Sympatric pairings of dryland grass populations, mycorrhizal fungi, and associated soil biota enhance mutualism and ameliorate drought stress

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.

Methods
Sources of plants, soil and inoculum

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.

Bouteloua gracilis 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 B. gracilis 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 et al., 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.

Experimental design

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. Bouteloua gracilis 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.

Watering treatments

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.

Plant performance

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.

AM fungal performance

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 et al., 1998).  Colonization by AM fungi and other root endophytes was determined using the gridline intersect method at 200 × magnification (McGonigle et al., 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.

1. 有证据表明，植物生态型及其共生丛枝菌根（arbuscular mycorrhizal, AM）真菌与其他相关土壤生物群落的分布，可能受必需土壤养分的有效性调控；且本地适配的共生体组合能更高效地获取限制性养分。本研究检验两项假说：其一，植物基因型适配其原生环境的水分有效性；其二，该适配过程涉及与包括AM真菌在内的本地土壤生物群落的共生关联。

2. 我们栽培了来自相对湿润与干旱生境的半干旱野牛草（*Bouteloua gracilis*）生态型，分别施加同域（sympatric）与异域（allopatric）土壤接种物，并设置中度与极端土壤干旱处理，以探究：1）不同程度的水分限制如何影响草本植物对土壤生物群落的响应；2）AM真菌结构与上述响应之间的关联。

3. 在极端土壤干旱条件下，干旱生境生态型的生长表现优于湿润生境生态型。两种生态型在任一干旱处理中，接种同域土壤生物群落时均表现最佳。同域组合可产生更多的AM真菌菌丝、丛枝与深色有隔内生真菌。相较于中度干旱处理，极端土壤干旱会强化同域共生对植物与真菌共生体的上述益处。

4. 我们的研究结果支持“AM共生助力野牛草生态型适配本地水分有效性”的假说。该结论基于以下观测：随着水分限制加剧，同域共生组合会产生更多AM真菌菌丝与丛枝，且泡囊数量更少。菌丝与丛枝的丰度与植物生长呈正相关，表明在植物与AM真菌的同域组合中，对真菌结构的资源分配已被优化，以最大化共生收益并最小化共生成本。这为植物与其关联AM真菌之间的协同适配可缓解干旱胁迫提供了有力证据。

5. 总结：本研究证实了本地适配的土壤源植物共生体在缓解水分胁迫中的作用。我们发现根系内AM真菌结构与植物生长表现存在关联。总体而言，源自同一生境的植物与真菌组合对植物生长的促进效应更为显著。

## 研究方法

### 植物、土壤与接种物来源
种子与土壤采集自两处相距25 km以内，但年降水量差异显著的生境。湿润生境（下称"湿生生境"）位于美国亚利桑那州科科尼诺县凯巴布高原西侧，为矮松-桧木林的半干旱草本下层群落，海拔2064 m，年降水量约43 cm（PRISM Climate Group）。干旱生境（下称"旱生生境"）位于凯巴布高原东侧，紧邻冲积排水区的半干旱草原，海拔1710 m，年平均降水量28 cm（PRISM Climate Group）。两处生境的土壤均源自凯巴布石灰岩；湿生生境土壤为黏化旱成土（argids），旱生生境土壤则为淡旱成土（orthents）与钙积旱成土（calcids）的镶嵌体。

野牛草（*Bouteloua gracilis*）种子的采集遵循《成功种子计划》（Seeds of Success）规程（http://www.nps.gov/planTs/sos/protocol/index.htm）。活土壤接种物采集自两处生境中野牛草的根际区域，在每个生境中，沿3条从随机起点（方位角分别为0°、90°与270°）布设的100 m样带进行取样。各生境内的土壤子样本被混合均质化。我们选择均质化各生境接种物的依据是，本研究旨在探究幼苗对不同生境平均土壤生物条件的响应，而非单一生境或超出采样范围的更大地理尺度的响应（即"C型设计"；Gundale等，2017，2019）。接种物土壤在实验前冷藏保存2周。

采用磷脂脂肪酸（phospholipid fatty acid, PLFA）与中性脂脂肪酸（neutral lipid fatty acid, NLFA）分析，测定两种接种物土壤中不同土壤生物的丰度。取5 g冻干接种物土壤，在柠檬酸盐缓冲液、甲醇与氯仿的单相混合体系（体积比0.8:2:1，pH 4.0）中涡旋提取脂质。AM真菌的生物量通过NLFA标记物16:1 ω5、20:1 ω9与22:1 ω13估算；其他真菌的生物量通过18:2 ω9,12c估算；细菌类群的生物量则通过革兰氏阳性与革兰氏阴性细菌的特征性PLFAs估算（Olsson等，1995）。分析结果显示，湿生与旱生接种物土壤中各类真菌（包括AM真菌）与细菌的丰度并无显著差异（支持信息表S1）。

实验前后均对湿生与旱生接种物处理中的土壤真菌群落组成进行了比较。取0.5 g土壤样本，采用PowerSoil DNA提取试剂盒（MO BIO实验室公司，美国加利福尼亚州卡尔斯巴德）提取基因组DNA。将基因组DNA归一化至2 ng/mL，稀释10倍后，采用通用真核ITS引物WANDA与AM真菌特异性引物AML2（针对小亚基（small subunit, SSU）rRNA基因）进行三次重复PCR扩增（Lee等，2008；Dumbrell等，2011）。纯化后的扩增产物通过PicoGreen荧光定量。采用8 bp双索引引物WANDA与AML2进行索引PCR。索引PCR产物采用1,1-羧基化磁珠溶液纯化、定量后，合并为最终样本文库。文库经纯化、浓缩后，采用基于Illumina DNA标准品的定量PCR在Illumina MiSeq系统（Illumina公司，美国加利福尼亚州圣地亚哥）上以双端2×300 bp模式进行测序。将正向读长修剪至250 bp以去除低质量尾部，采用QUIIME 1.9.1（Caporasso等，2010）中q20的最低质量阈值与默认参数进行分样拆分。序列分类学注释采用BLAST比对MaarjAM在线数据库（http,//maarjam.botany.ut.ee；2020年9月10日访问，Ōpik等，2010），相似性阈值设为90%，E值小于10^-4。相对丰度低于1%的分类群被标记为"其他"，其余物种则记录至属水平用于群落比较。多数物种仍未被鉴定，或仅能分类至目或科水平。

### 实验设计
实验单元（中宇宙，mesocosm）设置了植物与接种物来源的全部四种组合：两种同域组合（接种物与植物均来自湿生生境，或接种物与植物均来自旱生生境），以及两种异域组合（接种物来自旱生生境，植物来自湿生生境；或接种物来自湿生生境，植物来自旱生生境）。上述处理进一步与两个水分有效性水平交叉，以模拟两处源生境的水分限制严重程度。为了建立无同域/异域土壤生物时，植物在最接近原生境的干旱处理中的生长参照，我们设置了两种无菌接种物处理：湿生生境植物在中度干旱处理下种植于无菌土壤，旱生生境植物在极端干旱处理下种植于无菌土壤。

每种植物生态型、接种物来源与干旱处理的组合均设置9次重复，共得到72个中宇宙单元；加上2种无菌接种物处理各9次重复，总实验单元数为90。

中宇宙采用21 L塑料容器（43 cm×28 cm×18 cm）制作，底部钻有6个直径0.3 cm的排水孔。为消除两处生境土壤物理与化学特性差异的影响，我们将两处生境的土壤按1:1混合，经125℃蒸汽灭菌48小时，制备成标准化的通用土壤。本实验设计符合Gundale等（2017）定义的C型设计：从单一生境采集的种子随机获得的植物个体，分别暴露于代表该生境γ多样性的两种土壤生物群落条件下，且背景土壤条件一致。当研究目标为检测两组或多组受试对象的差异，且不关注样点内或区域空间变异时，该设计为优选方案（Cahill等，2017；Gundale等，2017，2019）。每个中宇宙装入约15 L灭菌通用土壤，表层覆盖1 cm厚的活接种物或灭菌（灭活）接种物。将野牛草种子以每中宇宙60粒的密度撒播于接种物表层，之后间苗至每中宇宙10株幼苗。将中宇宙随机摆放于温室内，以消除微气候变异的影响。

### 浇水处理
实验初期，所有中宇宙每周浇水3次，持续8周；之后改为每周浇水2次，持续4周，再开始干旱处理。每次浇水均将中宇宙浇至田间持水量，以确保植物定植所需的充足水分。我们并未模拟从水分充足到干旱的突兀过渡，而是基于田间持水量百分比模拟更自然的渐变过程，该过程模拟了植物在生长季中，融雪或夏季季风过后土壤水分逐渐减少的场景。田间持水量的质量通过在浇水后24小时称量10个随机选取的容器估算。之后每隔一天称量一个随机选取的容器，直至土壤质量阈值达到再次浇水至田间持水量的条件。中度干旱处理的初始阈值为田间持水量质量的60%；极端干旱处理的初始阈值为40%。每次浇水后，将两个阈值均降低5%。这一设置既逐步减少了植物可获得的水分，也延长了两次浇水之间的间隔。最终，两种处理均达到永久萎蔫点（约-1.5 MPa），实验终止时（8个月后）植物死亡率至少达90%。

### 植物生长表现
每两周测量所有容器内植物的高度，并通过目测植株绿色组织占比，监测植物衰老前的持续时间。绿色度基于颜色的目视评估。所有植株均未抽穗。实验终止时，剪取所有地上生物量，在60℃下烘干24小时后称重。根系生物量通过采集4个土芯（直径5 cm，深度18 cm）进行估算：清洗根系后烘干称重，通过单位体积土芯的根系重量，推算整个中宇宙体积内的根系生物量。

### AM真菌生长表现
实验结束时破坏性收获所有90个中宇宙的土壤与根系样本，进行分析。取10 g新鲜根系样本冷藏，用于检测真菌对根系的定殖情况。根系样本经5% KOH消解后，采用醋墨染色法（Vierheilig等，1998）染色。采用网格交叉法在200×放大倍数下，测定AM真菌与其他根系内生真菌的定殖率（McGonigle等，1990）。菌根根系定殖被区分为丛枝、泡囊与菌丝；深色有隔内生真菌（dark septate endophytes, DSEs）的定殖率也一并量化。

移除根系后，采用Sylvia（1992）的方法从土芯中提取AM真菌的土壤（外部）菌丝，在倒置复合显微镜下以250×放大倍数，采用网格目镜测微尺进行定量。在菌丝与网格线相交的点位计数菌丝，并将计数结果转换为每克土壤中的菌丝长度。根据形态与颜色，可将AM真菌菌丝与其他真菌菌丝区分开来。