DOI: 10.5061/dryad.gtht76hq9
title: Sympatric soil biota mitigate a warmer-drier climate for Bouteloua gracilis

description: Climate change is altering temperature and precipitation, resulting in
 widespread plant mortality and shifts in plant distributions. Plants
 growing in soil types with low water holding capacity may experience
 intensified effects of reduced water availability as a result of climate
 change. Furthermore, complex biotic interactions between plants and soil
 organisms may mitigate or exacerbate the effects of climate change. This
 three-year field experiment observed the performance of Bouteloua gracilis
 ecotypes that were transplanted across an environmental gradient with
 either sympatric soil from the seed source location or allopatric soil
 from the location that plants were transplanted into. We also inoculated
 plants with either sympatric or allopatric soil biotic communities to
 test: 1) how changes in climate alone influence plant growth, 2) how soil
 types interact with climate to influence plant growth, and 3) the role of
 soil biota in mitigating plant migration to novel environments. As
 expected, plants moved to cooler-wetter sites exhibited enhanced growth;
 however, plants moved to warmer-drier sites responded variably depending
 on the provenance of their soil and inoculum. Soil and inoculum provenance
 had little influence on the performance of plants moved to cooler-wetter
 sites, but at warmer-drier sites they were important predictors of plant
 biomass, seed set and specific leaf area. Specifically, transplants
 inoculated with their sympatric soil biota and grown in their sympatric
 soil were as large or larger than reference plants grown at the seed
 source locations, however, individuals inoculated with allopatric soil
 biota were smaller than reference site individuals at warmer, drier sites.
 These findings demonstrate complicated plant responses to various aspects
 of environmental novelty where communities of soil organisms may help
 ameliorate stress. The belowground microbiome of plants should be
 considered to more accurately predict the responses of vegetation to
 climate change.
description: Elevation gradient and collection of materials
 This study was conducted at six different sites within the
 Southwest Experimental Garden Array (SEGA) (https://sega.nau.edu/home) in Northern Arizona which is a collection of experimental sites situated across a climate gradient spanning 6oC. Detailed information about each site is listed in Table 1. Two sites in the middle of the gradient with abundant B. gracilis, Blue Chute (BC) and White Pockets Canyon (WPC), were selected as the source populations for seeds, thus allowing us to manipulate climate in both a warming and cooling direction. The BC and WPC seed sources represent two replicate populations of our study species.  The Seeds of Success protocol (https://www.blm.gov/sites/blm.gov/files/program_nativeplants_collection_quick%20links_technical%20protocol.pdf) was used to collect seeds of B. gracilis. Soil was collected from the same two source sites to be used for the sympatric soil treatment, and from the four transplant sites to be used for the allopatric soil treatment (Figure 1, Table S1). Live rhizosphere soil inoculum was collected from the rooting zone of B. gracilis along three 100 m transects established from a random origin (azimuths of 0°, 90° and 270°). At two transplant sites where B. gracilis was absent, live soil was collected from the rhizosphere of the dominant plant community of the site instead. Soil subsamples within each site were pooled together and mixed. We justify homogenizing inoculum from each site because we were interested in B. gracilis responses to the average soil biotic communities across sites, rather than within a single site or extrapolating to a broader geography than our sampling sites (a ‘type C’ design; Gundale et al., 2017, 2019). Inoculum soil was refrigerated 2 weeks until its use in the experiment. At each sampling location, background soil was collected along the same three transects by carefully digging into bare soil away from plants at depths between 0-60 cm. Background soil within each site was homogenized and steam sterilized at 125°C twice for 24 hours.   Design and Preparation of Experimental Units We used Steuwe & Sons 7.8L tree pots (model TP812) as experimental units. This pot size was selected to accommodate multiple years of growth of B. gracilis without getting pot-bound. Experimental units were planted into the two seed source sites to create sympatric reference plants or into the four possible transplant sites with warmer or cooler climates. Four combinations of soil and inoculum relative to each plant population and transplant site were generated (Figure 1). This design created three types of novel edaphic environments to compare to the fully sympatric reference plants and uncouple the abiotic and biotic components of the edaphic environment: sympatric inoculum and allopatric soil (SA), allopatric inoculum and sympatric soil (AS), and allopatric inoculum and allopatric soil (AA; Figure 1). Each treatment combination was replicated 10 times at each transplant site for each of the two plant populations, and 10 sympatric reference units were planted at the two seed source sites. This resulted in 170 experimental units for each of the two B. gracilis populations and a total of 340 experimental units (Figure 1; Table S1).    Each experimental unit, was filled with 7.5L of sterilized background soil and then covered with a 2 cm thick band (0.45L) of living inoculum soil. Bouteloua gracilis seeds were sprinkled onto inoculum soil at a density of 20 seeds per pot and covered with 1cm of sterilized background soil. Later, seedlings were thinned to one plant per mesocosm. Seedlings were grown in the greenhouse from November 2014 until late April 2015 under a standard nursery watering regime that maintained soil that was damp to the touch. This meant watering approximately every 48-72 hours to ensure that the seedlings did not experience drought stress that could induce premature mortality. The field experiment was initiated in early May, 2015 when 30cm by 30cm by 90cm deep holes were dug in the ground at each transplant site and whole experimental units, including the pots, were placed into the holes without disturbing the plants or soils in the pots such that the soil level inside and outside of each pot was approximately equal. Pots were used in the field to maintain the mesocosm as a whole unit to ensure soil abiotic and biotic properties remained manipulated for the duration of the experiment. Removing the pot would have allowed roots to explore multiple soil types, confounding the soil variable of this experiment.  At this point plants were six months old and fairly similar in size. This age of plant had matured enough roots to withstand some stress by manipulating climates. This was the initiation of the novel climates with transplant sites that are approximately 2°C (BP) and 3°C (WAL) warmer, and 2°C (LM) and 3°C (ARB) cooler compared to the source sites (Figure 1, Table 1). Ten experimental units were similarly planted back into their site of origins at BC and WPC. These units represented the climate and sympatric soil and inoculum environments that the seed sources were adapted to and were used as a frame of reference for all other treatments. All field plantings were completed on consecutive days. Measurements of plant performance Plant performance was measured at the end of the third growing season in November 2017. At the time of harvest, some plant mortality had been observed and the experiment was harvested at a time where dead plants still had remaining above ground biomass and decomposition had not yet begun. This allowed us to analyze biomass across all treatment units regardless of observed mortality in the field  Seeds were removed from plants whenever they were observed during the duration of the experiment, dried at 60°C for 24 hours and weighed. All remaining aboveground biomass was clipped, dried and weighed. Soil was carefully cleaned from roots by soaking and wet sieving, and clean roots were dried and weighed. Specific leaf area (SLA) is the ratio of leaf area to leaf mass and it has been shown to decrease with drought stress in a variety of plant species (McCoy-Sulentic et al. 2017). Under drought conditions plants tend to produce leaves with lower SLA to conserve resources. At the time of harvest, we collected green leaves to measure specific leaf area (SLA) using dry leaves as outlined in Garnier et al. (2001). All leaves were collected at 0900 on the day of sampling and were rehydrated for 6 hours in a dark room prior to measurements using WinRHIZO (Regent Instruments) to calculate leaf area. Statistical analysis Four-way repeated measures ANOVA were used to compare the effects of climate (i.e. transplant site), plant origin, soil inoculum origin, and soil origin on plant response variables. We used the four transplant sites as climate factors (i.e. +2C, +3C, -2C, -3C), the two plant populations for plant origin factors, sympatric vs. allopatric soil biota for the soil inoculum factor and sympatric vs. allopatric soil for the soil origin factor. All factors were included as fixed effect factors because we specifically selected these sites and controlled for these variables.  This model was repeated for plant biomass, root:shoot ratio, specific leaf area, and seed mass. The reference site was left out of the analysis to maintain a balanced design. Model assumptions were checked using the Shapiro-Wilk test of normality and the Levene’s test of heterogeneity of variance. All statistics were conducted in R (version 3.3.1). All data sets met all of the assumptions and no transformations were made. We compared mean and variation of each individual treatment combination to the reference site and other treatment combinations to detect overlapping 95% confidence intervals of each treatment to the all sympatric reference site. This approach utilized the reference site as a baseline and answers the question of whether plant performance varied with treatment. In each figure, if variance of individual treatments overlap the variance of the fully sympatric reference site then we conclude that there is no difference in plant performance for that variable. . Specifically, if a treatment has reduced growth relative to the reference site this indicates a negative effect of the treatment and if a treatment is greater than the reference site then the treatment had an advantageous effect.