DOI: 10.5061/dryad.bvq83bk74
title: Sympatric pairings of dryland grass populations, mycorrhizal fungi, and associated soil biota enhance mutualism and ameliorate drought stress

description: 1. There is evidence that the distribution of ecotypes of plants and their
 symbiotic arbuscular mycorrhizal (AM) fungi and other associated soil
 biota may be structured by the availability of essential soil nutrients;
 and that locally adapted partnerships most successfully acquire limiting
 nutrients. This study tests the hypotheses that plant genotypes are
 adapted to the water availability of their local environment, and this
 adaptation involves associations with local soil biota, including AM
 fungi.  2. We grew semi-arid Bouteloua gracilis ecotypes from
 relatively wet and dry sites, with either sympatric or allopatric soil
 inoculum under moderate and extreme soil drying treatments to examine 1)
 how varying degrees of water limitation influence grass responses to soil
 biota, and 2) the relationship between AM fungal structures and these
 responses.  3. Under extreme soil drying, the dry-site ecotype
 tended to perform better than the wet-site ecotype. Both ecotypes
 performed best in either drying treatment when inoculated with their
 sympatric soil biota. Sympatric pairings produced more AM fungal hyphae,
 arbuscules and dark septate fungi. Extreme soil drying tended to
 accentuate these apparent benefits of sympatry to both plants and fungal
 symbionts, relative to the moderate drying treatment.  4. Our
 findings support the hypothesis that AM symbioses help Bouteloua gracilis
 ecotypes adapt to local water availability. This conclusion is based on
 the observations that as water became increasingly limited, sympatric
 partnerships produced more AM fungal hyphae and arbuscules and fewer
 vesicles. The abundances of hyphae and arbuscules were positively
 correlated with plant growth, suggesting that in sympatric pairs of plants
 and AM fungi, allocation to fungal structures is optimized to maximize
 benefits and minimize the costs of the symbioses. This provides strong
 evidence that co-adaptation among plants and their associated AM fungi can
 ameliorate drought stress. 5. Synthesis: Our study documents the role of
 locally adapted soil borne plant symbionts in ameliorating water stress.
 We found a relationship between AM fungal structures in roots and plant
 performance. Generally, plants and fungi from the same site resulted in
 more positive effects on plant growth.
description: <i>Sources of plants, soil and inoculum
 </i> Seeds
 and soil were collected from two sites within 25 km of one another, but
 with very different annual precipitation. The wetter site (hereafter “wet
 site”) was a semi-arid grassy understory of a piñon-juniper woodland on
 the west side of the Kaibab Plateau (Coconino County, Arizona, USA) at an
 elevation of 2,064 m with approximately 43 cm of precipitation annually
 (PRISM Climate Group). The drier site (hereafter “dry site”) was a
 semi-arid grassland adjacent to an alluvial drainage on the east side of
 the Kaibab Plateau at an elevation of 1710 m with an average of 28 cm of
 precipitation annually (PRISM Climate Group). The soils at both sites are
 derived from Kaibab Limestone and the wet site soils are composed of
 argids while the dry site soils are a mosaic of orthents and
 calcids. <i>Bouteloua
 gracilis</i> seed was collected from the two sites using the Seeds
 of Success protocol (http://www.nps.gov/planTs/sos/protocol/index.htm). Live soil inoculum was collected from the rooting zone of <i>B. gracilis </i>along three 100 m transects established from a random origin (azimuths of 0˚, 90˚ and 270˚) at the wet and dry sites. Soil subsamples within each site were pooled together and mixed. We justify homogenizing inoculum from each site because we were interested in seedling responses to average soil biotic conditions 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. The abundance of different soil organisms in the two inoculum soils was determined using phospholipid fatty acid (PLFA) and neutral lipid fatty acid (NLFA) analysis. Lipids were extracted from 5 g of freeze-dried inoculum soil by vortex mixing in a one-phase mixture of citrate buffer, methanol, and chloroform (0.8:2:1: v/v/v, pH 4.0). The biomass of AM fungi was estimated from the NLFA 16:1 w5: 20:1 w9, and 22:1 w13, biomass of other fungi was estimated from 18:2 w9:12c, and biomass of bacterial groups was estimated signature PLFAs for gram positive and gram negative bacteria (Olsson <i>et al.,</i> 1995). This analysis indicated that the soil inoculum from the wet and dry sites had similar abundances of various fungal groups, including AM fungi, and bacteria (Supporting Information Table S1). The community composition of soil fungi in wet and dry inoculum treatments were compared before and after the experiment. Samples of soil were collected and DNA was extracted from 0.5 g of soil using a PowerSoil DNA Extraction Kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA). Genomic DNA was normalized to 2 ng/mL, diluted 10-fold and amplified in triplicate PCR using the universal ITS general eukaryotic primer WANDA and the AM fungal specific primer AML2 for the small subunit (SSU) rRNA gene (Lee et al. 2008; Dumbrell et al. 2011). Purified products were quantified with PicoGreen fluorescence. Indexing PCR was completed using 8 bp dual indexed WANDA and AML2 primers. Indexed PCR products were purified using a 1,1 carboxylated magnetic bead solution, quantified, and combined into a final sample library. The library was purified, concentrated, and quantified using quantitative PCR against Illumina DNA standards on an Illumina MiSeq System (Illumina, Inc., San Diego, CA) running in paired end 2 x 300 bp mode. Forward reads were trimmed to 250 bp to remove low quality tails and demultiplexing was carried out using a minimum quality threshold of q20 and default parameters in QUIIME 1.9.1 (Caporasso et al. 2010) Taxonomy was assigned to sequences using BLAST with 90% similarity and an E-value less than 10-4, against the online MaarjAM database (http,//maarjam.botany.ut.ee; accessed 10 September, 2020, Ōpik et al. 2010). Taxa that made up less than 1% of relative abundance were labeled as ‘other’, otherwise species were recorded to the genus level for community comparisons. Many species remained unidentified or classified only to order or family.   <i>Experimental design </i> Mesocosms were prepared with all four possible combinations of plant and inoculum origin, two sympatric combinations (inoculum and plants from the wet site, or inoculum and plants from the dry site) and two allopatric combinations (inoculum from the dry site with plants from the wet site, or inoculum from the wet site with plants from the dry site). These treatments were further crossed with two levels of water availability to mimic the severity of water limitation at the two source sites. To generate a frame of reference for the performance of plants without sympatric or allopatric soil organisms under the soil drying regime that most closely resembles their home site, we created two sterile inoculum treatments in which plants from the wet site were grown with sterile soil under a moderate drying regime and plants from the dry site were grown in sterile soil under extreme drying conditions. Each combination of plant ecotype, inoculum origin and drying regime was replicated 9 times, resulting in 72 mesocosms, plus, the two sterile inoculum treatments replicated 9 times for a total of 90 experimental units.             Mesocosms were constructed from 21 L plastic containers (43 cm x 28 cm x 18 cm) with six 0.3 cm diameter holes drilled into the bottom for drainage. In order to remove the effects of any variation in soil physical and chemical characteristics at the two different sites, we created a sterilized common soil using a 1,1 mixture of soil from the two sites that was steam sterilized at 125°C for 48 hours. Our experimental design matches type C in Gundale et al. (2017), because unique and variable sub-populations of plant subjects (a random draw of seeds collected from a site) are confronted with one of two soil biota conditions that represent the gamma diversity of each site, and the same background soil condition. This design is preferred when the goal is to detect differences among two or more groups of subjects, and when within-site or regional spatial variation is not a focus (Cahill et al. 2017; Gundale et al. 2017; Gundale et al. 2019). Each mesocosm was filled with approximately 15 liters of sterilized soil and topped with a 1 cm thick band of either live or sterilized (dead) inoculum soil. <i>Bouteloua gracilis</i> seed was sprinkled onto the inoculum soil at a rate of 60 seeds per mesocosm and later thinned to 10 seedlings per mesocosm. Mesocosms were placed in fully randomized spatial locations to account for microclimatic variation within the glasshouse.             <i>Watering treatments</i> Initially, all mesocosms were watered three times each week for eight weeks and then they were watered twice per week for four weeks before starting the drying treatments. Each watering event brought the mesocosms to field capacity to ensure adequate moisture for plant establishment. Rather than simulate an unrealistically abrupt transition from abundant moisture to dry conditions, we simulated a more gradual transition based on percent of field capacity. These transitions simulate what a plant may experience during the growing seasons as soil moisture diminishes after snowmelt or summer monsoons. Mass at field capacity was estimated by weighing ten randomly selected containers 24 hours after watering. Then, the mass of one randomly selected container was measured every other day, until a soil mass threshold indicated it was time to water again to field capacity. For the moderate drying treatment, we used an initial threshold of 60% of mass at field capacity. For the extreme drying treatment, we used an initial threshold of 40%. After each sequential watering, we decreased both of these threshold percentages by 5%.  This both gradually decreased the amount of water available to the plants and increased the length of time between watering events. Eventually, we reached permanent wilting point (approx. -1.5 MPa) in both treatments resulting in at least 90% mortality after 8 months when the experiment was terminated.   <i>Plant performance</i> Every two weeks, we measured plant height in all containers and the percentage of plant tissue that was green was monitored to estimate the length of time until plant senescence. Greenness was based on ocular estimates of color. No plants produced inflorescences.  At the termination of the experiment, all aboveground biomass was clipped, dried at 60°C for 24 hours and weighed. Root biomass was sampled by taking four soil cores (5 cm diameter and 18 cm deep). Roots were cleaned, dried and weighed and the weight of roots per volume of core was used to estimate root biomass in the total volume of the mesocosm.   <i>AM fungal performance</i> Soil and root materials obtained from destructive harvesting at the end of the experiment were analyzed from all 90 mesocosms.  A 10 g subsample of fresh root material was refrigerated until it could be examined for root colonization by fungi. Root samples were cleared with 5% KOH and stained with ink in vinegar (Vierheilig <i>et al.</i>, 1998).  Colonization by AM fungi and other root endophytes was determined using the gridline intersect method at 200 × magnification (McGonigle <i>et al.</i>, 1990).  Mycorrhizal root colonization was distinguished as arbuscules, vesicles and hyphae; dark septate endophytes (DSEs) were also quantified.  The soil-borne (external) hyphae of AM fungi were extracted from the soil cores after root removal, using the methods of Sylvia (1992),  and quantified using a gridded eyepiece graticule in an inverse compound microscope at 250 × magnification.  At points where hyphae intersected gridlines, hyphae were counted, and counts were converted to length of hyphae per gram of soil. Hyphae of AM fungi were distinguished from other fungal hyphae based on their morphology and color.
description: All variables are defined below.  Height = the
 total height of plants as measured from the soil surface to the maximum
 height of the tallest leaf blade or infloresence.  Pot
 = unique experimental unit identifier Time_until_brown
 = the length of time for plants to become 100% brown  in days from the
 onset of drought experiment.  week_brown = The week in
 which plants were determined 'seneseced' from 
 total_above_biomass = total biomass of shoots, leaves, and
 infloresences in grams below_ground_biomass = total
 biomass of  roots and plant belowground structures in grams
 Percent_hyphae = percent of Arbuscular Mycorhizzal Fungi  hyphae
 inside of plant roots Percent_arbuscules = percent of
 Arbuscular Mycorhizzal Fungi  arbuscules inside of plant roots
 per_vesicle = percent of Arbuscular Mycorhizzal Fungi  vesicles
 inside of plant roots Percent_DSE = percent of Dark
 septate endophytes inside of plant roots EMH = Amount
 of extra matrical hyphae in soil of experimnetal units in meters per gram
 of soil SoilP = Soil Phosphorous measured as total
 phosphorous in mg phosphorous per kilogram of soil  om
 = Soil organic matter as measured by loss on ignition in grams of
 carbon Soil = Soil inoculum source site where WPC =
 white pockets canyon field site and kane = Kane Ranch field
 site.  Plant= Source site of plant seeds where kane =
 Kane Ranch field site and WPC = White Pockets Canyon field site 
 Water= Watering treatment where dry = extreme drying and wet =
 moderate drying