Volume 221, Issue 1 p. 493-502
Free Access

Common garden experiments disentangle plant genetic and environmental contributions to ectomycorrhizal fungal community structure

Adair Patterson

Corresponding Author

Adair Patterson

Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ, 86011-5640 USA

Author for correspondence:

Adair Patterson

Tel: +928 2213174

Email:[email protected]

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Lluvia Flores-Rentería

Lluvia Flores-Rentería

Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ, 86011-5640 USA

Department of Biology, San Diego State University, San Diego, CA, 92182 USA

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Amy Whipple

Amy Whipple

Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ, 86011-5640 USA

Merriam-Powell Center for Environmental Research, Northern Arizona University, Flagstaff, AZ, 86011-5640 USA

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Thomas Whitham

Thomas Whitham

Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ, 86011-5640 USA

Merriam-Powell Center for Environmental Research, Northern Arizona University, Flagstaff, AZ, 86011-5640 USA

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Catherine Gehring

Catherine Gehring

Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ, 86011-5640 USA

Merriam-Powell Center for Environmental Research, Northern Arizona University, Flagstaff, AZ, 86011-5640 USA

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First published: 16 July 2018
Citations: 36


  • The interactions among climate change, plant genetic variation and fungal mutualists are poorly understood, but probably important to plant survival under drought. We examined these interactions by studying the ectomycorrhizal fungal (EMF) communities of pinyon pine seedlings (Pinus edulis) planted in a wildland ecosystem experiencing two decades of climate change-related drought.
  • We established a common garden containing P. edulis seedlings of known maternal lineages (drought tolerant, DT; drought intolerant, DI), manipulated soil moisture and measured EMF community structure and seedling growth.
  • Three findings emerged: EMF community composition differed at the phylum level between DT and DI seedlings, and diversity was two-fold greater in DT than in DI seedlings. EMF communities of DT seedlings did not shift with water treatment and were dominated by an ascomycete, Geopora sp. By contrast, DI seedlings shifted to basidiomycete dominance with increased moisture, demonstrating a lineage by environment interaction. DT seedlings grew larger than DI seedlings in high (28%) and low (50%) watering treatments.
  • These results show that inherited plant traits strongly influence microbial communities, interacting with drought to affect seedling performance. These interactions and their potential feedback effects may influence the success of trees, such as P. edulis, in future climates.


Extreme climate events, such as severe maximum and minimum temperature fluctuations, increased frequency of heat waves and cold fronts, and dramatic changes in precipitation, are projected to increase in frequency and severity throughout the 21st century (IPCC, 2014). For example, in the southwestern USA, temperatures increased by 0.9°C from 1886 to 2016, but are projected to increase by 4.8°C by the end of the century, resulting in chronic future precipitation deficits (USGCRP, 2017). The persistence of forest trees following such climate events is of particular concern, because trees can be foundation species that increase the stability and biodiversity of ecosystems (Dayton, 1972), and also provide services to the human sector, such as paper, lumber and food (IUFRO Task Force on Environmental Change, 2000). Forests across the globe are predicted to decline with climate change, principally as a result of drought (Allen et al., 2010; Anderegg et al., 2012). In a study of 226 tree species across 80 sites in semi-arid, tropical and temperate biomes around the world, Choat et al. (2012) found that 70% of species studied were susceptible to hydraulic failure under drought conditions. However, most of these studies have not considered the importance of intraspecific genetic variation within plants, as well as interactions between plants and their associated microbiomes, which could help to buffer plants from extreme drought events (Gehring et al., 2017).

Previous studies have shown that tree mortality differs within regional populations based on phenotypic variation in response to climatic stress (Klos et al., 2009; Sthultz et al., 2009a). Some individuals experience lower mortality and respond better to drought than others (Sthultz et al., 2009a; Franks et al., 2013). Recent studies have also shown that trees employ a range of mechanisms by which offspring may resemble their parents and acquire traits to cope with environmental stress (e.g. Cendán et al., 2013; Yakovlev et al., 2014). These mechanisms of inheritance go beyond the additive genetic variation in DNA sequence (narrow-sense heritability) to include parental effects transmitted by somatic or epigenetic mechanisms (Bonduriansky et al., 2012; Herman et al., 2014; Verhoeven et al., 2016). Intraspecific genetic variation, plasticity and transgenerational plasticity can all affect the ability of plants to withstand adverse changes in the environment (Bonduriansky et al., 2012; Herman et al., 2014; Whipple & Holeski, 2016). Intraspecific genetic variation in plants is also associated with changes in ectomycorrhizal fungal (EMF) communities. Replicated genotypes of Populus angustifolia planted in a common garden varied in their EMF species composition, with some genotypes dominated by ascomycetes and others by basidiomycetes (Lamit et al., 2016). However, few studies have experimentally manipulated precipitation in a common garden to understand how inherited plant phenotypic variation and EMF communities interact under climate stress.

Although many studies have focused on the effect of drought and climatic disturbance on dominant tree species, few have considered the interactions of mycorrhizal mutualists and their potential feedback effects on host plants. Mutualistic mycorrhizal fungi may be directly and indirectly influenced by climate change (Kauserud et al., 2012; Buentgen et al., 2015). EMF associate most often with woody plants, increasing host plant water and nutrient uptake from the surrounding soil and also providing protection against harmful pathogens and parasites in exchange for photosynthate (Smith & Read, 2008). Certain taxa of EMF have been shown to reduce water and nutrient stress on host trees (Lehto & Zwiazek, 2010; Pena & Polle, 2013). Likewise, drought can significantly alter the abundance, diversity and community composition of EMF (Swaty et al., 2004; Karst et al., 2014; Gehring et al., 2016). For instance, drought favors low biomass species with thinner mantles, shorter hypha and lower carbohydrate demands (Agerer, 2006; Karst et al., 2014). However, few studies have simultaneously examined EMF community shifts as a result of drought and corresponding host plant responses (but see Pena & Polle, 2013; Gehring et al., 2017).

In this study, we examined the EMF communities associated with seedlings of drought-tolerant or drought-intolerant individuals of a foundation species, pinyon pine (Pinus edulis Engelm.), occurring in an extreme environment affected by regional drought (Cobb et al., 1997; Breshears et al., 2005; Sthultz et al., 2009a). Within this system, differential chronic herbivory by the stem- and cone-boring moth (Dioryctria albovittella) on mature trees results in two identifiable phenotypes on the landscape: moth-resistant trees have an upright canopy architecture, whereas moth-susceptible trees are shrub-like as a result of prolonged consumption of their terminal shoots by D. albovittella (Whitham & Mopper, 1985; see icons in Fig. 1). Sthultz et al. (2009b) found three-fold higher mortality in moth-resistant trees than in moth-susceptible trees during a record drought, dramatically shifting the ratio of moth-resistant and moth-susceptible phenotypes at the stand scale. The offspring of surviving moth-resistant and moth-susceptible trees planted in a common garden showed mortality patterns similar to their maternal trees (Gehring et al., 2017). EMF communities also differed between mature moth-resistant and moth-susceptible trees, with moth removal and glasshouse experiments suggesting that these differences were inherited and not a result of differential herbivory (Sthultz et al., 2009b; Gehring et al., 2017). However, although these studies demonstrate complex relationships amongst host phenotype, susceptibility to herbivory, drought tolerance and EMF community composition, studies of seedlings in a natural setting have been limited and have not involved experimental manipulation of soil moisture to disentangle genetic and environmental effects.

Details are in the caption following the image
Nonmetric multidimensional scaling (NMDS) ordination of the ectomycorrhizal fungal communities associated with Pinus edulis seedlings of drought-tolerant (DT) maternal trees (shrubby tree icon) and drought-intolerant (DI) mothers (upright tree icon) subject to high water (large drops) and low water (small drops) treatments. Each point represents the centroid for each treatment group. Spatially distant points show less community similarity. Error bars denote ± 1SE.

To quantify the relative roles of maternal inheritance and drought on seedling performance and EMF communities, we examined the progeny of moth-susceptible/drought-tolerant (DT) and moth-resistant/drought-intolerant (DI) pinyon pine seedlings planted in a common garden in which soil moisture levels were manipulated to mimic high-stress, dry conditions and lower stress, moist conditions during ongoing drought. We measured the EMF communities and growth of these seedlings in order to test four hypotheses. Hypothesis 1: seedlings from DT mothers will recruit a different EMF community than seedlings from DI mothers. We expected to see similar differences to those observed in wild, mature pinyon trees, with DT trees associating predominantly with ascomycetes and DI trees associating with basidiomycetes (Sthultz et al., 2009b; Gehring et al., 2014). Members of these different fungal phyla vary in their ecology (Tedersoo et al., 2006) and genetic mechanisms employed to establish the symbiosis (Martin et al., 2010). Hypothesis 2: based on data from adult EMF communities observed over nearly 20 yr (Gehring et al., 2014), we also hypothesized that the EMF composition of seedlings from DI mothers would vary depending on water availability, whereas the EMF composition of seedlings from DT mothers would not. Hypothesis 3: the species richness, diversity and hyphal exploration of EMF communities will be greater in seedlings receiving higher supplemental moisture regardless of the drought tolerance category of their mothers (DI or DT). Greater precipitation is associated with EMF diversity at large spatial scales (Tedersoo et al., 2012), and declines in EMF species richness were observed following drought in P. edulis (Gehring et al., 2014). Increased hyphal complexity and exploration distance have been associated with EMF species that are able to extract greater volumes of water from surrounding soil (Agerer, 2006). Hypothesis 4: given that adult DI trees outperformed DT trees before drought (Gehring et al., 2017), we hypothesized that their seedlings would also outperform seedlings of DT trees in well-watered conditions, but that the reverse would be true in low moisture. Therefore, we measured the growth of DT and DI seedlings used for EMF assessments. Tests of these hypotheses are essential to understand how climate change (i.e. drought in this study) is likely to affect many arid land species and the communities they support. This knowledge can also be used in restoration programs that seek to utilize the natural genetic variation in plant species to mitigate climate change (e.g. O'Neill et al., 2008; Grady et al., 2015) and maintain biodiversity (Ikeda et al., 2014).

Materials and Methods

We studied Pinus edulis Engelm. seedlings from both DT and DI maternal trees growing in a common garden located near Sunset Crater National Park (35°23.154′N, 111°26.331′W, elevation 1902 m). This location is a semiarid volcanic landscape consisting of basaltic cinder, volcanic ash and soil deposits surrounded by numerous cinder cones and lava flows (Hooten et al., 2001). The dominant vegetation at the study site included P. edulis, P. ponderosa (ponderosa pine) and Juniperus monosperma (one-seed juniper), as well as an understory dominated by Fallugia paradoxa (Apache plume). Sunset Crater receives, on average, 170.3 mm of precipitation annually, and temperatures per month average < 10°C in the winter and can exceed 37°C in the summer (AZ Climate Division 2; National Park Service, Flagstaff, AZ, USA). Drought conditions prevailed during the time of EMF community and plant growth measurements based on the Palmer drought severity index (PDSI) (National Climate Data Center; https://www.ncdc.noaa.gov/temp-and-precip/drought/historical-palmers/).

The pinyon common garden was established in 2010 using seeds collected from DT and DI adult pinyons at a field site c. 0.8 km away from the garden. Maternal trees of each of the two drought tolerance groups were selected based on adult tree architecture (i.e. shrubby vs upright) and/or long-term records of moth herbivory. Seeds were surface sterilized in 10% sodium hypochlorite and weighed to ensure potential viability. Seeds with a weight of 0.30 g or above were considered to be potentially viable. Seedlings were germinated at the Northern Arizona University Research Greenhouse Complex in Flagstaff, AZ, grown for a year in sterile potting soil without mycorrhizal symbionts present and then transplanted to the garden site, a c. 2-ha area of native vegetation fenced to exclude vertebrate herbivores. Seedlings were spot checked before planting to ensure EMF contamination from the glasshouse had not occurred. The garden was constructed with an interspersed design with seedlings of DT and DI mothers planted randomly along eight transects. Each seedling was planted within half a meter of a nurse plant, F. paradoxa (Sthultz et al., 2007). As a result of the coarse texture of the soil, percentage soil moisture in May and June can be < 1%, whereas rooting zone soil temperatures can exceed 25°C (Swaty et al., 1998); therefore, we provided each seedling with two irrigation drip lines which supplied water during the driest months of the year, including May and June, a period of active seedling growth despite low moisture. After 2 yr of establishment with supplemental irrigation that achieved an average percentage soil moisture of 3.2%, seedlings were randomly assigned to different supplemental watering treatments. Each seedling was given either a high water treatment (three irrigation drip lines) or a low water treatment (one irrigation drip line). Supplemental water was administered only during the two driest times of the year flanking the summer monsoon season (May, June and October). Drip irrigation emitters were located within 15 cm of the base of the seedling, and seedlings were at least 0.5 m away from one another. During the watering periods described, water was administered twice weekly at a rate of 0.6 l min−1 per emitter for 12 min. Soil moisture (receiving no supplemental water) was measured gravimetrically at eight sites distributed throughout the garden in June, the driest month of the year. Soil moisture averaged 0.85% (0.19 SE) in June of 2012, 1.14% (0.17 SE) in June of 2013 and 0.35% (0.078 SE) in June of 2014. Soil moisture in the two moisture treatments was also gravimetrically measured at eight sites distributed throughout the garden at spots which emitters were installed as if a seedling was present (0.5 m from a Fallugia nurse), but no seedling was planted. In June 2012, soil moisture averaged 3.01% (0.25 SE) for the low water treatment and 4.30% (0.41 SE) for the high water treatment. Similarly, in June 2013, soil moisture averaged 2.82% (0.25 SE) for the low water treatment and 4.69% (0.41 SE) for the high water treatment, and, in June 2014, it averaged 1.81% (0.22 SE) for the low water treatment and 3.70% (0.32 SE) for the high water treatment, 24 h after watering. Although the high moisture treatment seedlings received three times as much water as the low moisture treatment seedlings, poor moisture retention in the coarse-textured volcanic soil meant that the difference in water treatments was less than three-fold after 24 h. Given the strongly negative PDSI values in 2012 and 2013 (May + June averages of −4.2 and −4.1, respectively, category = extreme drought), the 2-yr average soil moisture levels of 2.3% in the low water treatment represent moderate drought conditions based on the soil measurements of Swaty et al. (1998) during a year of similar average PDSI. Root sampling took place 1 yr after the differential watering treatments were implemented. The watering treatments were maintained after EMF sampling and all seedlings survived the sampling and had similar growth rates to unsampled seedlings (C. A. Gehring et al., unpublished data).

Sample collection and morphological analysis

In June 2013, we harvested roots from seedlings of DT and DI mothers of both high and low water treatments (Hypotheses 1–3) by collecting a minimum of 100 cm of fine roots (< 2 mm in diameter) at a depth of 0–30 cm, so as not to substantially damage the seedling. Although we aimed for a sample size of 10 seedlings per drought tolerance category within each watering treatment, difficulty in collecting sufficient roots on some seedlings limited our sample size to 17 for the high water treatment (= 9 for DT and = 8 for DI) and 15 for the low water treatment (= 7 for DT and = 8 for DI). We mapped the location of each of the sampled seedlings using a Garmin eTrex Legend HCx GPS unit. Seedlings from the same maternal seed source were sampled across treatments, but not within treatments. However, it was not possible to fully pair seedlings in high and low water treatments by maternal seed source. Pinyons grow very slowly and the study seedlings in both treatments only averaged 8.5 cm in height at the time at which their roots were collected 3 yr after planting. As our aim was to sample roots without causing seedling mortality, the quantity of roots collected was rather low and only 50–100 individual living EMF root tips could be collected per seedling (average = 90.1). These root tips were morphologically analyzed under a dissecting microscope at ×20 magnification and categorized for each seedling based on color, texture, hyphal quantity and structure (Agerer, 1991). Hyphal exploration type was assessed by observing each morphotype for emanating hyphae and the presence of rhizomorphs (Agerer, 2001; Tedersoo et al., 2012), in addition to utilizing the Agerer (2006) categorization of EMF genera. Roots were stored at −20°C until further analysis.

DNA extraction, amplification and Sanger sequencing

We extracted DNA from one to five root tips (depending on availability) of every fungal morphotype found on every seedling using DNeasy Plant Extraction Kits (Qiagen, Valencia, CA, USA). We performed polymerase chain reaction (PCR) under standard conditions to amplify the internal transcribed spacer (ITS) region of the rRNA of the fungal genome with the ITS1-F and ITS4 primer pair, as in White et al. (1990) and Gardes & Bruns (1993), using KAPA Taq Hotstart (Kapa Biosystems, Wilmington, MA, USA). Successfully amplified PCR product was purified and then cycle sequenced using BigDye Terminator Mix 3.1 (Thermo Fisher Scientific Inc., Waltham, MA, USA). Sequencing was performed on an ABI 3730xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) at the Environmental Genetics and Genomics Laboratory at Northern Arizona University. When amplification or sequencing of a morphotype of a seedling was unsuccessful, an additional root tip from that morphotype from that seedling was processed.

Seedling growth

To explore the relationship between seedling growth, soil moisture and drought tolerance category (Hypothesis 4), we measured annual cumulative shoot growth for each seedling from which ectomycorrhizas were collected in June 2013 and 2014. On each seedling, the length of every shoot of new growth from 2013 and 2014 was measured to within ± 0.1 cm and a total annual shoot growth was calculated. Because terminal buds in pinyon pine are set in the autumn of the previous year, with dependence on the previous year's conditions, and then elongate during the summer of the following year (Douglas & Erdman, 1967), we measured 2 yr of growth (2013 and 2014) to reflect the experimental conditions applied in 2012 and carried through 2014. Pinus edulis seedlings grow from multiple buds simultaneously rather than principally from a single terminal bud, and so the measurement of the elongation of all new shoots each year is a better representation of growth than is seedling height.

Data analysis

DNA sequences were aligned and trimmed in Bioedit (Hall, 1999) and then identified to the genus or species level using the Basic Logical Alignment Search Tool (Blast) (Altschul et al., 1990) and Unite (Kõljalg et al., 2013) databases. We considered sequence similarity of ≥ 98% to published sequences to be indicative of species-level identity and 95–97% to be indicative of genus-level identity (Kõljalg et al., 2013). To visualize the effects of drought tolerance class and water treatment on the EMF communities, we generated a non-metric multidimensional scaling (NMDS) ordination (Pc-ord 5; MjM Software, Gleneden Beach, OR, USA). We then ran a Permutational MANOVA (PERMANOVA) using Bray–Curtis similarity resemblance in Primer 6 (Primer-e Ltd, Ivybridge, UK) to assess the influence of DT and DI seed sources and watering treatments on EMF communities (Hypotheses 1 and 2). Both drought tolerance category and watering treatment were considered to be fixed variables. We then conducted an indicator species analysis of drought tolerance and water treatment to determine whether particular taxa contributed significantly to any community composition differences observed (Primer 6). We also conducted a Mantel test comparing seedling community composition and GPS position in the common garden to determine whether proximity of seedlings to one another influenced EMF community composition (Qiime 2; Caporaso et al., 2010). Species richness and a Shannon–Wiener diversity index were calculated with PC-ORD 5, and comparisons of community metrics among watering treatment and drought tolerance groups were assessed using a two-way analysis of variance in Spss (IBM SPSS v.20) (Hypothesis 3). Hyphal exploration type was evaluated using a MANOVA in Spss (IBM SPSS v.20). A two-way ANOVA was also used to analyze annual cumulative shoot growth in relation to watering treatment and drought tolerance category (DI and DT) (Hypothesis 4).


We morphologically typed 3019 EM root tips and generated 167 DNA sequences encompassing all four treatments. Total amplification success of extracted root tips was 97% and total sequencing success of amplified product was 91%. At least 25% of the EMF root tips from rare morphotypes were successfully sequenced. We observed a total of eight species of EMF in six genera in the communities of the 17 DI and 15 DT pinyon seedlings sampled in 2013 (Table 1). The taxa included two species of Geopora (Geopora pinyonensis; Geopora type C) and one species each of Rhizopogon, Tomentella, Amphimena, Suillus, Hebeloma and Astraeus. Four EMF species occurred in all experimental treatments – Geopora pinyonensis, Geopora type C, Rhizopogon and Tomentella. Seedling proximity in the garden was not significantly correlated with EMF community composition (= 0.03018, = 0.32).

Table 1. Ectomycorrhizal fungal species identified on Pinus edulis using internal transcribed spacer (ITS) sequences
GenBank accession numbera Species hypothesisc ID Fungal phylumd Hyphal exploration typee Query coverage (%)f Identity (%)g
MG593236 b SH026928.07FU Geopora pinyonensis A Short 90 98
MG593235 SH026932.07FU Geopora C A Short 86 99
MG593240 SH009917.07FU Tomentella sp. B Short 92 97
MG593238 SH032168.07FU Rhizopogon sp. B Long 96 98
MG593234 SH021907.07FU Amphinema sp. B Medium 96 92
MG593239 SH001728.07FU Suillus sp. B Long 94 97
MG593237 SH030703.07FU Hebeloma sp. B Medium 81 94
MG593233 SH010598.07FU Astraeus sp. B Long 98 97
  • a Accession numbers are from the current study.
  • b Bold type indicates that a species was observed in all experimental treatments.
  • c Species hypothesis refers to a portion of the UNITE database generated by clustering sequences at a 97–99% similarity threshold for more reliable identification and comparison across studies, as described in Kõljalg et al. (2013).
  • d Indicates Ascomycota (A) or Basidiomycota (B).
  • e Long, medium or short hyphal exploration type based on our observational measurements: Agerer (2006) and Tedersoo et al. (2012).
  • f Query coverage indicates the percentage of the query sequence that overlaps the reference sequence.
  • g Identity (%) indicates the similarity of the query sequence and the reference sequence across the length of the coverage area.

Hypothesis 1

Drought-tolerant seedlings will recruit a different EMF community than drought-intolerant seedlings. In support of Hypothesis 1, seedlings of DT and DI mothers differed in their EMF communities (pseudo F1,27 = 4.423, = 0.025; Fig. 1). Paired comparisons showed that DI and DT seedlings differed in EMF community composition regardless of watering treatment (high water treatment, DI vs DT, = 1.776, = 0.053; low water treatment, DI vs DT, = 2.141, = 0.028; Figs 1, 2). DT seedlings across both watering treatments associated with a largely Ascomycete community (mean DT low water 51.3% (0.18 SE); mean DT high water 76.7% (0.10 SE)), containing two species of Geopora. By contrast, DI seedlings varied in their communities depending on low water (mean 83.7% (0.12 SE) ascomycetes) and high water (mean 34.6% (0.17 SE) ascomycetes) treatments. Tomentella sp. dominated in DI seedlings within the high watering treatment, whereas G. pinyonensis dominated in the low watering treatment. Geopora C was also found in high abundance in DT seedlings of both watering treatments and was a significant indicator of drought tolerance (indicator value = 65.8, = 0.0004).

Details are in the caption following the image
Mean relative abundance of ectomycorrhizal fungal communities in drought-tolerant (DT) and drought-intolerant (DI) Pinus edulis seedlings with high and low water treatments. Blue bars indicate members of the Ascomycota, whereas all other colors indicate members of the Basidiomycota.

Hypothesis 2

The EMF communities of seedlings from DI maternal trees will vary depending on water availability, whereas the EMF communities of seedlings from DT maternal trees will not. In support of Hypothesis 2, variation in watering regime altered the EMF community of seedlings of DI trees, but not seedlings of DT trees. Watering treatment alone did not affect seedling fungal communities (pseudo-F1,27 = 0.469, = 0.419), as DT seedlings retained the same community in high and low water treatments (Figs 1, 2). However, there was a significant watering treatment by maternal lineage interaction (pseudo-F1,27 = 3.25, = 0.041). DI seedlings receiving high water had different communities than those receiving low water, but DT seedlings did not (paired comparisons: DI high water vs DI low water, = 2.030, = 0.044; DT high water vs DT low water, = 0.057, = 0.777).

Hypothesis 3

The species richness, diversity and hyphal exploration of EMF communities will be greater in seedlings receiving higher supplemental moisture regardless of their drought tolerance category. Our third hypothesis was not supported for any response variable. First, seedlings receiving high vs low water had similar species richness (F1,29 = 0.049, = 0.826; Table 2). Richness was also similar between DT and DI seedlings (F1,29 = 1.556, = 0.223; Table 2) and there was no significant interaction between drought tolerance group and watering treatment (F1,29 = 0.186, = 0.670). Second, although DT seedlings had 1.7 times the Shannon–Wiener diversity of DI seedlings (F1,29 = 4.486, = 0.044; Table 2), diversity did not vary between watering treatments (F1,29 = 0.110, = 0.743). In addition, there was no significant interaction between drought tolerance group and watering treatment (F1,29 = 1.598, = 0.217). Third, the EMF communities in all treatment groups, including high water treatments, were dominated by taxa with short hyphal exploration types, with few emanating hyphae and no rhizomorphs (Table 1; Fig. 3). Hyphal exploration type did not differ significantly as a result of watering treatment (F1,25 = 0.310, = 0.818), drought tolerance group (F1,25 = 0.696, = 0.563) or the interaction between the two (F1,25 = 1.914, = 0.153).

Table 2. The mean (SE) species richness and diversity of Pinus edulis ectomycorrhizal fungal species in the four experimental treatments
Experimental treatment Mean species richness (S) Mean Shannon–Wiener diversity index (H)
Drought intolerant, low water 1.62 (0.26) 0.07 (0.03)
Drought intolerant, high water 1.57 (0.20) 0.24 (0.10)
Drought tolerant, low water 1.83 (0.30) 0.43 (0.14)
Drought tolerant, high water 1.88 (0.26) 0.33 (0.11)
Details are in the caption following the image
Mean relative abundance of short, medium and long hyphal exploration types in drought-tolerant (DT) and drought-intolerant (DI) Pinus edulis seedlings receiving high and low water treatments.

Hypothesis 4

DI seedlings will have greater growth than DT seedlings in the high water treatment, but not in the low water treatment. Measurements of annual cumulative shoot growth in DT and DI seedlings did not support our hypothesis. DT seedlings grew larger than DI seedlings in both the high and low water treatments (Fig. 4). Although DI seedlings grew more with high water than low water, DT seedlings outperformed them in both moisture treatments. Drought tolerance category significantly affected shoot growth (F1,32 = 4.702, = 0.039), with DT seedlings growing 1.5-fold more on average, than DI seedlings. Watering treatment was marginally significant (F1,32 = 3.062, = 0.091), but there was no significant drought tolerance by watering treatment interaction, suggesting that the shoot growth of both seedling types responded similarly to increased soil moisture (F1,32 = 0.163, = 0.690).

Details are in the caption following the image
Mean annual cumulative shoot growth for 2013 (dark orange) and 2014 (light orange) of 5–6-yr-old Pinus edulis seedlings sampled for ectomycorrhizal fungi across the four experimental treatments. Error bars denote ± 1SE of the total growth for both years combined.


EMF richness during drought

Our finding of eight EMF species in the common garden seems to be low relative to the species richness observed in other species of pine (Ding et al., 2011; Reverchon et al., 2012; Garcia et al., 2016); yet, it is consistent with previous research on both adult and seedling P. edulis sampled during drought conditions. In three measurements of adult P. edulis over 16 yr, Gehring et al. (2014) observed 19 species of EMF, but only eight of these occurred in all years, suggesting that a core community was retained over time. Many members of this core community were found on the seedlings we studied, including members of the genera Geopora, Rhizopogon and Suillus. Also, a significant drop in EMF species richness was observed on adult trees following the onset of drought in the region Gehring et al. (2014), which was also experienced by seedlings in the present study. Pinyon seedlings grown in a glasshouse in field soil had a combined species richness of nine EMF species, also similar to our common garden results (Gehring et al., 2017). Direct comparisons of EMF communities of glasshouse-grown P. edulis analyzed using the methods employed in this study, and also analyzed using next-generation DNA sequencing of pooled root tips with the Illumina platform, both resulted in nine species, with nearly complete overlap in the species present (A. M. Patterson et al., unpublished data). These results indicate that low diversity is typical of this study system, perhaps because P. edulis is the only host for EMF across much of its range (Gehring et al., 2016), and potentially also because of the drought in the region as noted above (Gehring et al., 2014).

Seedlings inherit EMF community structure and growth traits from maternal trees

Our findings that seedlings of DI and DT trees grown in a common garden differed substantially in EMF community composition, but less markedly in species richness or diversity, are consistent with other studies. Genotypes of P. angustifolia did not differ significantly in species richness, but tree genotype explained 13% of the variation in EMF community composition (Lamit et al., 2016). Similarly, fast- vs slow-growing phenotypes of Picea abies showed comparable species richness, but differed in the relative abundance of certain EMF taxa (Velmala et al., 2014). In P. edulis, the EMF communities of adult DT and DI trees differed significantly at each of three sampling periods over 16 yr (Gehring et al., 2014), and reciprocal transplant glasshouse experiments showed that DI and DT seedlings acquired the EMF communities of their seed source trees, even when provided with inoculum from the alternate tree type (DI or DT) (Gehring et al., 2017). The combination of glasshouse, common garden and long-term field studies of DI and DT trees and their seedlings provides strong evidence of the inheritance of EMF community composition. However, further studies are necessary to directly compare the communities of parents and offspring in the field, as our studies were temporally separated and our common garden studies included supplemental watering, whereas studies with adult trees did not.

Seedlings of DT maternal trees showed significantly greater shoot growth than seedlings of DI maternal trees in the low water treatment. However, given that mature DI trees grew better than DT trees during non-drought years (Gehring et al., 2017), we expected seedlings of DI mothers to outperform seedlings of DT mothers in the high water treatment. Instead, DT seedlings grew larger than DI seedlings in both water treatments. One explanation for this discrepancy could be that our high supplemental water treatment did not mimic conditions during pre-drought years. As mentioned previously, the low water holding capacity of the soil at the study site made it difficult to achieve the high range of soil moisture observed before drought at the study site (6–10% soil moisture; Swaty et al., 1998). Also, our sample size was small because we only measured the growth of seedlings for which EMF measurements were also made. More comprehensive studies of growth are underway on a larger sample of seedlings. Despite these limitations, our common garden studies indicate that key traits in adult trees, EMF community composition and shoot growth, have a strong inherited component.

Shifts from ascomycete to basidiomycete dominance with water stress

Differences among DT and DI seedlings in EMF communities seem to be driven by ascomycete vs basidiomycete dominance, as observed in adult P. edulis. Ascomycetes dominate DT adult trees and seedlings, and basidiomycetes dominate adult DI trees and seedlings, under higher water conditions (Sthultz et al., 2009a; Gehring et al., 2014). Clones of mature deciduous riparian trees, Populus angustifolia, varied in their ratio of ascomycetes to basidiomycetes (Lamit et al., 2016). As in our study, the dominant ascomycete observed in P. angustifolia was a member of the genus Geopora, whereas the dominant basidiomycetes differed among studies. Ascomycetes, such as Geopora, can dominate in areas of intense drought (Gordon & Gehring, 2011), in alkaline soils with high salt concentrations (Ishida et al., 2008) and in sites disturbed by fire (Fujimura et al., 2004), indicating that they are important symbionts in a variety of extreme conditions. In support of this hypothesis, Geopora dominated the roots of both DT and DI seedlings in the low water treatment.

Drought-intolerant seedlings receiving high supplemental water were dominated by basidiomycetes, a result observed in other plant species (Kennedy & Peay, 2007; Leski et al., 2010). In a seedling-based nursery study, the EMF communities of Pinus sylvestris seedlings differed among five maternal lines, with some lines dominated by ascomycete fungi and others by basidiomycete fungi (Leski et al., 2010). Pinus sylvestris seedling performance, including growth and tissue nutrient concentration, was higher in seedlings with basidiomycete-dominated communities than those with ascomycete-dominated communities (Leski et al., 2010). This pattern is opposite to our observation that greater growth was associated with ascomycete dominance in seedlings of DT trees. However, P. sylvestris inhabits a different niche than P. edulis, occurring in cooler, moister environmental conditions; basidiomycete dominance may be correlated with better performance in seedlings under high moisture levels. In support of this suggestion, basidiomycetes, such as Suillus and Rhizopogon, colonized more root tips under higher moisture than lower moisture conditions in P. muricata seedlings (Kennedy & Peay, 2007). Although the dominant basidiomycete in our study, Tomentella, was found across all watering treatments in both drought tolerance categories, it was four times more abundant in high water treatment DI seedlings than in low water treatment DI seedlings, demonstrating consistency with other studies.

Soil moisture, hyphal exploration types, drought tolerance and EMF communities

Our finding that the EMF communities of DI seedlings differed between moisture treatments is consistent with several other studies. A previous study of the distribution of mycorrhizal fungi found that EMF colonization intensity was strongly influenced by seasonal precipitation on a global scale, suggesting the inhibition of colonization during drier periods (Soudzilovskaia et al., 2015). Experimental data support this relationship; when deciduous trees previously unaffected by severe chronic drought were subjected to rainfall exclusion, drought-stressed and control trees differed significantly in the relative abundance of the EMF community (Shi et al., 2002; Richard et al., 2011). Furthermore, Jarvis et al. (2013) found that precipitation had a strong impact on EMF communities among adult P. sylvestris. Significant community shifts were observed in which one dominant species, Piloderma sphaerosporum, decreased in abundance with increased rainfall, whereas a second dominant species, Elaphomyces muricatus, showed the opposite pattern. A second study of P. sylvestris along an altitudinal gradient also found that soil moisture strongly influenced EMF community composition (Jarvis et al., 2015). Likewise, significant community shifts were observed in two species of oak (Quercus montana and Quercus palustris), along a soil moisture gradient (Cavender-Bares et al., 2009). Members of the dominant genus Tuber varied across all treatments while members of Thelephoraceae including two species of Tomentella were found in highest abundance in the medium water treatment and less in the high and low water treatments (Cavender-Bares et al., 2009). In our study, Tomentella was present in various abundances in all treatment groups, suggesting a wide niche. Taken together, these results suggest that precipitation can play a large role in determining EMF community composition, and certain EMF species respond better to well-watered vs drought conditions. Our study is an important addition to current research as it represents a direct manipulation of soil moisture in an environment in which drought is known to have major impacts on tree populations (e.g. Mueller et al., 2005; Sthultz et al., 2009a).

In contrast with our findings with DI seedlings, the EMF communities of DT seedlings were similar in high and low watering regimes, demonstrating an interaction between inherited traits of trees, the environment and ectomycorrhizas. A similar interaction has been observed in adult P. edulis, in which the community of EMF on DT trees remained remarkably constant with the onset of drought in the region, whereas the communities of DI seedlings shifted from basidiomycete to ascomycete dominance with drought (Sthultz et al., 2009a; Gehring et al., 2014). Likewise, the removal of competing arbuscular mycorrhizal shrubs caused a shift in the EMF communities of DI trees, but not DT trees (Gehring et al., 2014). Experimental work suggests that the ability of DT trees to perform well during drought may be largely the result of associations with a Geopora-dominated EMF community (Gehring et al., 2017). Although the communities of DI trees shifted towards a more Geopora-rich community with increasing stress, their performance was consistently lower than that of DT trees in arid conditions (this study, Gehring et al., 2014), suggesting that DT and DI P. edulis may respond differently to the same genus of EMF.

Short- to medium-distance hyphal exploration types dominated all treatment groups, regardless of whether the fungi were ascomycetes or basidiomycetes. Although not observed in our study system, several previous studies have shown that the short-distance exploration type ascomycete, Cenococcum geophilum, exhibits higher abundance in areas of greater water stress (Jany et al., 2002; Bakker et al., 2006). Long-distance exploration types have been associated with greater efficiency in the transport of soil resources (Cairney, 1992), as well as resilience to soil disturbance (Agerer, 2001), and therefore are theorized to provide higher and more reliable nutrient and water services to their hosts (Koide et al., 2013). However, long-distance exploration types also have greater carbon requirements of their host than short-distance ones (Saikkonen et al., 1999). Only 15% of the total abundance of species found in this study were considered to be medium- to long-distance exploration types. As a result of the drought-stressed conditions, it is likely that, even under the high water treatment, a greater abundance of short-distance exploration types would be more beneficial and less costly to the host seedlings.

In conclusion, using a common garden in a natural field setting, we found strong associations between inherited variation in EMF community composition and drought tolerance in P. edulis. These results, based on a common garden field trial embedded in the native habitat of P. edulis, provide clear evidence of the importance of host plant phenotypic variation, genetics and microbial communities to climate change responses by plants. As the distributions of many arid land species have shifted over the last 50 years in the southwest (Brusca et al., 2013) and will probably shift even more with projected changes in future climate, these complex interactions may strongly influence seedling survivorship and performance in a changing environment.


Funding was provided by National Science Foundation (NSF) grants DEB DEB0816675 and DBI-1126840, an AZ NASA Space Grant and a NAU Hooper Undergraduate Research Award. We thank P. Patterson for help in the glasshouse, the Gehring–Haubensak–Hofstetter laboratory group for project feedback, L. Andrews for laboratory assistance, G. Kovacs for watering seedlings, the Evolution of Plasticity Reading Group and three anonymous reviewers for helpful suggestions on the manuscript.

    Author contributions

    A.P. and C.G. designed the study and analyzed the data; A.P., C.G. and L.F-R. collected field samples and data, and performed the research; C.G., A.W. and T.W. established the common garden; A.P. wrote the manuscript; all authors contributed to revisions.