects of AMF W and N2- xers in communities versus monocultures due to increased resource partitioning (Reynolds et al. 2003). However, this hypothesis was not supported. AMF and V W V Fig. 2 E ect of AMF inoculation on species diversity, as measured by N2- xers did not have interactive e ects on productivity in the Shannon Index (HЈ), in community microcosms communities or Panicum microcosms, although our ability to test for interactive eVects in Panicum microcosms was constrained by poor treatment eVectivity of associative W community shifts were established by MANOVA on axis N2- xing bacteria. Our results suggest that Larimer et al.’s scores, which indicated signiWcant overall eVects of AMF (2010) conclusions, from a meta-analysis of single-species W (F3,42 = 13.12, p < 0.0001) and N2- xers (F3,42 =7.83, responses, that the response of plants to dual inoculation p = 0.0003), but no interaction (F3,42 =0.73, p = 0.73). with microbial symbionts does not exceed the additive Shifts in community composition along axis 1 were due to eVects of either symbiont independently can also apply to W both AMF (F1,44 =14.6, p < 0.0001) and N2- xers (F1,44 = plant communities. 2.63, p = 0.012), with standardized canonical coeYcients We did observe signiWcant main eVects of AMF and V W indicating a stronger and opposite e ect of AMF treatment N2- xers on productivity. AMF increased the productivity W (1.387) versus N2- xers (¡0.722) on plant species abun- of community microcosms and Panicum monocultures. The W V dances (see species MANOVA results below). N2- xers e ect of AMF was greater in Panicum monocultures W V had a signi cant e ect on axis 2 scores (F1,44 =1.32, than in community microcosms, which may be a result of V p = 0.0065), but there was no e ect of AMF (F1,44 =0.35, Panicum’s strong positive response to AMF inoculation p = 0.15). Neither treatment had a signiWcant eVect on axis and the variable response of the other species included

3 scores (F3,44 =0.13, p =0.94). in our microcosms (Vogelsang et al. 2006; Wilson and 123 Oecologia (2012) 170:1089–1098 1095

(a) (b)

Fig. 3 NMS ordination of community microcosms showing main Wxers treatments. Similarly, Control in (b) includes both control and V W W W e ects of a AMF treatment and b N2- xers treatment on composition AMF treatments and N2 xers includes both N2- xers and AMF + N2- of community microcosms. Control in (a) includes both control and Wxers treatments W N2- xers treatments and AMF includes both the AMF and AMF + N2-

(a) (b)

Fig. 4 Rank abundance diagrams showing the response of species rosa (AT), Astragalus canadensis (AC), Ratibida pinnata (RP), Pani- W within 12 species microcosms to a AMF and b N2- xers (*p < 0.05, cum virgatum (PV). As in Fig. 3, Control in (a) includes both control W full statistics in “Electronic Supplementary Material” (ESM) Resource and N2- xers treatments and AMF includes both the AMF and W 1). Abbreviations are as follows: Elymus canadensis (EC), Sorgha- AMF + N2- xers treatments. Similarly, Control in (b) includes both W W strum nutans (SN), Desmodium canadense (DC), Symphyotrichum control and AMF treatments and N2 xers includes both N2- xers and W novae-angliae (SA), Liatris spicata (LS), Desmanthus illinoensis (DI), AMF + N2- xers treatments Monarda Wstulosa (MF), Echinacea purpurea (EP), Asclepias tube-

W V V Hartnett 1998). In contrast, N2- xers had no e ect in Pani- in communities, other studies have found positive e ects on W cum monocultures. Again, the lack of an N2- xer treatment plant community productivity, even within a single grow- eVect in Panicum monocultures is not surprising because ing season, due to reduced competition for nitrogen and Azospirillum colonized all treatments and Panicum does rapid transfer of nitrogen to other species (Marty et al. not associate with rhizobia. We did observe a negative 2009; Temperton et al. 2007; van der Heijden et al. 2006a). V W W main e ect of N2- xers on productivity in community However, other pathways for nitrogen input from N2- xers microcosms, indicating that the costs of maintaining this into ecosystems, such as decomposition of Wne roots and symbiosis during the establishment of these communities leaf litter, may have positive eVects over longer time scales outweighed the beneWts of increased nutrient inputs (Schulze than our experiment tested (Hogh-Jensen and Schjoerring et al. 1999; Warembourg and Roumet 1989). Although we 1997; Walley et al. 1996). Consequently, we may have V W W V W found a negative e ect of N2- xing bacteria on productivity been able to observe bene cial e ects of N2- xing bacteria 123 1096 Oecologia (2012) 170:1089–1098 had our experiment been conducted over a longer period of Our Wndings are consistent with others that have found time. Further, nitrogen addition generally increases the ben- that many legumes require rhizobia to grow and persist eWcial eVects of AMF on plants (Hoeksema et al. 2010) so with competitive dominants (van der Heijden et al. 2006a; that long-term nitrogen inputs in communities through the Wurst and van Beersum 2009). In our case, two legumes, legume–rhizobia symbiosis could lead to AMF–rhizobia Astragalus and Desmanthus, both had increased biomass W synergies. in association with N2- xing bacteria, although a third We also expected synergistic eVects of AMF and legume, Desmodium, tended to decrease in biomass. We W W N2- xers on plant community composition and diversity. For expected N2- xing bacteria to increase total productivity W example, AMF and N2- xers might be expected to have due to increased nitrogen inputs, but we found reduced pro- V W interactive e ects on legumes, which can be highly respon- ductivity in the N2- xers treatment. This can largely be sive to both groups of microbial symbionts (van der attributed to the 44 % decline in productivity of Sorgha- V W Heijden et al. 2006a; Wilson and Hartnett 1998). Further, strum. We did not detect any e ect of N2- xers on Pani- W W the potential for AMF and N2- xers to mediate resource cum, the other C4 grass included in our study. The N2- xers partitioning could lead to synergistic eVects on diversity. treatment included free-living Azospirillum in addition to However, we did not Wnd evidence for interactive eVects of rhizobia, but our treatment conWrmation data indicate that W AMF and N2- xers in our analyses of diversity, overall all treatments were similarly colonized by Azospirillum. plant community composition (NMS), or response of indi- Rhizobia and the legumes they associate with thus appear vidual species within communities. As suggested above, to be the most likely drivers of the observed decline in Sor- V W synergistic e ects may require more time for development ghastrum yield in microcosms treated with N2- xers. One than aVorded in one growing season. Alternatively, it is possibility is that Sorghastrum experienced increased possible that opportunities for resource partitioning or other above- or belowground competition from the two legumes W complementary species interactions were constrained by whose productivity was enhanced in the N2- xer treatment. the physical size of our microcosms. Although Sorghastrum overtopped the legumes by the end We found support for our second hypothesis, that AMF of the experiment, it is possible that light competition was W and N2- xers would shift the composition of grassland important early on, at the seedling stage. Alternatively, pre- plant communities. Major shifts in community composition vious studies have reported reduced abundance of grasses occurred as a result of inoculation with AMF, with domi- as a result of phosphorus limitation associated with the nance of the communities shifting from Elymus towards presence of legumes (Thomas and Bowman 1998). Sorghastrum. Along with this increase in Sorghastrum, Increased productivity of legumes and perhaps increased seven other species increased in biomass in the presence of demand for phosphorus associated with the presence of rhi- AMF. This shift increased the evenness of the plant com- zobia may thus have increased competition for phosphorus munities, explaining the increase in diversity (HЈ) associ- and decreased productivity in our experiment. Sorghastrum W W V ated with AMF. N2- xers did not signi cantly a ect and other C4 prairie grasses are sensitive to phosphorus diversity, but did aVect plant community composition limitation, explaining their strong dependence on AMF through increases in the legumes Astragalus and Desman- (Hartnett et al. 1993; Hetrick et al. 1986). Therefore, Sor- thus, and decreases in Sorghastrum and Liatris. ghastrum’s positive response to AMF and negative V W Previous work has reported mixed results for the e ect response to N2- xers may both be driven by demand for of AMF on species diversity. When the dominant plant is phosphorus. non-mycorrhizal, as in European calcareous grasslands, The consistent, positive main eVect of AMF on both AMF promote diversity by increasing subordinate species Panicum and 12-species communities in our experiment and increasing the evenness of the plant community (Grime supports further consideration of this symbiont as a means et al. 1988). In contrast, previous work in tallgrass prairie to improve the sustainability of biofuel crops and increase has found that when the dominant plants (e.g., C4 grasses) diversity and ecosystem services in prairie restoration and are mycorrhizal, AMF reduce diversity (Hartnett and remediation. In addition to improved productivity, AMF Wilson 1999). In our study, the dominant species in unin- have other beneWts including increased carbon inputs into oculated control microcosms was Elymus, a C3 grass with a the soil and improved soil aggregation (Jakobsen and weak response to AMF (Wilson and Hartnett 1998). The Rosendahl 1990; Wilson et al. 2009). Although our experi- increase in diversity we observed supports the general ment found reduced plant community productivity in W framework that AMF can promote diversity when subordi- response to the presence of rhizobial N2- xers, long-term nate species are more dependent on mycorrhizae than nitrogen inputs from legumes and associated rhizobia have dominants, but AMF suppresses diversity when dominant the potential to improve the overall sustainability and pro- species are mycorrhizal (Hartnett and Wilson 2002; Urcelay ductivity of a site over time (Hogh-Jensen and Schjoerring and Diaz 2003; Vogelsang et al. 2006). 1997). 123 Oecologia (2012) 170:1089–1098 1097

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