Exotic invasive plants increase productivity ... - Wiley Online Library

Journal of Ecology 2016, 104, 994–1002

doi: 10.1111/1365-2745.12584

Exotic invasive plants increase productivity, abundance of ammonia-oxidizing bacteria and nitrogen availability in intermountain grasslands Morgan Luce McLeod1,2*, Cory C. Cleveland3, Ylva Lekberg2,3, John L. Maron4, Laurent Philippot5, David Bru5 and Ragan M. Callaway6 1

Wildlife Biology, University of Montana, Missoula, MT 59812, USA; 2MPG Ranch, Missoula, MT 59801, USA; Department of Ecosystem and Conservation Sciences, University of Montana, Missoula, MT 59812, USA; 4Division of Biological Sciences, University of Montana, Missoula, MT 59812, USA; 5INRA, UMR 1347 Agroécologie, Dijon, France; and 6Division of Biological Sciences and Institute on Ecosystems, University of Montana, Missoula, MT 59812, USA 3

Summary 1. Exotic plant invasion is often associated with dramatic increases in above-ground net primary productivity and soil nitrogen. However, most evidence for these increases comes from correlative studies of single species, leaving open the question of whether invasive plants drive these processes and whether they are consistent among invaders. 2. We combined field surveys and measurements within experimental plantings to examine how plant productivity, soil nitrogen and the abundance of ammonia-oxidizing bacteria (AOB) change in response to invasions by four exotic species. 3. The relationship between plant productivity and soil nitrate differed among native and invasive species, suggesting a fundamental disparity in the effects of natives and invaders on ecosystem processes. In field surveys, dense patches of all invasive species had higher abundances of AOB than native-dominated sites. Three of the four invasive species had higher productivity, soil nitrate concentrations and rates of potential nitrification as compared to nearby native-dominated communities. In our experimental plantings, we found that two invasive species drove increases in soil nitrate and one invader caused increased productivity after a single season. 4. Synthesis. Our results highlight the importance of the N cycling soil microbial community in how exotic invasive plants alter ecosystem function and show that shifts in function can occur rapidly. Key-words: ANPP, ammonia-oxidizing bacteria, Centaurea stoebe, Bromus tectorum, ecosystem, exotic invasion, Euphorbia esula, invasion ecology, grassland, nitrogen cycling, Potentilla recta

Introduction Net primary productivity (NPP) of ecosystems is thought to operate near the maximum that local environmental and ecological constraints allow (Running 2012). Yet paradoxically, ecosystems invaded by exotic plants often exhibit substantially higher above-ground net primary productivity (ANPP) and soil nitrogen (N) concentrations than the native communities they replace (Vitousek 1990; Ehrenfeld 2003; Rout & Callaway 2009). In a meta-analysis, Liao et al. (2008) found that exotic plant invasion was associated with 83% more ANPP, 17% higher soil nitrate (NO 3 ) and 53% higher net

*Correspondence author. E-mail: [email protected]

nitrification rates across ecosystems and invader life histories as compared to non-invaded systems. Links between elevated ANPP, soil N and plant invasion challenge current ideas about local-to-global scale constraints on ANPP. However, previous studies have been limited in several important ways. First, most studies have focused on a single invasive species (see Liao et al. 2008), with few studies examining multiple invasive species with unique traits and life histories within a single ecosystem, thus limiting broader generalizations. Secondly, most studies have been field based and correlative; thus, the effects of invaders on ecosystems are potentially confounded by disproportionate invasion of nutrient-rich sites (but see Maron & Marler 2008; Maron et al. 2014a). Finally, few studies have examined the potential contribution of key N cycling microbial groups,

© 2016 The Authors. Journal of Ecology © 2016 British Ecological Society

Invasive plants increase ANPP, AOB and soil N 995 such as ammonia-oxidizing bacteria (AOB), to altered ecosystem function following invasion (but see Hawkes et al. 2006). Nitrogen availability constrains ANPP in many ecosystems (Elser et al. 2007; LeBauer & Treseder 2008). Atmospheric N is abundant, but conversion to biologically available N forms is energetically costly, which generally results in low N supply rates (Vitousek & Howarth 1991). Under low N conditions, rates of N recycling (i.e., via mineralization during decomposition) are also generally low, further enhancing N limitation (Parton et al. 2007). N availability and loss rates are also affected by nitrification, a limiting step in N cycling (Hart et al. 1994; Van der Heijden, Bardget & Van Straalen 2008; Cabello, Roldan & Moreno-Vivian 2009). The first step in nitrification is ammonia oxidation, which in turn is controlled by the abundance and activity of ammonia-oxidizing bacteria (AOB) and thaumarchaea (AOA). Above-ground processes and plant traits have been thought to explain links between invasion, elevated ANPP and soil N. For example, fundamental trait differences among invaders and natives such as species-specific differences in timing, quality, or amount of energetically rich litter or root inputs may increase N availability (Baker 1965; Liang, Zhang & Zhao 2006; Castro-Dıez et al. 2014). The intensity of herbivore and pathogen attack also often differs among invasive and native species (Keane & Crawley 2002; Agrawal et al. 2005; Kulmatiski et al. 2008; Tallamy, Ballard & D’Amico 2010; Schaffner et al. 2011). This may result in higher ANPP in invaded ecosystems simply because consumers remove less biomass. However, gains in ANPP associated with invasion are usually much higher than generalized estimates of tissue loss to consumers (Keddy 2007). Further, the direct effects of herbivory on plant litter quality are not consistent among species (Bardgett et al. 1998). Below-ground processes and microbial communities have been less explored, yet may represent a key link between invasion and elevated ANPP and soil N. Shifts in timing or quality of litter or below-ground carbon input (e.g., via root exudates) by invaders could promote short-term turnover of soil microbial biomass (Kuzyakov, Friedel & Stahr 2000) or enhance soil organic matter mineralization (Ehrenfeld 2001), resulting in greater fluxes of soil N from organic to inorganic pools. Invasion is often associated with shifts in the abundance or activity of microbial groups (Batten et al. 2006; Broz, Manter & Vivanco 2007; Callaway et al. 2011) such as arbuscular mycorrhizal fungi (Lekberg et al. 2013) and/or pathogens (Kulmatiski et al. 2008). Thus, invasive plants have the potential to both alter and benefit from microbially regulated soil processes (Philippot et al. 2013). The role of microbial functional groups in soil N cycling (Van der Heijden, Bardget & Van Straalen 2008) and the mechanics of this cycling are well understood (Hart et al. 1994; Cabello, Roldan & Moreno-Vivian 2009). However, few studies have explicitly considered the potential contribution of the soil N cycling bacterial community to increased ANPP and N cycling with invasion (Hawkes et al. 2006, Booth 2003; Evans et al. 2001), and in turn, how invasion might alter the

composition, abundance and function of these microbial groups (Rout & Callaway 2009). Here, we investigated whether increased ANPP and N availability is associated with invasion by four exotic species common to intermountain grasslands of the northern Rocky Mountains and explored the potential contribution of increased abundance of AOB to observed shifts in ecosystem function. We addressed three main questions: (i) Is exotic plant invasion in our system generally associated with increased ANPP and soil N availability? (ii) Do invasive plants cause these changes? and (iii) Do shifts in ecosystem function correspond with shifts in N cycling microbial groups? To address these questions, we compared ANPP, N dynamics and AOB abundance in paired patches of adjacent invaders and natives in the field and assessed rapid shifts in ANPP and N dynamics in experimentally established invader monocultures and native mixtures.

Materials and methods FOCAL INVASIVE SPECIES

We examined the impacts of four invasive plant species (Bromus tectorum, Centaurea stoebe, Euphorbia esula and Potentilla recta) that represent different plant families and life-history strategies, within a common habitat. Bromus tectorum (cheatgrass) is a widespread, highly invasive annual grass distributed throughout the American West. Bromus tectorum has been associated with increased AOB and increased N cycling rates and pool sizes (Svejcar & Sheley 2001; Sperry, Belnap & Evans 2006). Centaurea stoebe (spotted knapweed) is a highly invasive, perennial forb that is abundant throughout much of the Rocky Mountain West. Euphorbia esula (leafy spurge) is a deep-rooted, perennial forb, invasive throughout grasslands of the Northern Great Plains and Rocky Mountains. Finally, Potentilla recta (sulphur cinquefoil) is a perennial forb distributed throughout the Northern United States with heavy infestations from the Mountain West to the Great Lakes region (Rice 1999). Invasion by each of these species has been correlated with decreases in native species richness (Ortega & Pearson 2005), and C. stoebe and P. recta have been shown experimentally to have large impacts on native communities (Maron & Marler 2008). NATURALLY INVADED FIELD SITES

Our field sites were in grasslands in the Missoula (46°51’45”N, 114° 00’42”W, 972 m) and Bitterroot (46°40’48”N, 114° 1’40”W, 1024 m) valleys in western Montana, USA. Mean annual precipitation is ~ 350 mm, and the rainiest months are May and June (http:// www.ncdc.noaa.gov). Soils consisted of fine to gravelly, loamy Mollisols. We utilized a paired plot design to examine differences between invaded and native plots both within and among target invasive species. For each of the four target invasive species, plots were established in eight densely invaded naturally occurring mono-dominant patches. Patches ranged in size from 5 9 5 m to greater than 25 9 25 m. Each of the 32 invaded patches was paired with an adjacent patch that was dominated by a mix of native species and in which none of the invasive species occurred. Paired plots were matched for elevation, aspect, slope and landscape position to minimize difference in environmental factors. The maximum distance between invaded and paired native plot edges was 15 m, and we

© 2016 The Authors. Journal of Ecology © 2016 British Ecological Society, Journal of Ecology, 104, 994–1002

996 M. L. McLeod et al. avoided disturbed areas. Soil and plant biomass samples were taken 28 May – 4 June and 28 June – 3 July 2012 respectively, in three 0.25 m2 plots located within each patch.

FIELD SOIL SAMPLING AND BIOGEOCHEMICAL ANALYSIS

We took eight soil cores with an Oakfield probe (0–10 cm depth) and combined them to create one composite soil sample for each of the three plots within our 32-paired patches. Our composite samples were then subsampled, and subsamples were frozen within 24 h at 80°C for subsequent analysis of ammonia-oxidizing thaumarchaeal (AOA) and bacterial (AOB) abundance. We subdivided the remaining soil of the composite samples for the following biogeochemical analyses: inorganic N pools, net and potential nitrification rates, soil chloroform labile C estimate of microbial biomass, pH and total soil carbon (C) and N. Gravimetric water content was determined for all soil samples, and results are reported on a lg/g oven dry soil basis for each nutrient. To determine inorganic N pools, we extracted inorganic N using 2M KCl (Hart et al. 1994). Fifteen g of soil was added to 100 mL of 2M KCl, shaken on an orbital shaker for one hour, allowed to settle overnight, filtered and along with an extraction blank, frozen until þ analysis. Concentrations of NO 3 and ammonium (NO4 ) in filtrates were determined colorimetrically using a Synergy 2 Microplate Reader (BioTek, Winooski, VT, USA) after Weatherburn (1967) and Doane & Horwath (2003) for NOþ 4 and NO3 , respectively. We determined net nitrification rates using the buried bag field method (Hart et al. 1994). During initial soil sampling, an additional intact soil core was collected, placed inside of a breathable polyethylene bag, loosely closed using a twist tie to allow gas exchange but not liquid water flow and reburied. After 28 days, we collected bagged soil cores from the field. Pre- and post-incubation samples were extracted with 2M KCl and analysed as described above. We determined nitrification potential for each plot using the shaken slurry method (Hart et al. 1994). Nitrification potential is an indirect estimate of nitrifier abundance that normalizes abiotic factors such as water availability, pH, NOþ 4 availability and uptake by plant roots, as this variability can mask differences among sites and treatments. For this analysis only we combined the three plot samples to create a mixed sample for each patch. We added 15 g of sample to 100 mL of 1 mM phosphate buffer solution (pH 7.2) and 1.5 mM (NH4)2SO4 in a 250 mL flask which was loosely covered and shaken continuously. We then sampled and centrifuged aliquots and froze the supernatant at 2, 4, 22 and 24 h. Determination of NO 3 from aliquots was performed using micro-plate analytical technique described above. We used these four sampling points to calculate the rate of potential nitrification, or amount of NO 3 generated through time, for each sample. Soil microbial biomass was measured as chloroform labile C and was determined by extracting two subsamples. To one 15-mL subsample, we added 50 mL 0.5 M K2SO4, shook for one hour on an orbital shaker, settled overnight, filtered and frozen until analysis. A second subsample was fumigated for five days with ethanolfree chloroform (Horwath & Paul 1994) and then extracted in the same manner as above. Determination of non-purgeable organic carbon (NPOC) and total N (TN) was determined using a Shimadzu TOC-V TN Analyzer (Shimadzu Corporation, Kyoto, Japan). We determined chloroform labile C and N estimate of microbial biomass by subtracting un-fumigated from fumigated TN and NPOC values.

All remaining soil was air-dried and ground to a fine powder using mortar and pestle. We measured pH on this air-dried soil in 0.01 M CaCl2 using a 1:1 (w/v) soil: liquid ratio (Accumet Dual Channel pH/ Ion/Conductivity Meter, Pittsburgh, PA, USA). Soil total C and N were determined by combustion using an elemental analyser (CE Elantech, Lakewood, NT, USA).

PLANT BIOMASS ANALYSIS – FIELD PLOTS

We estimated ANPP at our sites by measuring peak standing biomass. We harvested all plant biomass produced in the current year within each 0.25 m2 plot by clipping at ground level. Harvesting plant biomass in at this time period captures biomass produced by dominant perennial native grasses and invasive forbs in our system, but may miss a relatively small proportion of biomass produced by early season ephemeral forbs, some of which are annual. Biomass was dried 70 °C for 72 h and weighed. A subsample was ground and total C and N determined by combustion (CE Elantech).

ABUNDANCE OF AOB AND AOA – FIELD PLOTS

We measured changes in the soil microbial community involved in N cycling by real-time PCR quantification of the abundance of AOB and AOA in soils from the paired invaded and native field plots. Genomic DNA was extracted from approximately 0.25 g of soil from each sample using the Mo Bio Powersoil kit (Carlsbad, CA, USA). Soil from one plot within each patch (n = 8 for each invader) was analysed. Extracted DNA suspended in 100 lL of sterile elution buffer was stored at 80 °C. Quantification of the bacterial and thaumarchaeal ammonia oxidizers was performed according to Leininger et al. (2006) and Tourna et al. (2008) using the genes encoding catalytic enzymes of ammonia oxidation (bacterial and thaumarchaeal amoA) as molecular markers. Reactions were carried out in an ABI prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Quantification was based on the increasing fluorescence intensity of the SYBR Green dye during amplification. The realtime PCR assay was carried out in a 15-ll reaction volume containing the SYBR green PCR Master Mix (Absolute QPCR SYBR Green Rox Mix; Thermo, Villebon-sur-Yvette, France), 1 lM of each primer, 250 ng of T4 gene 32 (QBiogene, Strasbourg, France) and 2 ng of DNA. Two independent quantitative PCR assays were performed for each gene. Standard curves were obtained using serial dilutions of linearized plasmids containing the studied genes. PCR efficiency for the different assays ranged between 83 and 85%. Two to three no-template controls were run for each quantitative PCR assay, and no-template controls gave null or negligible values. The presence of PCR inhibitors in DNA extracted from soil was estimated by mixing a known amount of plasmid DNA with soil DNA extract or water prior to qPCR. The measured cycle threshold (Ct) values obtained when quantifying the added plasmid DNA were not significantly different between the different soil DNA extracts and the controls with water, which indicates that no inhibition occurred.

EXPERIMENTALLY MANIPULATED PLOTS

To determine whether invaders caused shifts in productivity and ecosystem function observed in field studies, we also collected soil and plant biomass from within experimental plots located at the MPG Ranch in Florence, MT (46°40’48.92”N, 114°1’40.73”W).


Invasive plants increase ANPP, AOB and soil N 997

We used a generalized linear mixed model (GLIMMIX module, SAS ver. 9.1; SAS Institute Inc., Cary, NC, USA) to determine how invasion status affected all measured response variables including ANPP, þ NO 3 , NO4 , net nitrification, nitrification potential and microbial biomass. Invasion status and species were fixed factors, and site and patch were random factors. This model accounts for potential spatial autocorrelation of patches, as well as the paired nature of the field study. We then used orthogonal contrasts to test a priori hypotheses regarding invader native pairs at species level. Distributions of means were checked for normality using generalized chi-square/DF fit test. Data were log-transformed when necessary to satisfy assumptions of normal distribution of means and homoscedasticity. The magnitude of the effect of invasion on below-ground processes may vary with the biomass of the invader because greater amounts of biomass produced above-ground may correlate with greater plant source soil inputs. To investigate potential differences in relationship between ANPP and below-ground function between invaders and natives, we used ANCOVA to determine how ANPP and invasion status affected soil NO 3 , including species identity in the model (R, version 1.40, 2011). For the garden experiment, we used an ANOVA with species as a fixed factor and block as a random factor to compare differences in þ ANPP, NO 3 , NO4 and net nitrification across five plant communities (four invaded monocultures and mixed native community). Tukey’s post hoc analysis was used to determine differences among species (a = 0.05).

ANPP AND NITROGEN CYCLING IN THE FIELD

On average, invaded patches produced 74% greater ANPP than nearby native patches (Fig. 1a; FINVADED = 33.47, d.f. = 1,28, P < 0.0001), but not all invasive plants responded to invasion in the same way. Bromus tectorum-dominated patches produced 71% more biomass (results of GLIMMIX contrast difference of invader least squares means; t = 3.57, d.f. = 46.66, P = 0.003), C. stoebe 63% more biomass (t = 3.90, d.f. = 46.49, P = 0.001) and E. esula 200% more biomass (t = 6.66, d.f. = 46.51, P < 0.0001), in comparison with paired native patches. Biomass in P. recta -invaded patches was not higher than paired native patches (t = 0.23, d.f. = 42.25, P = 0.82). Soil NO 3 concentrations were 103% higher in invaded patches in comparison with native patches (Fig. 1b; FINVADED = 34.23, d.f. = 1,53, P < 0.001). Bromus tectorumdominated patches were associated with a 42% increase in (a)

Aboveground net primary productivity (g m–2 year –1)

STATISTICAL ANALYSES

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These plots were located near our field sites and were surrounded by a similar native community to that in our native field plots. Plots (2 9 2 m) were established in spring 2011 and were planted with monocultures of 64 individuals of the four invasive species discussed above, or even mixtures of the following native species; Pseudoroegneria spicata, Elymus elymoides, Koelaria macrantha, Bouteloua gracilis, Penstemon strictus, Linum lewisii, Erigeron speciosus, Gaillardia aristata and Achillea millefolium. Plots were planted in a randomized block design (n = 5) for a total of 25 plots on tilled soil that previously hosted an introduced forage grass (Agropyron cristatum) and ruderal exotic weeds (B. tectorum, Sisymbrium altissimum, Erodium cicutarium, Poa bulbosa) prior to multiple applications of 0.473 L Roundup PowerMAXâ herbicide (Monsanto Company; 0.84 kg glyphosate ha1). Seedlings for transplant were grown in the glasshouse for six weeks from locally collected seed and inoculated with a mixture of soil collected under target invasive species and representative native plants to ensure that microbes that normally associate with the target species were present (see Data S1, Supporting Information, for details). Experimental plots received supplemental water during the first year when small plants were establishing, but no water was added thereafter. For the duration of the experiment, we removed all non-target species by hand weeding within plots and by applying Roundup PowerMAXâ (as above) to areas outside of plots. In mid-June 2012, we randomly located two 0.25 m2 subplots in each of the 25 plots and collected soil and estimated ANPP by collecting above-ground plant peak biomass following the protocol described above for the field study. Soil samples were subsampled for the following analyses: inorganic N pools, net nitrification and chloroform labile C estimate of microbial biomass. However, we did not include an estimate of ANPP for E. esula due to substantial herbivory by Apthona lacertosa and A. nigriscutis, two biological control insects.

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Fig. 1. Mean (1 SE) above-ground net primary productivity (a) and soil extractable NO 3 (b) of Bromus tectorum, Centaurea stoebe, Euphorbia esula and Potentilla recta patches and respective paired native-dominated patches in the field. Asterisks denote significant differences among invaded and paired native patch means using orthogonal contrasts (a = 0.05).


998 M. L. McLeod et al.

R E L A T I O N S H I P B E T W E E N A N P P A N D S O I L N O 3 FOR INVADERS VS. NATIVES

In invaded patches, ANPP was positively correlated with soil NO (Fig. 4; FINVADED = 30.71, d.f. = 1,29, P < 0.001, 3 R2 = 0.51). In contrast, in soil occupied by native species, there was no significant relationship between ANPP and soil NO 3 (Fig. 4; FNATIVE = 1.56, d.f. = 1,30, P = 0.221, R2 = 0.05). In an ANCOVA, the interaction between ANPP and invaded vs. native treatment was significant (FTREATMENT = 4.50, d.f. = 1, P = 0.038), indicating that the relationship between NO 3 and ANPP differed between invaded and native patches. In a

subsequent ANCOVA of invaded patches only, we found that there was no significant effect of invader identity on the relationship between NO 3 and ANPP (FINVADER = 0.43, d.f. = 3, P = 0.73). ABUNDANCE OF AOB, AOA AND CHLOROFORM LABILE C ESTIMATE OF MICROBIAL BIOMASS IN THE FIELD

The absolute abundance of AOB in invaded patches was 4.7 times higher than in native patches (Fig. 5a; FINVADED = 66.79, d.f. = 1,28, P < 0.0001). At the species level, B. tectorum-dominated patches demonstrated an increase of AOB of 620% (t = 5.80, d.f. = 44.21, P < 0.0001), C. stoebe 330% (t = 5.55, d.f. = 44.16, P < 0.0001), E. esula 282% (t = 4.46, d.f. = 47.43, P < 0.0001) and P. recta 225% (t = 3.54, d.f. = 41.17, P < 0.001) as compared to native patches. The absolute abundance of AOA in invaded patches

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NO 3 (t = 4.00, d.f. = 56, P < 0.001), C. stoebe with a 124% increase (t = 3.96, d.f. = 57, P = 0.0008) and E. esula with a 193% increase (t = 5.81, d.f. = 57, P < 0.0001), relative to paired native patches. Soil NO 3 concentrations beneath P. recta did not differ from those in paired native soil samples (t = 1.03, d.f. = 54.01, P = 0.31). Invasion was associated with a significant increase in soil NOþ 4 in E. esula dominated patches only (t = 3.44, d.f. = 57, P = 0.01, Table S1); no other species showed this pattern. Nitrification potential of soil patches across all species in the field was 59% higher than in native patches (Fig. 2; FINVADED = 13.48, d.f. = 1,30, P < 0.001). Nitrification potential was 52% greater in C. stoebe-dominated patches (t = 1.93, d.f. = 47.31, P = 0.059), patches of E. esula had 88% greater nitrification potential (t = 3.63, d.f. = 47.44, P < 0.001) and P. recta 94% greater nitrification potential (t = 2.86, d.f. = 43.43, P < 0.01) than paired native patches. The nitrification potential in soil beneath B. tectorum did not differ from that in native soil (t = 0.28, d.f. = 47.77, P = 0.78). Net nitrification rates in the soil of invaded patches did not differ significantly from those in native soil for any of the species studied (Table S1).

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Fig. 2. Mean ( 1 SE) nitrification potential of Bromus tectorum, Centaurea stoebe, Euphorbia esula and Potentilla recta and respective paired native patches in the field. Asterisks denote significant differences between the paired means (a = 0.05).

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Fig. 3. Mean ( 1 SE) above-ground net primary productivity (a) and soil extractable NO 3 (b) of Bromus tectorum, Centaurea stoebe, Euphorbia esula and Potentilla recta versus natives in experimental plots. Different letters represent significant differences in post hoc comparisons (a = 0.05).


Invasive plants increase ANPP, AOB and soil N 999 Invaded Native

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Annual aboveground net primary productivity (g m–2 year –1) Fig. 4. Relationships of ANPP and soil extractable NO 3 in the field plots. Invader identity was not significant in invaded treatment. ANPP was not significant in native treatment. ANPP of exotics explained 51% of variation in soil extractable NO 3 . Inset shows relationships of soil extractable NO 3 and ANPP in the field plots with the invader plots constrained to range of ANPP values measured for natives.

did not differ significantly from that in native patches (Fig. 5b; FINVADED = 1.11, d.f. = 1,28, P = 0.30). At the species level, P. recta -dominated patches showed a near-significant increase of AOA of 186% (Fig. 5b; t = 1.92, d.f. = 43.85, P = 0.06) as compared to native patches. Invasion was associated with a significant increase in chloroform labile C estimate of microbial biomass in E. esula (t = 2.99, d.f. = 47.43, P = 0.003) dominated patches (Table S1); no other species showed this pattern. EXPERIMENTAL PLOTS

Overall, there was a 57% increase in ANPP in invaded plots relative to native plots in the common garden, but this varied significantly among species and the overall effect was driven by C. stoebe (Fig. 3a; Fspecies = 53.28 d.f.=3,16; P < 0.001). Centaurea stoebe had higher ANPP than natives (Fig. 3a; t = 6.75, d.f. = 4.95, P = 0.001), whereas B. tectorum had lower ANPP than native plots (Fig. 3a; t = 6.98, d.f. = 4.98, P < 0.001). Potentilla recta did not significantly increase ANPP, and E. esula biomass values are not reported because an introduced biological control beetle, Aphthona sp., heavily grazed these plots. ANPP in planted native plots averaged 727.3 148.5 g m2, roughly seven times higher than the average ANPP of native patches in field conditions (Fig. 3a; see Fig. 1a for comparison). Soil NO 3 concentrations in garden native plots averaged 3.7 2.7 lg g1, similar to soil NO 3 concentrations native patches in the field. Soil NO 3 concentrations in invaded soil showed a similar (but more subtle) pattern to what was observed in the field. The increases in NO 3 within invaded plots varied significantly by species and was driven by E. esula (Fig. 3b; t = 7.58, d.f. = 7.64, P < 0.001).

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Fig. 5. Mean (1 SE) relative abundance of amoA genes (copies per gram soil) from ammonia-oxidizing bacteria (AOB) (a) and ammoniaoxidizing archaea (AOA) (b) in soils under patches of Bromus tectorum, Centaurea stoebe, Euphorbia esula, Potentilla recta and paired native plots for each invader in the field. Asterisks denote significant difference in mean between the invaded plot and native paired plot.

Centaurea stoebe also exhibited significantly higher amounts of NO 3 (Fig. 3b; t = 3.06, d.f. = 5.38, P = 0.03) and B. tectorum and P. recta did not significantly alter NO 3 . Net nitrification rates in the experimental garden were negligible, and averaged 0.003 0.05 lg g1 day1, and did not differ among invaded and native plots (Table S2; FINVADED = 2.72, d.f. = 4, P = 0.06).

Discussion ANPP is both constrained by (Elser et al. 2007; LeBauer & Treseder 2008) and influences (Vitousek 1990; Wardle et al. 2004) N availability in temperate terrestrial systems. Exotic invasion in our system was highly associated with increased ANPP and soil N, as has been shown in other systems (Liao et al. 2008). Three of our four invasive species, B. tectorum, C. stoebe and E. esula, had significantly greater ANPP and soil NO 3 than paired native communities in field plots. Our findings support a large number of previous studies of single


1000 M. L. McLeod et al. species of grassland invaders (see supplementary table Liao et al. 2008) and suggest that increases in ANPP and soil N associated with invasion may be somewhat consistent across species in a single system. However, the magnitude of these increases varied among species. Euphorbia esula, the most productive species in our study, was associated large increases in ANPP, soil NO 3 concentrations and nitrification potential. By contrast, P. recta invasion was associated with a large increase in nitrification potential, but no significant increase in ANPP or soil NO 3. The ANPP and soil NO values we observed in E. esula 3 patches were higher than previously reported values for many other invasive forbs (see supplementary table Liao et al. 2008), indicating that this species may just be especially productive with a disproportionately large effect on ecosystem function. Our findings suggest that above-ground factors such as species identity and associated life history or traits are important in determining the magnitude of shifts in ecosystem function following invasion. Plant traits such as tissue stoichiometry or novel compounds (Macel et al. 2014) have been associated with invasion; some of which seem to promote greater acquisition or more rapid N cycling (Penuelas et al. 2010). However, in our study, plant traits alone did not appear to predict ecosystem responses. For example, the C:N ratio of B. tectorum was higher than that of native biomass, whereas E. esula had a lower C:N ratio than native biomass (Table S1), but both species were associated with higher ANPP and higher soil N availability than native patches. This indicates that additional above-ground variation in phenology, nutrient requirements, uptake rates and plasticity as well as below-ground factors such as carbon allocation to soil microbial communities might influence ecosystem function following invasion. Herbivore preferences may also have contributed to differences in ANPP and soil NO 3 concentrations in our study. The enemy release hypothesis suggests that native generalist herbivores may prefer and consume greater amount of native plant biomass as compared to invader plant biomass (Keane & Crawley 2002; but see Parker & Hay 2005). We did not observe evidence of significant herbivory on native plants in our study, and in addition, it is not clear why an increase in ANPP via escape from consumers would correspond with an increase in soil NO 3 and AOB abundance. Also, the mean increase in ANPP (74%) we found was larger than the mean consumption rate for grasslands in general (44%; Keddy 2007), suggesting that the activity of herbivores alone did not account for differences in ANPP among invaded and native patches. To our knowledge, our results are among the first to identify a strong positive relationship between ANPP and soil NO 3 for invaders, but not for natives (Fig. 4). We chose to focus on ANPP as an independent variable, with soil NO 3 as a response variable. However, plant productivity has the potential to both alter and benefit from microbially regulated soil processes (Wardle et al. 2004; Philippot et al. 2013). In our study, ANPP accounted for 51% of the variation in NO 3 in soils occupied by invasive plants, but there was no such

relationship in soils beneath native species, suggesting that invaders may influence ecosystem function in fundamentally different ways than natives. Increased ANPP and soil N with exotic invasion are well documented, but much less is known about the second question addressed by our study; do invaders cause these increases? We sampled experimental plots and found that two of our invaders, C. stoebe and E. esula, caused increased soil NO 3 and a single invader, C. stoebe, caused increased ANPP just one year after establishment. This suggests that some invasive species may alter ecosystem function after a single year, rather than invaders preferentially colonizing nutrient-rich sites. Species-specific differences between field and experimental results may be partially attributed to time since establishment. Immediately after establishment, phenotypic plasticity (Price, Qvarnstr€ om & Irwin 2003), or the ability of an organism to alter its phenotype in response to its environment, may play a large role. Potentilla recta, for example, had no effect on ANPP in the field but significant effects in the experimental garden. Our experimental plots had slightly higher soil N concentrations than native field patches, and phenotypically plastic species, such as P. recta may have used this resource to grow large in the first year, and perform more as expected in subsequent years. Also, we found native mixtures in the experimental plots produced much higher biomass as compared to field natives, which may have dampened differences in ANPP and N cycling between natives and invaders in the experimental plots. Centaurea stoebe, for example, increased ANPP and NO 3 in both the field and garden, but B. tectorum had strong effects on ANPP in the field only. The more consistent and relatively stronger impacts of exotics in field plots may be due to the invaders being present longer, with ample time for feedbacks (Kulmatiski et al. 2008) between plants, soil properties and the soil microbial community, to develop. Below-ground processes and microbial communities represent underexplored, yet potential key links between invasion and elevated ANPP and soil N. The third question addressed by our study was, does the abundance of N cycling AOB also increase in the presence of invaders? One of the most striking findings of our study was a large increase in AOB abundance in soil associated with all invasive species (Fig. 5a). Our findings are consistent with an experimental garden study by Hawkes (et al. 2005) who also reported increased abundance of AOB in planted plots of exotic grasses. The abundance of AOA (Fig. 5b) at our sites did not increase in invaded patches, indicating that AOB were likely drivers of increased nitrification and soil NO 3 in our system (see also Di et al. 2009). We suspect that the increase in AOB abundance paralleled an overall shift in microbial community composition (as opposed to an overall increase in the size of the microbial community), as we saw no significant increase in total microbial biomass (Table S1) except for. E. esula. However, increased AOB abundance did not always translate to shifts in ecosystem function. For example, P. recta, the least productive invader in our study, exhibited an unexpected combination of increased AOB abundance and nitrification potential, but no measurable increase in soil NO 3 or ANPP.


Invasive plants increase ANPP, AOB and soil N 1001 This suggests that increased abundance of AOB alone may not translate to increased AOB activity, and unrealized nitrification potential in this case may be due to limitations other than amount of N. Increased AOB abundance and activity coupled with high ANPP of B. tectorum, C. stoebe and E. esula in our study may enhance N availability. The activity of chemoautotrophic AOB is not directly limited by access to plant-supplied carbon (C), but can be limited by access to ammonium (NOþ 4 ), oxygen, soil moisture, favourable temperature and pH (Hart et al. 1994; Myrold 2005). Access to C does, however, limit the bacterial and fungal decomposers that supply AOB with NOþ 4 and greater access to C could result in increased mineralization and NOþ 4 supply rates. There are several potential sources of increased C following invasion. First, higher quality litter on the soil surface following invasion could supply decomposers with an additional source of labile C, but we rarely saw persistent litter layers decomposing at the soil surface in our system. Alternatively, Funk & Vitousek (2007) hypothesized that some invaders might exhibit higher nutrient use efficiency than the natives they out-compete, and this trait would allow invaders to allocate more C below-ground. Decomposers could access this C via increased root exudates (Kuzyakov et al. 2000), or via increased decomposition of fine plant roots. Finally, in our system, the persistent, dense pool of standing biomass of invaders may also moderate the physical soil environment through shading, resulting in a longer time period with favourable temperature and moisture for C and N cycling. However, increased biomass alone does not explain elevated AOB abundance associated with our least productive invader, P. recta. Our findings of increased AOB abundance and increased nitrification potential coupled with no change in ANPP or soil NO 3 indicate that invasion itself may result in a shift in the overall composition of the soil microbial community, resulting in a greater proportion of AOB. The mechanics of these shifts remain unclear, and evidence for such shifts in microbial community composition associated with invasion is few (but see Batten et al. 2006; Broz, Manter & Vivanco 2007), more work is needed in this area. Our results advance the current body of literature showing positive relationships between exotic invasion, increased soil NO aggblom 3 (Evans et al. 2001; Kourtev, Ehrenfeld & H€ 2002; Hawkes et al. 2006), potential nitrification (Lee, Flory & Phillips 2012) and ecosystem productivity (Maron & Marler 2008). This is primarily because we have documented causal relationships and identified key microbial groups that can help to explain the process. The mechanisms by which the soil biota in a region interact with native and exotic invasive plant species remain unclear (see Vila et al. 2011; Maron et al. 2014a,b), and it is equally unclear how these interactions feedback to increase ANPP. However, our results indicate that a diverse suite of invaders can cause rapid increases in productivity and soil N, and that these shifts are associated with broad, consistent, increases in AOB abundance. Furthermore, the different relationship between ANPP and soil NO 3 for natives and invaders (Fig. 4) indicates that invasive plant

species have fundamentally different effects on ecosystem processes than native species. When exploring broad questions about invasive success, consideration of below-ground processes is crucial.

Acknowledgements RMC and JLM thank the National Science Foundation DEB 0614406 and the NSF EPSCoR Track-1 EPS-1101342 (INSTEP 3), and MM and YL thank MPG Ranch for funding. The Wildlife Biology Program of the University of Montana gave additional support. Field and laboratory support provided by Becky Fletcher, Kevin Moore and Ben Sullivan. Yvette Ortega provided statistical guidance. Laurie Marczak and two anonymous referees provided comments on earlier versions of this manuscript. Authors have no conflict of interests to declare.

Data accessibility Data associated with this study are deposited in the Dryad repository (http:// dx.doi.org/10.5061/dryad.dd490) (McLeod et al., 2016).

Author contributions MM and RC designed the study, MM collected and analysed data. DB performed genetic analysis of microbial DNA. MM wrote first draft of manuscript and all authors contributed substantially to revisions.

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Supporting Information Additional Supporting Information may be found in the online version of this article: Data S1. Details regarding experimental garden plot establishment and species identity. Table S1. Means (SE) of all measured variables for all invaded and native field plots. Bold text denotes significant differences (P < 0.05) among invaded and native means for that species. Table S2. Means (SE) of all measured variables of Bromus tectorum, Centaurea stoebe, Euphorbia esula, and Potentilla recta versus natives in experimental plots. Different letters represent significant differences in post hoc comparisons (a = 0.05).
