Research Article. Keywords: Context-dependency; coexistence; environmental stress; plant microbe interactions; prairie; water availability.

Transcription

1 Research Article Interactions between plants and soil microbes may alter the relative importance of intraspecific and interspecific plant competition in a changing climate Anna P. Hawkins* and Kerri M. Crawford Department of Biology and Biochemistry, University of Houston, Houston, TX 77204, USA Received: 30 March 2018 Editorial decision: 11 June 2018 Accepted: 18 June 2018 Published: 20 June 2018 Associate Editor: Jean H. Burns Citation: Hawkins A, Crawford K Interactions between plants and soil microbes may alter the relative importance of intraspecific and interspecific plant competition in a changing climate. AoB PLANTS 10: ply039; doi: /aobpla/ply039 Abstract. Interactions between plants and soil microbes play an important role in structuring terrestrial ecosystems by influencing plant growth and competitive ability. Studies have shown that abiotic conditions such as varying nutrient levels or environmental stress can alter the direction and magnitude of plant microbe interactions. Given this context-dependency, it is possible that the effects of changing climates, including changing water availability, could alter the outcome of plant microbe interactions, which could in turn affect interactions among plant species. We tested whether water availability mediated the effect of soil microbes on pairwise plant interactions in the Texas coastal prairie using a controlled greenhouse experiment with three plant species: Schizachyrium scoparium, Rudbeckia hirta and Plantago lanceolata. To test for an interaction between water availability and soil microbes, plants were grown in either live or sterile soil treatments and with high, medium and low water availability. We found that the presence of soil microbes generally increased the strength of intraspecific competition relative to interspecific competition, but this effect depended on water availability. In the presence of microbes, as water availability decreased the strength of intraspecific competition generally increased. Our results suggest that soil microbes may play a role in stabilizing coexistence by increasing conspecific negative density dependence, especially in drier environments. Keywords: Context-dependency; coexistence; environmental stress; plant microbe interactions; prairie; water availability. Introduction A growing body of research suggests that soil microbial communities can play a key role in structuring plant communities (Reynolds et al. 2003; Bever et al. 2010, 2015; Classen et al. 2015). Soil microbial communities contain a diversity of plant-antagonistic and plant-beneficial microbes, and plants respond to these soil communities in species-specific ways (Bever 1994). These species-specific responses help set the stage for soil microbes to influence plant community composition (Bever et al. 1997; Classen et al. 2015; van der Putten et al. 2016; Smith-Ramesh and Reynolds 2017). For example, differences in plant responses to soil microbial communities may affect the relative abundance of plant species (Klironomos 2002; Mangan et al. 2010) and soil *Corresponding author s address: anna.p.hawkins@gmail.com The Author(s) Published by Oxford University Press on behalf of the Annals of Botany Company. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. AoB PLANTS The Author(s)

2 microbes often less negatively affect non-native plant species than native plant species (Reinhart and Callaway 2004, 2006; Kulmatiski et al. 2008), possibly because non-native species have escaped from pathogens present in their native ranges (Keane and Crawley 2002). Soil microbes can also indirectly influence plant performance by mediating interactions among plant species (Grime et al. 1987; van der Putten and Peters 1997; van der Heijden et al. 1998; Birhane et al. 2014; Emam et al. 2014). Soil microbes that provide species-specific benefits can allow their hosts to gain competitive advantages over other plant species, as can occur with mycorrhizal fungi (Allen and Allen 1984; Hartnett et al. 1993; Hartnett and Wilson 1999; Smith et al. 1999). Alternatively, mycorrhizae can increase diversity and decrease competitive inequalities between species (Grime et al. 1987; van der Heijden et al. 1998; Wagg et al. 2011; Jiang et al. 2017). Antagonistic soil microbes may also exacerbate or weaken the effects of competition (van der Putten and Peters 1997; Petermann et al. 2008; Bever et al. 2015; Albornoz et al. 2016), depending on the plant species examined and the magnitude of their responses to the soil microbes. However, other experiments have found that soil microbes have no effect on plant competition (Bever 1994; Casper and Castelli 2007), or that competition overwhelms the effects that soil microbes have on plant performance (Crawford and Knight 2017). The context-dependency of plant microbe interactions may help explain some of the variation in how plant interactions respond to soil microbes. Soil microbial communities can respond rapidly to changes in the environment, which may cause changes in the outcome of plant microbe interactions and plant plant interactions mediated through microbes (Meisner et al. 2013; Classen et al. 2015; van der Putten et al. 2016). For example, increased nitrogen deposition can indirectly alter interactions between plant species (Larios and Suding 2015), and microbial communities can quickly respond to changes in precipitation, helping their hosts tolerate the novel environmental conditions (Lau and Lennon 2012). Even without a change in microbial community composition, changes in the environment can alter how individual components of the microbial community influence plants. One of the best-known examples of this is the tendency for nutritional mutualists such as mycorrhizae to become parasitic under high nutrient conditions (Johnson et al. 1997; Hoeksema et al. 2010). As the climate changes, it is increasingly important to understand how environmental factors can alter plant communities. While there are many aspects of the environment that are predicted to be affected by climate change, there is mounting evidence that water availability can influence interactions between plant species and between plants and their associated soil microbiota (van der Putten et al. 2016). In the absence of other factors, drier conditions generally favour drought-tolerant plant species when they compete with drought-intolerant plant species (Lopez et al. 2013; Napier et al. 2016). Water availability can also influence the strength and direction (i.e. competitive to facilitative) of interactions in plant communities (Kadmon 1995; Bu et al. 2013). Below-ground, water availability can change the structure of soil microbial communities and how they interact with plants. Drought can decrease the biomass of soil pathogens (Augspurger 1984) and beneficial microbes such as arbuscular mycorrhizal (AM) fungi and rhizobia (Augé 2001; Compant et al. 2010). Despite changes in abundance, some evidence suggests that soil mutualists and pathogens may have a stronger impact on plant performance in drier conditions. Mutualists can help plants cope with stressful conditions (Kivlin et al. 2013; Pischl and Barber 2017) and some pathogens can have a more detrimental effect on plant performance when plants are already stressed (Jactel et al. 2012; Kolb et al. 2016). When changes in the composition of the soil microbial community or in plant microbe interactions differentially influence plant species, it can lead to changes in plant community composition. For example, legacy effects of drought and rainfall on soil microbial communities have been found to decrease native biomass but have no effect on the biomass of non-natives (Meisner et al. 2013). Despite evidence that water availability influences plant plant interactions and plant soil interactions, few studies have examined whether water availability mediates the effect of soil microbes on plant interactions. If drought decreases the abundance of soil microbes, it might be expected that microbes will play a weaker role in mediating plant interactions in dry conditions than wet conditions. Alternatively, if dry conditions increase the effect of soil microbes on plant performance, soil microbes may play a stronger role in mediating plant interactions in dry conditions than wet conditions. To increase our ability to predict how plant microbe interactions will change with changing climate, it may be especially important to understand how particular groups of soil microbes respond to water availability (van der Putten et al. 2016). For instance, if decreased water availability increases the potency of species-specific soil pathogens, then drought may magnify the effects of intraspecific competition. Alternatively, if drought decreases the effectiveness of beneficial soil microbes, then plant species reliant on those beneficial associations may experience stronger interspecific competition in dry conditions. Because the relative strength of intraand interspecific competition influences coexistence 2 AoB PLANTS The Author(s) 2018

3 (Chesson 2000), microbe-mediated changes in plant interactions caused by water availability could influence plant diversity. Therefore, it may be of particular importance to understand these complex interactions to better predict how plant communities will change in future climates (van der Putten et al. 2016). To test how water availability shifts microbe-mediated interactions, we conducted a laboratory experiment using three plant species commonly found in the coastal prairie of Texas. Water availability can be highly variable in the coastal prairie, and periods of drought and rainfall are expected to become more severe with climate change (Jiang and Yang 2012; IPCC 2014; Shafer et al. 2014). Therefore, understanding how water availability interacts with soil microbes to influence interactions among plants may be important for predicting future changes in plant community composition in this system. Specifically, we asked the following questions: (i) Do soil microbes alter the relative strength of intraand interspecific plant competition? (ii) Does the effect of soil microbes on plant competition depend on water availability? We hypothesized that changes in water availability would shift the relative importance of intraversus interspecific competition by altering the interactions between plants and the soil microbial community. Methods We examined how water availability and soil microbes influenced intra- and interspecific competition among three plant species using a factorial experimental design consisting of three watering treatments (low, medium and high) crossed with two soil treatments (live and sterile). We ran this experiment in the laboratory and fully randomized the locations of all pots at the start of the experiment. The three plant species differed in their origins and functional groups: Plantago lanceolata, a non-native, weedy forb; Rudbeckia hirta, a native forb; and Schizachyrium scoparium, a native grass. These three species co-occur in the Texas coastal prairie and all form associations with AM fungi. Arbuscular mycorrhizal fungi, though generally mutualistic, can become parasitic to their plant hosts under certain environmental conditions (Johnson et al. 1997; Hoeksema et al. 2010). Seeds for each species were purchased from commercial suppliers: R. hirta and S. scoparium from Prairie Moon Nursery (Winona, MN, USA) and P. lanceolata from Sheffield s Seed Company (Locke, NY, USA). To prepare sterile seedlings, we surface-sterilized the seeds by agitating them in 10 % bleach solution for 5 min and thoroughly rinsing them with de-ionized water. We germinated the sterile seeds in autoclaved (two cycles for 90 min at 121 C with a 24-h resting period between cycles) potting soil (SunGro Metromix 250; Agawam, MA, USA). Seedlings grew for 3 weeks before we transplanted them into the pots used in the experiment. In February 2015, we collected soil from the coastal prairie at the University of Houston Coastal Center. To obtain a representative sample of the prairie s soil microbial community, we sampled the top 30 cm of soil from multiple locations within the prairie. The three focal species were present and common at each collection site. However, of the three species, only S. scoparium is dominant in the plant community. Some collection sites had more woody encroachment of Chinese tallow (Triadica sebifera) and wax myrtle (Morella cerifera), but the composition of grasses and forbs at the different sites remained relatively stable. Other species present at the collection sites include: Rubus trivialis (southern dewberry), Tripsacum dactyloides (eastern gamagrass), Berchemia scandens (Alabama supplejack), Helianthus annuus (common sunflower) and Andropogon glomeratus (bushy bluestem). We homogenized the soil samples after passing them through 2-mm mesh sieves to remove rocks and large roots. It is possible that homogenizing the soil artificially reduced variance in soil microbe effects among our replicates (Reinhart and Rinella 2016; Rinella and Reinhart 2018). For instance, if there is a specific class of microbe that occurs only in one part of the prairie, it would be present in all of our pots. However, the soil microbial community present in the homogenized soil allows us to assess how soil microbes in the prairie, in general, influence plant interactions, even if it does not allow us to assess potential variation in these responses. A portion of the soil was separated from the bulk and stored at 3 C to use as live inoculant. To create a sterile background soil, we mixed the remaining soil with sand to a 7:3 ratio and autoclaved the mixture twice for 90 min at 121 C with a 24-h resting period between cycles (Crawford and Knight 2017). We used sand rather than another substrate in order to minimize the addition of mineral nutrients to the prairie soil. We filled sterile D16 Deepots (262 ml capacity, Stuewe & Sons, Inc., Tangent, OR, USA) with sterile background soil (sterile soil treatment) or with sterile background soil plus 20 ml live inoculum (live soil treatment). We added the live inoculum to sterile soil rather than using a whole live soil treatment to minimize differences in soil nutrient availability caused by soil sterilization (Davitt et al. 2011). The inoculum was added around the root zone of the seedlings and capped with sterile soil to prevent splash contamination during watering. To establish the competition treatments, we planted two plants of either the same or different species in each pot. We replicated each treatment combination seven AoB PLANTS The Author(s)

4 times, except for species pairs containing S. scoparium, which were replicated five times due to low germination. We randomized the pots and placed them under growth lights (Virtual Sun T5 HO fluorescent lights; Ontario, CA, USA) in the lab. Lights remained on for 12 h day 1. For the first week after planting, all the plants received 60 ml of water three times throughout the week. Any plants that died during this time were replaced. After 1 week, we watered the plants according to their assigned watering treatments. Prior to the start of this experiment, we grew six individuals of each study species in a greenhouse and subjected them to different watering regimes. We observed that P. lanceolata was the least drought tolerant of all three species; therefore, we calibrated our final watering treatments for this experiment based on P. lanceolata s tolerance. Our medium level of water availability was 60 ml of water applied three times per week. Our high and low water availability treatments increased or decreased the amount of water by 33 %, respectively, and were also watered three times per week (modified from Davitt et al. 2011). After 7 weeks, we harvested and dried above-ground plant biomass. We were unable to collect below-ground plant biomass because the high clay content of the soil caused it to solidify, especially in the low water availability treatment, making it difficult to separate roots from the soil without destroying them. However, we were able to collect a small sample of roots to quantify colonization by AM fungi. We stained the root samples with 0.05 % trypan blue according to the procedure from the International Culture Collection of (Vesicular) Arbuscular Mycorrhizal Fungi ( methods/mycorrhizae/staining-roots). We mounted the stained roots on slides and quantified the frequency of occurrence of AM fungal structures in 60 non-overlapping views observed at 400 magnification (Mack and Rudgers 2008). Statistical analyses To measure the outcome of plant competition in our treatments, we calculated the relative competition intensity, RCI, for each species (Grace 1995). RCI is calculated as follows: RCI = ( Pmono -Pmix)/ P mono, where P mono is the average biomass of the species in monoculture and P mix is the biomass of the species in mixed culture (Mangla et al. 2011). This index controls for differences in intrinsic growth rates among plant species, which can mask the effects of competition when species differ greatly in size (Connolly et al. 2001). A negative RCI indicates that the species experienced stronger intraspecific competition than interspecific competition, while a positive RCI indicates that the species experienced stronger interspecific competition than intraspecific competition. To determine the treatment effects on RCI, we used separate mixed models for each species with the fixed effects of soil treatment (live, sterile), water availability (low, medium, high), competitor identity and all possible interactions (PROC GLM; SAS 9.4). Separate models were analysed for each species to avoid non-independence of species growing in the same pot (i.e. pots did not contribute two values to a single model). To determine if RCI was significantly different from zero, we calculated 95 % confidence limits for the least squares mean for each treatment combination (PROC GLM; SAS 9.4). When confidence limits did not bound zero, we inferred statistically significant intraspecific (negative RCI) or interspecific (positive RCI) competition. To determine treatment effects on above-ground plant biomass, we used separate generalized linear models for each species with fixed effects of soil treatment (live, sterile), water availability (low, medium, high), competitor identity and all possible interactions (PROC GLM; SAS 9.4). Separate models were analysed for each species to avoid non-independence of species growing in the same pot (i.e. pots did not contribute two values to a single model). Furthermore, the two biomass values per pot for the individuals in the conspecific competitor treatment were averaged prior to analysis. Biomass data for each species were log transformed to improve normality prior to analysis. Following analyses, Tukey s HSD was used to determine which treatment means were significantly different from one another (PROC GLM; SAS 9.4). We used AM fungal colonization data from intraspecific competition pots to test the efficacy of the soil sterilization treatment and to test whether plant identity and watering treatment influenced percent AM fungal colonization of roots. We analysed AM fungal colonization data using a general linear model with the fixed effects of soil treatment (live or sterile), plant identity, water availability and all possible interactions (PROC GLM; SAS 9.4). Results Soil microbes and water availability differentially influenced how the three plant species experienced competition. Overall, the presence of soil microbes tended to increase the strength of intraspecific competition relative to interspecific competition, particularly in the dry treatments. Of the three species, P. lanceolata was the best competitor and S. scoparium was the worst competitor. In the sterile soil treatments, neither P. lanceolata nor R. hirta experienced a difference in intra- and interspecific competition (i.e. RCI was not significantly different from zero) (Table 1; Fig. 1). In contrast, both 4 AoB PLANTS The Author(s) 2018

5 experienced increased intraspecific competition in live soil treatments, but this was only statistically significant for P. lanceolata which experienced 100 times greater intraspecific competition in live soil compared to sterile soil (Table 1; Fig. 1). The low water availability treatment increased the strength of intraspecific competition for both P. lanceolata and R. hirta (Fig. 1). In the low water availability treatment, P. lanceolata experienced nine times greater intraspecific competition than in the medium water availability treatment and R. hirta switched from weakly positive RCIs in the medium and high water treatments to a significantly negative RCI. Interestingly, soil microbes mediated the effect of water availability on RCI for P. lanceolata, which experienced three times and two times greater intraspecific competition in the low and high water treatments, respectively, in the live soil treatments (Table 1; Fig. 1). Additionally, competitor identity influenced the RCI of P. lanceolata (Table 1). In live soil, intraspecific competition was generally stronger when P. lanceolata was competing with R. hirta than with S. scoparium (Fig. 1). The RCI of S. scoparium was relatively unaffected by water availability and soil microbes. However, it was affected by competitor identity. Interspecific competition was stronger than intraspecific competition when S. scoparium was competing with P. lanceolata, but not when it was competing with R. hirta (Table 1; Fig. 1). Examination of the biomass data that go into the calculation of RCI provides some insights into the observed responses. Among the three plant species, P. lanceolata produced the most biomass, and its biomass responded positively increased water availability (Table 2; Fig. 2). It produced ~70 % more biomass in the medium and high water availability treatments than the low water availability treatment (P = 0.01 for both comparisons). Competitor identity also influenced P. lanceolata s biomass, but this effect depended on the soil treatment (Table 2). It produced the least biomass when competing against itself, and it produced the most biomass when competing against R. hirta but only in the live soil treatment (Fig. 2). In live soil, P. lanceolata s average biomass was almost 100 % greater when competing with R. hirta than when competing with itself (P = ) (Table 2; Fig. 2). Like P. lanceolata, R. hirta also produced the least biomass in the low water treatment (~40 % less than the high water availability treatment, P = 0.02). It also produced the least biomass when competing with P. lanceolata, but only when water availability was medium or high (Table 2; Fig. 2). Finally, S. scoparium produced the least biomass of the three species. The biomass of S. scoparium was negatively affected by the live soil treatment, where it produced almost 50 % less biomass than in the sterile soil treatment. It also produced the least biomass when in competition with P. lanceolata (Table 2; Fig. 2). It produced almost twice as much biomass when competing with itself than with P. lanceolata (Fig. 2; P < ). While our soil sterilization treatment did not completely remove AM fungi, soil sterilization reduced root colonization by AM fungi by over 30 % (Table 3). Plants in the live soil treatment averaged root colonization of 66 % while plants in the sterile soil treatment averaged 45 %. We also found an effect of plant species identity on root colonization (Table 3). Monocultures of P. lanceolata were colonized the most (84 % ± 0.04), followed by R. hirta (56 % ± 0.06) and S. scoparium (26 % ± 0.04). Discussion We found that water availability influenced how soil microbes affected plant competition. While our three plant species differed in their responses to the Table 1. Results from general linear models testing the effects of water availability, soil treatment (live or sterile), and competitor identity on relative competition intensity (RCI) for Schizachyrium scoparium, Rudbeckia hirta, and Plantago lanceolata. P-values in bold are significant at the P < 0.05 level. S. scoparium R. hirta P. lanceolata d.f. F P d.f. F P d.f. F P Water availability 2, , , Soil treatment 1, , , < Competitor 1, , , Water Soil 2, , , Water Competitor 2, , , Soil Competitor 1, , , Water Soil Competitor 2, , , AoB PLANTS The Author(s)

6 Figure 1. Average relative competition intensity (RCI) ± SE for each species (A, B, and C) in live and sterile soil across the three water availability treatments (white, low water availability; light grey, medium water availability; dark grey, high water availability). Asterisks (*) refer to interactions that are significantly (P < 0.05) different from zero. Negative RCI indicates stronger intraspecific competition than interspecific competition, and positive RCI indicates stronger interspecific competition than intraspecific competition. experimental treatments, soil microbes tended to strengthen intraspecific competition relative to interspecific competition, and this effect was generally stronger in drier conditions. This was especially true for the strongest competitor, P. lanceolata, which was also the most colonized by AM fungi. The weakest competitor, S. scoparium experienced stronger interspecific competition than intraspecific competition, and it was relatively unaffected by either soil microbes or water availability. Because intraspecific competition was generally stronger in the presence of soil microbes, we conclude that soil microbes may play a role in generating coexistence among the species, especially under drier conditions. Several non-exclusive mechanisms may explain why soil microbes interact with water availability to cause a shift in competition. Both plant species that experienced stronger intraspecific competition in the live soil treatment produced the least amount of biomass in the low water availability treatment, and both showed a trend for competition with conspecifics to further lower biomass in those conditions. If increased conspecific density increases the negative effects of species-specific pathogens (Schnitzer et al. 2011) and pathogens decrease plant performance more severely when they are water stressed (Jactel et al. 2012), then pathogens may be driving the observed patterns. Alternatively, if below-ground mutualists strengthen competition for limiting resources (Hartnett and Wilson 1999; Smith et al. 1999) and if conspecifics are more ecologically similar than heterospecifics (Burns and Strauss 2011), then mutualists may be driving the observed patterns. However, another explanation is necessary to explain why the live soil treatment also increased intraspecific competition for P. lanceolata in the high water treatment. Some evidence points to increased soil pathogen proliferation under wetter conditions (Augspurger 1984; Kolb et al. 2016). If this is the case in this system, and the pathogens specific to P. lanceolata increased in the high water treatment, then increased conspecific density could lead to higher pathogen loads and decreased plant performance. Our experimental design does not allow us to test these potential mechanisms, but understanding how water availability shifts interactions between plants and their soil mutualists and pathogens is an area of research that holds much potential for predicting changes in plant community composition in changing climates (van der Putten et al. 2016). Non-native plant species generally respond differently to soil microbes than native plant species. Nonnative species can escape from co-evolved pathogens when colonizing a new range (Reinhart and Callaway 2004, 2006) and there is evidence that they can form novel beneficial associations in invaded ranges (van der Putten et al. 2007). Therefore, it may be expected that non-native species will gain a competitive advantage 6 AoB PLANTS The Author(s) 2018

7 Table 2. Results from general linear models testing the effects of water availability, soil treatment (live or sterile) and competitor identity on above-ground biomass for Schizachyrium scoparium, Rudbeckia hirta and Plantago lanceolata. P-values in bold are significant at the P < 0.05 level. S. scoparium R. hirta P. lanceolata d.f. F P d.f. F P d.f. F P Water availability 2, , , Soil treatment 1, , , Competitor 2, < , , Water Soil 2, , , Water Competitor 4, , , Soil Competitor 2, , , Water Soil Competitor 4, , , Table 3. Results from general linear models testing the effects of soil treatment, plant identity and water availability on root colonization by AM fungi. Only monoculture pots were analysed. P-values in bold are significant at the P < 0.05 level. d.f. F P Soil treatment 1, < Plant identity 2, < Water availability 2, Soil Plant ID 2, Soil Water 2, Plant ID Water 4, Soil Plant ID Water 4, over native species in live soils. While we are not able to generalize our results to all non-native species, we did find intriguing responses for the one non-native species we included in our study. Our non-native species, P. lanceolata, did increase in biomass when grown in the live soil treatment relative to the sterile soil treatment while the two native species did not. Soil microbes also increased the relative strength of intraspecific to interspecific competition for P. lanceolata, suggesting that it experienced weaker competition from native species. However, the effect was not strong enough to increase interspecific competition for the native species in the live soil treatment, suggesting that soil microbes may not drive competitive exclusion by the non-native species. It should also be noted that P. lanceolata is not a problematic invader. It is a weedy species that is originally native to Eurasia and has been introduced around the world. At the site this study took place, the University of Houston Coastal Center, P. lanceolata is common but does not dominate the community. Therefore, it may not have the same interactions with soil microbes as commonly studied, problematic non-native plant species. Relative to some studies, the effects of soil microbes on competition that we documented were weak. One potential reason for this discrepancy is that our sterile soil treatment failed to completely remove microbes from the soil, as evidenced by mycorrhizal colonization of roots. It is unclear whether this is from incomplete sterilization of the soil or contamination during the experiment. In any case, the decrease in soil microbial abundance or shifts in community composition influenced plant performance, but we are unable to determine whether microbial abundance, composition or both influenced our results. Another potential explanation for the relatively weak effect of water availability on microbe-mediated plant interactions is the natural variation in soil moisture found in the Texas coastal prairie ecosystem. Texas coastal prairies experience seasonal flooding and droughts, with heavy rainfall in the spring and periodic droughts during the summer. Therefore, it may be expected that soil microbial communities and plants have adapted to withstand the range of these conditions or maintain diversity associated with varying environmental conditions (Vavrek et al. 1996; Lau and Lennon 2012). If adaptation to variable precipitation does weaken the effects of soil microbes on plant competition, water availability may have a stronger effect on microbe-mediated plant competition in ecosystems that do not experience such a wide range of natural variation in precipitation. From our results, one might conclude that S. scoparium is unable to coexist in prairie ecosystems because it experiences high interspecific competition; however, S. scoparium is abundant in coastal prairies. Our study focused on the effects of water availability and soil microbes for competition at the seedling stage. While AoB PLANTS The Author(s)

8 Figure 2. Average above-ground biomass ± SE for each species (A, B, and C) with each competitor in live and sterile soil across the three water availability treatments (low, medium, high). the seedling stage is an important determinant of colonization in prairies, other life history stages are obviously important for generating patterns of community structure and dynamics in this system. For instance, the high seedling mortality of S. scoparium has been supported by other studies, but S. scoparium s long lifespan may contribute to its dominance in the coastal prairie by masking its high seedling mortality (Lauenroth and Adler 2008). A fruitful, but much more difficult, avenue for future studies would be to track how water availability and soil microbes influence population growth rates of species in natural environments, which would allow better predictions for overall populationlevel performance of species (Maron and Crone 2006; Pearson et al. 2017). Finally, the effects of soil microbes may be stronger in a study that explicitly tests plant soil feedback. Plant soil feedback theory posits that plants alter soils in species-specific ways that differentially feedback to alter plant performance (Bever et al. 1997). Pairwise feedback values can be used to infer whether soil microbes promote or deter coexistence between plant species and can be quantified by growing plants in soils cultured by conspecifics and heterospecifics (Bever et al. 1997). 8 AoB PLANTS The Author(s) 2018

9 While our experiment did test whether water availability and soil microbes interact to influence plant plant interactions, it did not test how plant soil feedback is influenced by water availability. Plant soil feedback experiments are a promising avenue for future studies examining how environmental factors influence plant coexistence through altered plant microbe interactions, but few plant soil feedback experiments have explicitly addressed how abiotic or biotic factors influence feedback (Smith-Ramesh and Reynolds 2017). Conclusions By testing how water availability and soil microbes interact to influence plant competition, we found that soil microbes generally increased the strength of intraspecific competition relative to interspecific competition and water availability mediated this effect, with intraspecific competition generally increasing under drier conditions. Our results suggest that soil microbes may play a role in species coexistence by increasing conspecific negative density dependence, and that the importance of soil microbes for facilitating coexistence may be stronger in drier environments. We also found evidence that the single non-native species in our study benefited more from soil microbes than the two native species, but that this effect was not strong enough to shift the direction (e.g. stronger inter- and intraspecific competition) of competitive interactions for the native species. In sum, our work suggests that a better understanding of how plant microbe interactions are modified by the abiotic environment holds promise for predicting plant community dynamics across environmental gradients and under a changing climate (van der Putten et al. 2016). Sources of Funding This experiment was conducted with funds from the University of Houston. A.P.H. and K.M.C. were supported with funds from the University of Houston. Contributions by the Authors A.P.H. initially posed the central questions and the authors worked together to design the experiment. A.P.H. ran the experiment in the lab and took measurements/collected data. The authors analysed the data together. A.P.H. wrote the original manuscript. K.M.C. edited for content and provided guidance on structure and style. Conflict of Interest None declared. Acknowledgements The authors would like to thank M. Busch, H. Slinn and H. Locke for providing helpful discussion and commentary on the ideas presented in this manuscript. We would also like to thank all the undergraduate students in the Crawford Lab, especially J. Trinh, C. Laurea, S. Berger and C. Perez, for their assistance in the laboratory. We would like to thank the University of Houston Coastal Center and our colleagues Dr S. Pennings, Dr D. Wiernasz and Dr A. Armitage for their advice on this project. 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