TOWARD PREDICTION IN THE RESTORATION OF BIODIVERSITY
Effects of between-site variation in soil microbial
communities and plant
-soil feedbacks on the
productivity and composition of plant communities
Jonathan T. Bauer*
,1, Noah Blumenthal
2, Anna J. Miller
2, Julia K. Ferguson
3and
Heather L. Reynolds
11Department of Biology, Indiana University, 1001 E. 3rd St., Bloomington, IN 47405, USA;2Indiana University
Bloomington, Bloomington, IN, USA; and3Independent Researcher, USA
Summary
1. A critical challenge in the science and practice of restoration ecology is to understand the drivers of variation in restoration outcomes. Soil microbial communities may have a role in explaining this variation due to both site-to-site variation in the composition of soil microbial communities and due to variation that can arise due to plant-soil feedbacks. We tested the relative importance of between-site variation in soil microbial community compo-sition and plant-soil feedbacks in shaping plant community composition and ecosystem function.
2. We used a standard two-phase plant-soil feedback design. Soil inoculum was collected from four tallgrass prairie sites. Then, soils were conditioned separately with nine plant spe-cies, and conditioned soils were used to inoculate prairie community mesocosms. In a sepa-rate experiment using soil from an additional site we tested conditioned soil samples for the abundance of arbuscular mycorrhizal fungi (AMF) and rhizobia.
3. Site of soil origin and plant-soil feedbacks both had effects on the composition and pro-ductivity of our plant communities, and the magnitudes of these effects were similar. We also found changes in the abundance of AMF and rhizobia due to plant-soil feedbacks and that AMF abundance were associated with differences in plant community composition.
4. These results indicate that the composition of soil communities due to site-to-site variation and plant-soil feedbacks are both important determinants of plant community composition and productivity. Our results also suggest that AMF and rhizobia are key microbial func-tional groups underlying plant-soil feedback effects.
5. Synthesis and applications.Site-to-site variation in soil communities can explain some vari-ation in restorvari-ation of plant communities. Since plant-soil feedback effects of restored plant species do not overcome this variation, knowledge of soil microbial communities present at a site prior to initiation of restoration efforts may improve predictability of restoration out-comes, and reintroduction of some components of the soil community may be necessary to achieve restoration goals. Additionally, by understanding variation due to plant-soil feed-backs, restoration practitioners can choose plant species for reintroduction that will create favourable soil conditions, including promoting microbial mutualists. Plant-soil feedbacks should also make it possible to increase heterogeneity in soil microbial communities, leading to increases in beta diversity in plant communities.
Key-words: ecosystem services, legacy effects, mycorrhizae, plant-soil feedback, priority effects, restoration, rhizobia, soil microbial communities, tallgrass prairie
Introduction
Soil microbial communities are known to be important drivers of the diversity and function of plant communities (Reynolds et al. 2003). Soil microbes can vary between sites, and these differences in soil microbial communities can lead to dramatic differences in restoration outcomes (Wubs et al. 2016). Additionally, different plant species can change the composition of soil microbial communities
through plant-soil feedbacks, and these feedbacks can
shape plant community composition and ecosystem func-tion (Mangan et al. 2010; Maron et al. 2011). Thus, dif-ferences between sites and plant-soil feedbacks both have the potential to cause variation in restoration outcomes. However, little is known about the relative importance of
between-site variation vs. plant-soil feedbacks, or their
potential interactions, for explaining variation in plant community composition and function.
Variation in soil microbial composition between sites can have important effects on plant communities. Evi-dence for this comes from studies of agricultural land-use legacies and the natural recovery or restoration of these areas. For example, intensive agriculture can alter the composition of soil communities, including bacteria, fungi, and nematodes (Kardol et al. 2005; Fitzsimons, Miller & Jastrow 2008; Fierer et al. 2013), and, as a result, inoculation with arbuscular mycorrhizal fungi (AMF) (Maltz & Treseder 2015; Middleton et al. 2015), rhizobia (Tlusty, Grossman & Graham 2004; Thrall et al. 2005), or whole soil communities from undisturbed sites (Carbajo et al. 2011; Middleton & Bever 2012) can all influence the outcomes of ecological restoration of aban-doned agricultural fields. Sites at different stages of recov-ery since being farmed show increasing abundance of AMF, improving soil structure and greater carbon seques-tration (Duchicela et al. 2013), and sites of different suc-cessional ages support different soil communities which can reinforce the dominance of late-successional plant species (Van Der Putten, Vandijk & Peters 1993; De Deyn
et al. 2003; Van de Voorde, van der Putten & Bezemer
2012).
Within a site, soil communities change in response to different plant species, resulting in plant-soil feedbacks. The importance of feedback to community structure is
supported by studies finding a positive association
between the strength of plant-soil feedback experienced
by a species and that species’ relative abundance
(Kliro-nomos 2002; Mangan et al. 2010). Plant-associated
changes in soil communities are also likely to have con-sequences for co-occurring species (Bever 1994), and this variation in a species’ response to other species’ soils may create heterogeneity in plant communities (Fukami
& Nakajima 2013). Additionally, plant-soil feedbacks
among early colonizers of a restoration site may create priority effects with long-lasting impacts on plant com-munity composition (Kardol et al. 2007; Grman & Sud-ing 2010).
The changes in soil community composition that cause differences in plant community composition remain poorly understood. Microbial mutualists such as mycorrhizal fungi and rhizobia are likely to have central roles in plant-soil feedbacks due to their considerable effects on plant com-munity structure and function (Bauer et al. 2012; Van der Heijden et al. 2016). Plants vary substantially in their response to these symbioses (Bauer et al. 2012), and differ-ent plant species affect the relative abundance of these microbial functional groups (Eom, Hartnett & Wilson 2000). As a result, feedbacks involving plants and microbial mutualists can alter plant community composition (Stinson et al.2006; Vogelsang & Bever 2009; Fitzsimons & Miller 2010; Bever, Broadhurst & Thrall 2013).
Past work has strongly supported the hypotheses that site-to-site variation in the soil community and plant-soil feedback effects have important effects on plant community composition and ecosystem function. However, the relative importance of site-to-site variation versus plant-soil feed-back effects and their potential interactions are not well understood. Understanding the relative importance of these factors will help explain variation in restoration outcomes and contribute to more predictive restoration practice (Brudvig et al. 2017). If variation in the composition of soil communities between restoration sites has strong, persistent effects on plant community composition and productivity, management of soil microbial communities may be neces-sary to reach restoration goals at some sites. On the other hand, if plant-soil feedbacks overwhelm these initial effects, understanding plant-soil feedbacks may be more important for designing seed mixes for restoration and understanding successional trajectories of restored communities. We designed experiments to test the relative importance of between-site variation in soil microbial community compo-sition vs. variation due to plant-soil feedback effects within a site for the productivity and composition of tallgrass prairie. We also test the relative contributions of changes in
the density of AMF and rhizobia as drivers of plant-soil
feedback in this context.
Materials and methods
We conducted two parallel experiments. In the main experiment, we tested the relative importance of site-to-site variation in the composition of soil microbial communities as compared to changes in soil communities due to plant-soil feedbacks. To gain insight on the role of key microbial functional groups in within-site feedback effects, we carried out an additional experiment to examine plant-soil feedbacks at a single site and measured the abundance of mycorrhizal fungi (AMF) and rhizobia in soil asso-ciated with different plant species.
SI TE VS. PLAN T- S O IL F E E D B A C K E X P E R I M E N T
and both have been protected for at least 30 years. Cressmoor is owned and managed by the Shirley Heinze Land Trust, and Bei-secker Nature Preserve is owned and managed by the Indiana Department of Natural Resources. The two restored sites, Grei-ner Nature Preserve and Conrad Nature Preserve, were restored by the Shirley Heinze Land Trust and The Nature Conservancy 7 years before the study. These were both established on former agricultural fields, where agricultural legacy effects are expected to alter both the composition and function of the soil community (Fitzsimons, Miller & Jastrow 2008; Fierer et al. 2013). It is also possible that between-site differences in plant communities could cause differences in soil communities.
S OIL C ONDI TI ONI NG P H A SE
Soils from the four sites were used to inoculate seedlings of nine tallgrass prairie plant species. Plants were then allowed to ‘con-dition’ the soil for one full growing season (8 months) in Indi-ana University’s greenhouse facilities under ambient light conditions and with daily watering. From each site, live soil was collected from 25 locations regularly spaced within a 25 m9 25 m area, then sieved through a 4-mm mesh to remove litter and homogenize the inoculum within site. Double-Deepots (1 L total volume, Stuewe & Sons, Inc. Tangent, OR) were lay-ered with 800 L of sterile background soil, 100 mL of soil inoculum from one of the sites and a 100-L cap of sterile back-ground soil. The sterile backback-ground soil consisted of Crider ser-ies silt-loam top-soil mixed 50 : 50 with sand to facilitate drainage. To remove existing biota from the sterile background soil, this soil mixture was heated to approximately 90 C for 2 h using an electric soil sterilizer (Model SS-60; Pro-Grow Supply, Brookfield, WI, USA), allowed to cool for 24 h, then heated to 90°C again for 2 h. Each pot was planted with a single seedling of one of nine tallgrass prairie species that had been germinated in sterilized potting soil (Metro Mix 360; Sun Gro Horticulture, Vancouver, Canada). With 4 sites, 9 plant species and 10 repli-cates, this resulted in 360 experimental units in the soil condi-tioning phase of this experiment. Plant species included: Elymus canadensis, Panicum virgatum, Schizachyrium scoparium, Sporobolus heterolepis(Poaceae), Rudbeckia hirta, Coreopsis trip-teris, Parthenium integrifolium (Asteraceae), Monarda fistulosa (Lamiaceae) and Tradescantia ohiensis (Commelinaceae). These species were chosen because they are all relatively common spe-cies in our study system and they represent a diversity of life-histories and variation in mycorrhizal responsiveness (Koziol & Bever 2015). Plants were overwintered in cold frames. After overwintering, soils and roots were harvested for use as inocu-lum in the second phase of the experiment.
T ES T P H A S E
In the second phase of the experiment, the response of our study plant species to the conditioned soils from each site was tested in community mesocosms. An initial layer of 9 L of sterile back-ground soil was added to 15-L treepots (Stuewe & Sons, Inc.). A second layer of 1 L of conditioned inoculum mixed with 3 L of sterile background soil was added, and mesocosms were then capped with 1 L of sterile background soil. Soil used for inocu-lum originated from a single pot from the conditioning phase of the experiment for each planted mesocosm so that conditioned soils remained independent replicates. An additional 10 control
mesocosms were planted entirely in sterile background soil. Char-acteristics of the sterile background soil were as described above for the conditioning phase. Seedlings of each of our plant species were added to the pots in an evenly spaced 39 3 grid. Each soil treatment was replicated 10 times and the arrangement of each plant species within this grid was randomized for each block (10 blocks total containing one replicate of each treatment). Meso-cosms were kept under greenhouse conditions as above. The test phase of the experiment consisted of a total of 363 pots [4 sites9 9 conditioning treatments 9 10 replicates (some treat-ments were limited to nine replicates due to mortality during the conditioning phase)] and included a total of 3267 plants. Plants grew for 4 months, then above-ground biomass was harvested, dried and weighed.
AM F A N D R H IZ O B IA E X P E R I M E N T
This experiment used soil from a single additional remnant prairie site (Iroquois State Game Area (ISGA), IL). Soils were conditioned following the methods in experiment 1, including eight of the plant species from experiment 1 (excluding Sporobo-lus) and including two legume species, Baptisia alba and Melilotus officionalis. Both legume species commonly occur in tallgrass prairie where Baptisia is a native species and Melilotus is a com-mon introduced species. In this experiment, we tested conditioned soil from these 10 plant species for the AMF and rhizobia. To quantify abundance of these microbes, 100 mL of conditioned soil from each replicate were mixed evenly with 300 mL of our sterile background soil (described above), and used to fill three ‘stubby cone-tainers’ (Stuewe & Sons, Inc.). These were planted either with sorghum (Sorghum bicolor), an introduced grass com-monly used as an AMF host species, or Baptisia or Melilotus seedlings. As above, each replicate of conditioned soil was tested separately to maintain independence of replicates within the soil conditioning treatments. After 4 weeks, sorghum seedlings were harvested and the roots were analysed for total colonization by mycorrhizal fungi and the abundance of vesicles and arbuscules (following Mcgonigle et al. 1990). Baptisia and Melilotus seed-lings were also harvested and the total number of nodules on the roots was counted. We conducted a test phase of experiment 2 as described above for experiment 1, where community mesocosms composed of the 10 conditioning species used in experiment 2 were grown in inocula conditioned by each of those 10 species. This experiment was grown for 6 months before above-ground biomass was harvested.
ST ATIST ICA L A N ALYSIS
calculate pairwise plant-soil feedback interaction coefficients for each possible pair of species included in our experiment (Is,
fol-lowing Bever, Westover & Antonovics 1997). We calculated interaction coefficients for species pairs, but it should be noted that our species pairs were grown in a plant community context. For the AMF and rhizobia experiment, we tested the response of rhizobia and AMF to soil conditioning treatments using separate ANOVAs with soil conditioning treatments and replicate as fixed effects and the percentage of roots that con-tained AMF hyphae, vesicles or arbuscules and total numbers of nodules formed on Melilotus or Baptisia as response vari-ables. Plant-soil feedback effects on total above-ground produc-tivity of the mesocosms were tested using ANOVA with soil conditioning treatments and replicate as fixed effects. AMF abundance, as measured by colonization of sorghum seedlings and rhizobia abundance, as measured by nodule formation on Baptisia and Melilotus, were included as covariates. All of the above analyses were conducted in SAS 9.4 using proc glm. In
all cases, sterilized controls were excluded from the analysis so that the effect size of natural variation in soil community com-position could be assessed more accurately, but sterile controls are retained in the figures for comparison. We analysed changes in community composition in the AMF and rhizobia experiment using NMDS ordination with the vegan package in
R. The relative contribution of AMF and rhizobia was tested
using the envfit function, which determines the correlation of
predictor variables with the location of our mesocosms within the ordination space.
Results
O V ER A L L
Our main experiment revealed that both site and plant
-soil feedback treatments had significant main effects on productivity and species composition in our experimental mesocosms. There were no interactive effects of site and
soil conditioning on either productivity (F24, 351= 071,
P= 085) or species composition (F216,2763= 090,
P= 084). Most species experienced significant pairwise
plant-soil feedback interactions, and the strength and
direction of plant-soil feedbacks experienced by a species
depended on the other species it was being compared to. The strength of plant-soil feedbacks experienced by pairs of plant species was consistent across study sites. The additional experiment demonstrated that both AMF and rhizobia responded to soil conditioning by different plant species.
SI TE EF FEC TS
The site where soil was originally collected had a signifi-cant effect on productivity (Table 1), and the soils from the two remnant prairies supported a more productive plant community than soils from the two restored prairies (Fig. 1a). Site also had a significant effect on the composi-tion of plant communities (Table 2). Six of our nine plant species showed a significant response to the site soils were collected from (Table 3, Fig. 2). Of these, three plant spe-cies, Elymus, Coreopsis and Parthenium, were more pro-ductive in soils from remnant prairies than soils from
Table 1. ANOVA results for the response of total productivity to site and soil conditioning treatments
d.f. F P g2 Model 44 1772 <00001 072 Site 3 203 00001 0056 Soil conditioning 8 367 00004 0027 Site9 soil conditioning 24 071 085 0016 Block 9 7473 <00001 062 Error 351
(a) (b)
restorations, but the three other species that responded significantly to site, Rudbeckia, Tradescantia and Sporobo-lus, did not show a consistent difference between remnant and restored prairies.
P L A N T -S O I L F E E D B A C K EF F EC T S
Soil conditioning treatments also had a significant effect on the productivity of our mesocosms (Table 1). Rudbeck-ia-conditioned soils supported the most productive plant community, which produced 35% more biomass than the least productive soils, those conditioned by Tradescantia (Fig. 1b). The difference between the most productive and least productive soil conditioning treatments was similar to the difference between the most and least productive site effects (Table 1, Fig. 1).
Soil conditioning treatments had significant effects on plant community composition within our mesocosms (Table 3). Tradescantia had the strongest effects overall. Seven of our nine study species were the least productive in Tradescantia-conditioned soils. This explains the rela-tively low productivity of plant communities in
Trades-cantia-conditioned soils, although Elymus and
Tradescantia were both most productive in
Tradescantia-conditioned soils. All of our study species showed signifi-cant responses to soil conditioning treatments (Fig. 3), and for all species except Elymus, soil conditioning treat-ments were a more important predictor of their biomass than site effects (Table 3).
IN TE R A CT ION C OE FF IC IE NT S
For all species except Sporobolus, feedback interactions (Is)
were significant with one or more other plant species. Within a species, the direction of plant-soil feedbacks var-ied between species pairs for five species (Fig. 4). For exam-ple, Rudbeckia experienced negative feedbacks with Elymus and Monarda, but positive feedbacks with Tradescantia.
A M F A N D R H I Z O B I A FE E D B AC K S
In our second experiment, we found that AMF and rhizo-bia had significant responses to soil conditioning treat-ments (Figs 5 and 6). AMF responses were evident as
differences in root colonization by hyphae (F10,61= 385,
P= 00004) and the abundance of vesicles (F10,61= 231,
P= 0022) in our sorghum test plants. The abundance of
Table 2. MANOVA results for species responses to site and soil conditioning treatments, including numerator (dfn) and denomi-nator (dfd) degrees of freedom
d.f. (dfn, dfd) F P Site 27, 903 312 <00001 Soil conditioning 72, 2448 325 <00001 Site9 soil conditioning 216, 2763 09 084 Block 81, 2763 723 <00001
Table 3. Follow-up ANOVAs from MANOVA analysis (Table 2) of species responses to site and soil conditioning treatments
d.f. F P g2 Coreopsis tripteris
arbuscules followed similar patterns, but differences were marginally significant (F10,61= 186, P = 0069). Rhizobia
also responded to soil conditioning treatments, as indi-cated by significant variation in the formation of nodules
on Melilotus test seedlings (F10,72= 257, P = 0010).
However, Baptisia seedlings did not show significant dif-ferences in the total formation of nodules based on soil conditioning treatments (F10,72= 130, P = 025).
As in our first experiment, we observed significant effects of soil conditioning treatments on the productivity and composition of plant communities. Productivity effects were associated with changes in the abundance of AMF and rhizobia (Table 4). As stated earlier, we found that plant species tended to show strong responses to soils conditioned by Tradescantia as compared to soils condi-tioned by other species. This was apparent in our NMDS ordination of plant community composition, where
meso-cosms inoculated with Tradescantia-conditioned soils
formed a relatively distinct group (Fig. 7), but there was nearly complete overlap within the NMDS between meso-cosms inoculated with soils conditioned by each of the other species. We also found that AMF and rhizobia abundance were correlated with variation in plant com-munity composition. AMF abundance showed the
stron-ger correlation (r2= 026, Fig. 7). Nodule formation on
Baptisia test plants was not significantly correlated with
variation in plant community composition, but nodule formation on Melilotus was significantly correlated with
community composition (r2= 14%). Even though AMF
and rhizobia abundance were correlated with NMDS scores, our MANOVA found that only soil conditioning treatments were statistically significant (Table 5).
Discussion
We found that site-to-site variation in the composition of soil microbial communities affected the productivity and
composition of plant communities. Within sites, plant-soil
feedback treatments also affected plant community pro-ductivity and composition. The strength of the effects of
site and plant-soil feedback treatments on plant
commu-nity productivity was similar, but feedback treatments had stronger effects on the productivity of individual plant species, and thus on plant community composition. For
the one site examined, plant-soil feedback effects were
associated with large changes in the AMF and rhizobia. Together, these results indicate that variation in the com-position of soil communities between sites can contribute to variation in species composition and changes in pro-ductivity of plant communities, and these effects can
persist despite strong effects of plant-soil feedbacks. Within sites, plant-soil feedbacks result in plant species-specific changes in the composition of the soil community that can further influence plant community composition and function and contribute to heterogeneity within com-munities. Our results also suggest that shifts in AMF and
rhizobia due to plant-soil feedback effects can contribute
to plant community properties such as composition and productivity.
Both site and plant-soil feedback treatments had effects on the productivity of the plant communities in our meso-cosms, and these effects were almost identical in their magnitude. For example, soil inocula from Cressmoor prairie (remnant prairie) supported the most productive plant community, which was 35% more productive than plant communities grown in soil inocula from Greiner Nature Preserve (restored prairie). Similarly, plant-soil feedback effects resulted in 35% greater productivity in the most productive soil conditioning treatment as com-pared to the least productive. We note that after
collect-ing soil in the field we homogenized soil before
inoculating plants in the first phase of our experiment. We did this so that each experimental replicate would interact with a representative sample of the soil microbial communities that occur at a site. However, it is also likely that this reduced the variance associated with site effects (Reinhart & Rinella 2016). So, in the context of ecological
restoration, it is possible that within-site variation will lead to greater heterogeneity in plant community compo-sition and productivity than our results suggest. On the other hand, our results strongly suggest that the variation in plant communities resulting from any between-site vari-ation in soil microbial communities will persist despite the changes that the restored plant community may have on
microbial communities through plant-soil feedbacks.
The signature of site effects on most individual plant species was not as strong as on measures of total plant community productivity. Nevertheless, these results may have important consequences for ecological restoration. The outcomes and trajectories of restored communities are notoriously unpredictable (Matthews & Spyreas 2010; Brudvig 2011). Our results indicate that differences in soil communities between sites can contribute to this site-to-site variation in the composition of plant commu-nities. These differences in soil microbial communities may arise due to differences in land-use history, soil properties or other aspects of site history, including between-site differences in plant community composition that may cause differences in soil microbial communities.
Plant-soil feedback effects of newly restored species may
further modify these soil communities, and plant-soil
feedbacks could have a role in shaping the outcomes and trajectories of restored plant communities (see also Bauer, Mack & Bever 2015). However, in our study,
0 0·5 1 1·5 0 0·5 1 0 0·05 0·1 0·15 0 2 4 6 8 10 0 0·5 1 1·5 0 0·5 1 0 1 2 3 4 0 1 2 3 4 0 0·5 1 1·5 2 2·5
plant-soil feedback effects did not overwhelm or interact with the effects from between-site differences in soil microbial communities.
The strongest soil conditioning effects in both of our experiments were observed in Tradescantia-conditioned soils, which generated the lowest plant community produc-tivity (main experiment) and overall low AMF abundance and a distinct plant community composition (experiment 2). Yet although it was the strongest driver, Tradescantia was not the only driver of plant and soil community prop-erties. The grasses E. canadensis, S. scoparium and S.
het-erolepis produced soil inocula that generated low plant
community productivity, and the reverse was true of the forbs P. integrifolium, Coreopis tripteris, M. fistulosa and R. hirta. Abundance of AMF and rhizobia, as gauged by hyphal root colonization, vesicles and nodule formation, also varied due to plant species other than Tradescantia, with M. fistulosa, P. virgatum and S. heterolepis tending to reduce AMF and Rhizobia abundance. Overall, these
results support the potential for plant-soil feedbacks to
result in heterogeneity in the composition of soil and plant communities, and this heterogeneity could increase within-or between-site beta diversity of plant communities. Fur-ther support for plant-soil feedbacks as a source of
vegeta-tion heterogeneity comes from our calculations of
interaction coefficients. All species, except Sporobolus,
experienced significant plant-soil feedbacks. However, the
strength and direction of the feedbacks experienced by a species was dependent on the species to which it was being compared. Both positive and negative feedbacks can have important effects on plant diversity, but they operate on different scales. Positive feedbacks lead to the exclusion of interacting pairs of plant species and reduce alpha diversity, but it is possible for positive plant-soil feedbacks to increase heterogeneity within a site by increasing the patchiness of a plant community and increasing beta diversity (Bever, Westover & Antonovics 1997; Molofsky et al. 2002). Nega-tive feedbacks prevent competiNega-tive exclusion of competing plant species and can increase alpha diversity by promoting species coexistence (Bever, Westover & Antonovics 1997). Thus, the variability we observed in the strength and direc-tion of plant-soil feedback experienced between pairs of species indicates that both positive and negative plant-soil feedbacks may be operating simultaneously and contribut-ing to both species coexistence and community heterogene-ity.
Despite significant effects overall, we expected stronger and more consistently negative plant-soil feedback effects.
Meta-analysis of plant-soil feedback studies has indicated
that negative plant-soil feedbacks are expected to
predom-inate in native plant communities (Kulmatiski et al. 2008). However, many of the pairwise feedback interac-tions that we observed were positive or non-significant. This is especially surprising, given that negative plant-soil
–2·5 –1·5 –0·5 0·5 1·5 2·5 –2·5 –1·5 –0·5 0·5 1·5 2·5 –2·5 –1·5 –0·5 0·5 1·5 2·5 –2·5 –1·5 –0·5 0·5 1·5 2·5 –2·5 –1·5 –0·5 0·5 1·5 2·5 –2·5 –1·5 –0·5 0·5 1·5 2·5 –2·5 –1·5 –0·5 0·5 1·5 2·5 –2·5 –1·5 –0·5 0·5 1·5 2·5 –2·5 –1·5 –0·5 0·5 1·5 2·5
Fig. 4. Strength of plant-soil feedback (S.E.) experienced by pairs of species (Is, following Bever, Westover & Antonovics 1997).
feedback did predominate in a previous study in this sys-tem testing feedback effects on individual seedlings of similar species (Bauer, Mack & Bever 2015). The majority of microbial feedback studies test feedbacks on a single seedling, whereas our measures of feedback were made in a plant community context. In communities, it is possible that other interactions, such as resource competition or differences in relative growth rate, moderate the strength
of microbially mediated plant-soil feedbacks (Kulmatiski
et al.2016). It is also possible that in high diversity
com-munities strong plant-soil feedback effects are diluted by
heterospecific neighbours (Maron et al. 2011; Schnitzer, Klironomos & HilleRisLambers 2011). Future work which compares the importance of microbially mediated
plant-soil feedbacks to resource competition and other
mechanisms of abiotic feedback will yield insights into the
relative importance of plant-soil feedbacks as drivers of
plant community structure and function.
The effects of soil conditioning we observed in experi-ment 2 were associated with changes in the density of microbial mutualists. Our results are consistent with the well-established importance of AMF as drivers of plant-soil feedbacks (Bever 2002; Fitzsimons & Miller 2010), with AMF density being correlated with changes in plant com-munity composition (Fig. 7). Species-specific responses are also consistent with an important role for AMF. Only two of our study species are generally unresponsive to AMF (Elymus and Tradescantia; Bauer et al. 2012; Koziol & Bever 2015), and these species were the only species posi-tively affected by Tradescantia-conditioned soil, which
(a)
(b)
(c)
contained very low AMF abundance. Rhizobia density is also correlated with differences in species abundance (Fig. 7), although in our statistical analyses these effects appear to be explained by main effects of soil conditioning on plant community composition (Table 4). Presumably, shifts in the abundance of AMF and rhizobia also occurred along with changes in other components of the soil commu-nity. So, it is likely that other components of the soil
com-munity are important in explaining plant-soil feedback
effects, and understanding the biotic and abiotic
mecha-nisms underlying plant-soil feedbacks remains an important
research priority (Van der Putten et al. 2016).
Our tests of the abundance of AMF and rhizobia were limited to one remnant prairie site. However, it is possible that changes in the density and community composition of AMF and rhizobia also have a role in explaining differ-ences in plant communities between sites. Agricultural
(a)
(b)
Fig. 6. Mean response of Rhizobia (S.E.) to soil conditioning treatments, as mea-sured by (a) total number of nodules formed on Baptisia test plants and (b) total number of nodules formed on Melilo-tus test plants. Ba= Baptisia alba, Ct= Coreoposis tripteris, Ec= Elymus canadensis, Mf= Monarda fistulosa, Mo= Melilotus officionalis, Pi = Parthe-nium integrifolium,Pv= Panicum virgatum, Rh= Rudbeckia hirta, Ss = Schizachyrium scoparium, To= Tradescantia ohiensis, ‘SC’= sterile control.
Table 4. ANOVA results for AMF & rhizobia experiment testing the effects of AMF abundance, rhizobia abundance and soil conditioning treatments on total productivity
d.f. F P g2 Model 22 223 001 05 AMF 1 255 01166 0026 Baptisianodules 1 781 00074 00796 Melilotusnodules 1 715 00102 00729 Soil conditioning 10 206 00466 02101 Block 9 122 03065 01117 Error 307 −0·2 0·0 0·2 0·4 0·6 −0·4 −0·3 −0·2 −0·1 0·0 0 ·1 0·2 0 ·3 NMDS1 NMDS2 Ec Pv Ss Mo Ba Rh To Mf Ct Pi AMF nodulesBA nodulesMO
fertilization favours less mutualistic microbial communities (Johnson 1993), and persistent soil disturbance negatively impacts communities of mycorrhizal fungi (Helgason et al. 1998). Our two most productive sites were unploughed remnant prairies, but our two restored prairies were in row-crop agriculture as recently as 9 years before our study. Some components of the soil community appear to be able to recolonize following restoration of former agri-cultural fields (Kardol et al. 2005; Bach et al. 2010; Card & Quideau 2010), but other are likely to be dispersal lim-ited (Jangid et al. 2010; Middleton & Bever 2012). In our case, it appears that components of the soil community that are likely to support primary productivity are missing from these restored communities. It is likely that several underlying causes contribute to between-site differences in soil microbial communities. Prior to the initiation of restoration efforts, differences in land-use legacies, abiotic soil properties and previous plant community composition may all influence soil microbial communities that then shape the initial outcomes of ecological restoration. Plant-soil feedbacks that occur early in restoration may further shape these between-site differences in soil communities. Further research that identifies the underlying causes of between-site variation in soil microbial communities and the components of the soil community that are absent from restored sites is needed so that active reintroductions of soil biota can be used to improve the restoration of these communities.
If restoration is to achieve its full potential as a means to reverse biodiversity loss and mitigate declines in ecosystem function, it is critical that we improve our understanding of the variability in restoration outcomes (Brudvig et al. 2017). Our results show that variation between sites in the composition of soil microbial communities can cause varia-tion in plant community composivaria-tion and productivity of re-established plant communities. Since these effects per-sist following soil conditioning treatments, understanding existing variation in soil microbial communities could allow better predictability of restoration outcomes. If important components of the soil community are absent, then active reintroduction of soil microbes may be necessary to achieve restoration goals (e.g. Wubs et al. 2016). However, strong
effects of plant-soil feedbacks suggest that plant species
may be chosen for restoration based on their effects on soil communities. For example, in our experiment, Tradescantia
and Rudbeckia are early successional species that are often abundant early in a restoration, and these species differ strongly in their effects on AMF density. These differences
in plant-soil feedback effects could be used to increase
heterogeneity in soil communities and increase within-site beta diversity by favouring non-mycorrhizal and AMF-dependent plant species in different patches. Furthermore, most late-successional plant species in our study system are highly responsive to AMF (Koziol & Bever 2015), and these fungi have a key role in maintaining productivity and other ecosystem functions (Bauer et al. 2012; Koziol & Bever 2015). Consequently, when designing seed mixes for restoration, including species like Rudbeckia that establish quickly and support high densities of AMF may also pro-mote the development of high-functioning late successional communities.
Overall, in this experiment, we demonstrate strong effects of soil communities on plant community productivity and composition. Differences in soil communities between sites contribute to differences in plant community composition and function between sites. Within sites, plant species can alter the abundance of AMF and rhizobia. The resulting feedbacks have important consequences for plant commu-nity composition and productivity. These feedbacks have the potential to contribute to site diversity both by increas-ing heterogeneity in plant and soil communities and by pro-moting the coexistence of some species on smaller scales.
Authors’ contributions
J.T.B. and H.L.R. developed the ideas and designed the experiment. J.T.B., N.B., A.J.M. and J.K.F. established the greenhouse experiments and collected data. J.T.B. analysed the data. J.T.B. led the writing of the manuscript, H.L.R. contributed substantially to revisions, and all authors contributed to editing and gave final approval for publication.
Acknowledgements
Funding for this experiment was provided to J.T.B. by the Indiana Acad-emy of Science and the Agriculture and Food Research Initiative Competi-tive grant no. 2012-67011-19765 from the USDA National Institute of Food and Agriculture. We appreciate the support of the Indiana chapter of The Nature Conservancy, the Indiana Department of Natural Resources, and the Shirley Heinze Land Trust for their assistance with finding suitable study sites, granting us permission to collect soils from their preserves and for protecting these sites.
Data accessibility
Data available from the Dryad Digital Repository https://doi.org/10.5061/ dryad.r43d0 (Bauer et al. 2017).
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