Plant genetics and interspecific competitive interactions determine ectomycorrhizal fungal community responses to climate change

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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/mec.12503 This article is protected by copyright. All rights reserved.

Received Date : 01-Apr-2013 Revised Date : 04-Jul-2013 Accepted Date : 27-Jul-2013 Article type : Original Article Plant genetics and interspecific competitive interactions determine ectomycorrhizal fungal

community responses to climate change

Catherine Gehring1, Dulce Flores-Rentería1,2, Christopher M. Sthultz1,3,

Tierra M. Leonard1, Lluvia Flores-Rentería1, Amy V. Whipple1, Thomas G. Whitham1

1Department of Biological Sciences and Merriam-Powell Center for Environmental Research,

Northern Arizona University, Flagstaff, AZ 86011-5640

2Current address: Museo Nacional de Ciencias Naturales-CSIC C Serrano, 115 dup 28006,

Madrid, España

3Current address: Department of Math, Science and Technology, University of Minnesota,

Crookston, MN 56716

Corresponding Author: Catherine Gehring, Department of Biological Sciences, Northern

Arizona University, Flagstaff, AZ, USA, fax number: (928) 523-7500, e-mail address:

[email protected]

Running title: Drought, competition and soil fungi

Key words: climate change, fungi, ecological genetics, species interactions

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This article is protected by copyright. All rights reserved.

Abstract

Although the importance of plant-associated microbes is increasingly recognized, little is known

about the biotic and abiotic factors that determine the composition of that microbiome. We

examined the influence of plant genetic variation, and two stressors, one biotic and one abiotic,

on the ectomycorrhizal (EM) fungal community of a dominant tree species, Pinus edulis. During

three periods across 16 years that varied in drought severity, we sampled the EM fungal

communities of a wild stand of P. edulis in which genetically-based resistance and susceptibility

to insect herbivory was linked with drought tolerance and the abundance of competing shrubs.

We found that the EM fungal communities of insect susceptible trees remained relatively

constant as climate dried, while those of insect resistant trees shifted significantly, providing

evidence of a genotype by environment interaction. Shrub removal altered the EM fungal

communities of insect resistant trees, but not insect susceptible trees, also a genotype by

environment interaction. The change in the EM fungal community of insect resistant trees

following shrub removal was associated with greater shoot growth, evidence of competitive

release. However, shrub removal had a seven-fold greater positive effect on the shoot growth of

insect susceptible trees than insect resistant trees when shrub density was taken into account.

Insect susceptible trees had higher growth than insect resistant trees, consistent with the

hypothesis that the EM fungi associated with susceptible trees were superior mutualists. These

complex, genetic-based interactions among species (tree-shrub-herbivore-fungus) argue that the

ultimate impacts of climate change are both ecological and evolutionary.

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Introduction

Recent methodological advances have allowed unparalleled exploration of the microbial

communities associated with macro-organisms. Studies of the plant microbiome are rapidly

advancing our understanding of the importance of this “second plant genome” to plant health,

particularly in crops and model species such as Arabidopsis (Berendsen et al. 2012; Hirsch &

Mauchline 2012). Likewise, more limited data demonstrates that microbes can mediate host

responses to global changes including biological invasions (Inderjit & van der Putten 2010) and

climate change (Lau & Lennon 2012). Plants significantly influence the communities of

microbes with which they associate, leading to feedbacks that influence plant establishment,

diversity and successional dynamics (Kulmatiski et al. 2008; van der Putten et al. 2013).

Understanding the forces that structure microbial communities is thus critical to identifying

plant-microbe feedbacks and improving our predictions of the role of the plant microbiome in

ecosystems.

A key issue emerging in studies of plant-associated microbes is the importance of

intrinsic properties of organisms, such as species or genotype, relative to external environmental

factors in shaping microbial community composition and diversity (Berendsen et al. 2012).

Abiotic environmental factors such as nutrient concentration, temperature and moisture have

well documented effects on the community composition of root-associated microbes

(Heinemeyer et al. 2004; Murray et al. 2010; Lundberg et al. 2012). Herbivores, plant neighbors,

and other microbes can have similarly strong effects on plant-associated fungi (Murray et al.

2010; Berendsen et al. 2012). Likewise, plant species and genotype also influence the

composition of their associated bacteria and fungi (Ishida et al. 2007; Aira et al. 2010; Leski et

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al. 2010). While these studies provide important insights, they frequently examine intrinsic and

environmental properties in isolation from one another, limiting our ability to predict the

importance of plant-microbe interactions to issues such as climate change (but see Lau & Lennon

2012; deVries et al. 2012). Studies in which intrinsic properties such as plant genotype and their

numerous linked traits (e.g., Todesco et al. 2010) vary along with abiotic and biotic

environmental variables are rare, but are needed to establish the relative influence of plant

genotype versus environment on microbial communities, and to describe their interactions.

We assess environmental and plant genetic contributions to the community composition

of the EM fungi associated with pinyon pine (Pinus edulis Engelm) growing in woodlands

experiencing increasingly arid conditions (Seager et al. 2007). We focus on EM fungi because

they contribute significantly to the soil resource acquisition, growth and stress tolerance of their

host plants (Hoeksema et al. 2010), and their abundance and species composition can be highly

responsive to environmental variables (Lilleskov et al. 2002; Richard et al. 2011). We focus on

drought because of its importance to trees world-wide (Choat et al. 2012), to diverse forest

species in the American West (van Mantgem et al. 2009) and particularly to P. edulis, which has

suffered massive drought-related mortality (Mueller et al. 2005; Garrity et al. 2013). We assess

interspecific competition as a biotic environmental variable because of its importance in

structuring plant communities (Coomes & Grubb 2000), and the likelihood that dwindling or

more temporally variable precipitation will alter competitive interactions among plants (Chesson

et al. 2004). In addition, variation in the communities of symbiotic fungi may play a central role

in plant competition if they differentially access soil resources and thereby influence resource

partitioning among plants (Bever et al. 2010, Engelmoer et al., THIS ISSUE). Limited field

studies show that plant-plant interactions can influence and be influenced by mycorrhizal fungi

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(McHugh & Gehring 2006; Becklin et al. 2012), suggesting that these fungi may play a key role

in belowground competition.

We incorporated intraspecific genetic variation into our study by focusing on co-

occurring P. edulis that vary in their genetically-based resistance and susceptibility to insect

herbivory by the stem-boring moth (Dioryctria albovittella)(Mopper et al. 1991). A central and

realistic complexity of this system is that other plant traits are directly and indirectly linked to

insect resistance and susceptibility, which cascades to affect tree architecture and associated

microclimate, the density of competing shrubs, drought tolerance and EM fungal community

composition (Fig. 1). For example, insect susceptible mature P. edulis suffered chronic herbivory

while resistant trees did not , resulting in the distinct upright, open growth forms of resistant

trees and the shrub-like, dense growth forms of susceptible trees (Whitham & Mopper 1985).

Experiments demonstrated that the shrub phenotype was due to herbivory as the removal of

moths from insect susceptible trees resulted in a gradual change in architecture from closed

shrubs to open canopy trees (Fig. 1). These distinct architectural phenotypes grow intermixed

within a population where the dense canopy of susceptible trees and the open canopy of insect

resistant trees have distinct microclimates (Classen et al. 2005). The dense shading of insect

susceptible trees is associated with far fewer shrub competitors relative to resistant trees (Fig. 1).

Pinyons also vary in drought tolerance; insect resistant trees suffered 68% mortality, while only

21% of insect susceptible trees died during record drought (Fig. 1). These patterns were

confirmed experimentally with seedlings grown from insect resistant and insect susceptible seeds

(Sthultz et al. 2009a). Mutualisms with mycorrhizal fungi were also affected; censuses

conducted ten years apart showed that insect resistant and insect susceptible trees differed in EM

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fungal community composition and insect removal experiments demonstrated that these

differences were due to plant genetics rather than herbivory (Sthultz et al. 2009b)(Fig 1).

Because so many ecologically important traits are linked to genetically-based insect

resistance and susceptibility, including drought tolerance and competitor density, we combined a

three year competition experiment of P. edulis with shrubs and 16 year long-term contrasts of

insect resistant and susceptible trees as climate dried to understand how these two factors

influenced an important fungal mutualist community. Combining such multiple effects is rare,

but important to understand the ultimate consequences of drought on the fate of a dominant tree

that characterizes much of the American southwest. Furthermore, because findings based on

single factors can differ from those found when multiple factors are included in a study and long-

term studies often reverse the findings of short-term studies (Bailey & Whitham 2007), our

combination of genetics-based drivers of the EM fungal community and P. edulis performance

represents a realistic and novel integration of interacting factors.

We utilize this study system to test two hypotheses: 1. Ongoing drought is associated

with changes in the EM fungal communities of insect resistant trees but not insect susceptible

trees resulting in a convergence of communities in response to severe climate conditions. While

our previous studies spanned pre-drought to peak drought conditions over a ten year period

(Sthultz et al. 2009), here we expand those studies to include another six years of post-peak

drought conditions in which the fundamental relationships may have changed. Our study is novel

because previous work on the temporal dynamics of mycorrhizal fungal communities has

focused on relatively short (seasonal or annual time scales) or comparatively long (successional)

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time scales with little emphasis on intermediate time scales over which the effects of recent

climate change in the western US have been observed (Nara et al. 2003; Richard et al. 2011). 2.

Removal of co-occurring shrubs will have a greater net effect on the EM fungal community and

growth of insect resistant trees than insect susceptible trees. Our three year competitor removal

study was initiated during drought conditions at an adjacent study site in the same environment,

allowing us to add a realistic layer of biocomplexity into our understanding of the factors that

shape the EM fungal community, which in turn can feed back to affect plant performance. Tests

of these hypotheses are of general interest as plant genetics-based interactions among a few

players have the potential to affect the ecology and evolution of a larger community and

ultimately help us understand the broader implications of climate change.

Materials and Methods

Study sites

We studied insect resistant and insect susceptible P. edulis and their associated fungi at a long-

term research site near Sunset Crater National Monument (35° 23′ 25″N, 111° 25′ 40.8″ W,

2050m elevation, designated site S) and at a lower elevation site ~5km away (35° 23′26.9″ N,

111° 23′ 23.3″W, 1886m elevation, designated site H). This region of Arizona is semi-arid; site S

averages ~ 320 mm of precipitation yr−1 (Selmants & Hart 2010). Climate data is not available

for site H, but the vegetation and soils at both sites are similar. Soils at both sites are dominated

by 1200-yr-old cinder deposits that include basaltic ash, cinders and lava flows and belong in the

US Department of Agriculture Soil Taxonomic Sub-Group of Typic Ustorthents (Selmants &

Hart 2010). These soils have low water holding capacity and low levels of several nutrients

(Sthultz et al. 2009a). Dominant tree species at both sites are P. edulis and one-seed juniper

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(Juniperus monosperma Engelm). Several species of shrub are also present with Apache plume

(Fallugia paradoxa D. Don Endl. ex Torr.) being the most abundant (McHugh & Gehring 2006).

At low elevation sites like S and H, shrubs facilitate the growth and survival of juvenile pinyons

(Sthultz et al. 2007), but these interactions shift to competition as trees mature (McHugh &

Gehring 2006). Pinus edulis is the only host for EM fungi at these sites, with the exception of the

occasional Ponderosa pine (Pinus ponderosa Douglas ex C. Lawson)(Haskins & Gehring 2005).

Hypothesis 1: Ongoing drought is associated with changes in the EM fungal communities of

insect resistant trees but not insect susceptible trees

We tested our first hypothesis by comparing the EM fungal communities of insect

resistant and insect susceptible pinyons sampled during three years of a 16 year period

encompassing wet and dry years. We included two groups of insect susceptible pinyons: 1) a

group of trees exposed to natural levels of herbivory, and 2) a group of trees in which insect

herbivory has been experimentally reduced by an annual application of a systemic insecticide

beginning in 1983 (Whitham & Mopper 1985). Susceptible insect removal trees allowed us to

determine the potential effect of drought on the EM communities of insect susceptible trees,

independent of herbivory. The first year sampled, 1994, occurred at the end of a period of wet

years, while the second two years, 2004 and 2010, occurred during a period of ongoing drought.

Although the climate of the southwestern US is characterized by fluctuations in annual

precipitation, the 14 year period from 1979-1994 was wet, on average, with a mean water year

(Oct 1-Sept 30) Palmer Drought Severity Index (PDSI) of 2.09, and only two years with a

negative PDSI (NOAA National Climatic Data Center, PDSI of Arizona Division 2;

http://www.ncdc.noaa.gov/). PDSI ranges from 6.0 to −6.0, with values between 1 and −1

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indicating average conditions and more negative values indicating drought. In contrast, the

fourteen year period between 1995 and 2010 was dry, with an average PDSI of -0.85 and seven

years of negative PDSI values including four below -3.0. These persistently dry conditions

resulted in extensive mortality of P. edulis in northern Arizona (Mueller et al. 2005). Sthultz et

al. (2009b) previously compared the EM fungal communities of resistant, susceptible and

susceptible moth removal trees in 1994 and 2004. Over this 10 year period of significant climate

drying, they observed changes in the EM community composition of insect resistant, but not

insect susceptible or insect removal trees. The 2004 sampling occurred shortly after the 2002

extreme drought (growing season PDSI of -5.484) and this climate event may have been

associated with a short-term change in the communities of resistant trees. Our additional

sampling as drought continued helped us to address this possibility. We predicted that ongoing

drought would be associated with further changes in the EM fungal communities of insect

resistant trees, but that the communities of insect susceptible and susceptible insect removal trees

would remain the same.

We used the data of Sthultz et al. (2009b) for 1994 and 2004 and followed their methods

for 2010 sample collection and analysis. The molecular methods used to assess fungal

communities advanced between 1994 and 2004 and Sthultz et al. (2009b) detailed the differences

in methods used during those two years. For the 2010 samples, we followed the 2004 methods

and describe them briefly here, along with the key differences between the 1994 and 2004

methods. We sampled the same long-term study trees in which insect resistant versus insect

susceptible status was determined using annual censuses of D. albovittella herbivory conducted

since 1983 (Whitham & Mopper 1985). The study trees were ~75-80 years old in 2010,

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reproductively mature at least since their selection for study in the 1980’s, and extended over 0.8

km2. The EM fungal communities of mature trees ranging in age from 50-160 yrs old sampled at

the same time were similar to one another, so it is unlikely that tree age influenced EM fungal

community composition. There was extensive continuity in sampling and morphotyping as

Gehring was involved in all three sampling periods 16 years apart. We collected fine roots

(<2mm diameter) from the north side of each tree at a depth of 0-30cm. We classified between

75-100 living EM fungal root tips per tree based on morphology and stored the EM root tips in

1.5ml microcentrifuge tubes at -20 ºC until molecular analysis. We sampled a total of 25 trees, 8-

9 per treatment, with the same trees being sampled during multiple years. We accounted for this

lack of independence in our statistical analyses.

We extracted the DNA from a minimum of two- three root tips of each morphotype from

each tree using DNeasy Kits (Qiagen, Valencia, CA, USA) in 2004 and 2010. We used the mini-

prep method of Gardes & Bruns (1993) to extract DNA from the 1994 samples. DNA extraction

and amplification success was similar for samples collected during all years, averaging >90%.

We amplified the internal transcribed spacer (ITS) region of the fungal genome, located between

the 18S and 28S rRNA, using PCR (polymerase chain reaction) with the ITS1F and ITS4 primer

pair (Gardes & Bruns 1993). We designed an additional set of primers for our initial screening in

2010 due to early problems with amplification, LFR170F 5’-

CATTTTGAGCTGCATTCCCAAACAACTC-3’ and LFR930R 5’-

CGCATCGATGAAGAACGCAG-3’. However, we sequenced multiple representatives of all

morphotypes with both sets of primers prior to final identification. Morphotypes were

characterized by a single species of EM fungus, except for the smooth, red-brown morphotype

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that characterizes the genus Geopora. Multiple closely related species of Geopora are found on

P. edulis (Gordon & Gehring 2011); additional sequencing was done to estimate the relative

abundance of the Geopora species if multiple species were found in the initial screening. Sthultz

et al. (2009b) conducted PCR-RFLP analysis prior to sequencing but we omitted this step,

electing to sequence representatives of all morphotypes from all trees instead. We conducted

DNA sequencing using an ABI 3730 Genetic Analyzer at NAU. We assembled forward and

reverse DNA sequences in BioEdit version 7.0.5.3 (Hall 1999) to create a consensus sequence

that was used in a BLASTn search on the NCBI and UNITE websites (Altschul et al. 1990;

Abarenkov et al. 2010). We used percentage query coverage, percentage maximum identity, and

bit score data to identify the closest match of our fungi to those in GenBank. We re-sequenced

poor quality samples and only used high quality sequences for BLASTn analysis, ranging from

500 to 700 bp in length with query coverage ranging from 94% to 100%. We considered

sequence similarity of >98% to published sequences indicative of species level identity and 94–

97% indicative of genus level identity. The names of some species reported in Sthultz et al.

(2009b) were modified based on new data (Table 1). Some of these changes altered estimates of

species richness, so values reported here do not exactly match those of Sthultz et al. (2009b) for

1994 and 2004. We cross-referenced nomenclature and phylogenetic placements with Index

Fungorum (http://www.indexfungorum.org, accessed March 8, 2013.

Variation in the prevalence of the two major lineages of EM fungi, the Ascomycota and

Basidiomycota, was observed between insect resistant and insect susceptible P. edulis by Sthultz

et al. (2009b), so we compared the percentage of EM fungi belonging to the division

Ascomycota in the three groups of trees by calculating the percentage of the community of each

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tree that consisted of members of the Ascomycota. The importance of the Ascomycota as EM

fungi is increasingly recognized (Tedersoo et al. 2006), but we still understand little about how

they differ functionally from the Basidiomycota.

Hypothesis 2: Removal of co-occurring shrubs will have a greater net effect on the EM

fungal community and growth of insect resistant trees than insect susceptible trees

We tested this hypothesis at study site H using a competitor removal experiment aimed at

the dominant shrub and likely competitor in the system, Fallugia paradoxa. In August 2007, we

identified a set of mature trees, 24 insect resistant and 24 insect susceptible, within a 1km2 area

based on the architectural differences that result from chronic insect herbivory. We corroborated

that these differences in architecture were associated with insect herbivory by measuring shoot

mortality due to Dioryctria albovittella as in Whitham & Mopper (1985). The trees were similar

in size as the trees used in Hypothesis 1. We measured the abundance of F. paradoxa shrubs

associated with each tree by counting the number of shrub stems within the rooting zone of the

tree, an area estimated by root system exposure studies to be half of a crown diameter beyond the

dripline. Although other species of shrub were found at the site, only F. paradoxa grew in

association with the study trees. We collected root samples from a subset of the trees (n= 6 per

group) to assess EM community composition prior to shrub removal using the methods described

for Hypothesis 1. We also obtained pre-treatment data (2006 and 2007) on growth by measuring

the length of ten randomly selected shoots per tree on ten trees per group. The presence of bud

scale scars allowed us to distinguish 2006 and 2007 growth, but data for the two years was

averaged to obtain a single pre-treatment measurement.

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In early fall of 2007, we removed shrubs from the rooting zone of 12 of the insect

resistant trees and 12 of the insect susceptible trees. We assigned trees to treatments (shrubs

removed or shrubs intact) so that shrub abundance was the same for the removed and intact

groups within a tree type (insect resistant or insect susceptible). We pulled out the shallowly-

rooted smaller shrubs while larger shrubs were sawed off at the base. Fallugia paradoxa can

resprout from cut stumps, so we cut sprouts periodically during the following six months. We

made no further measurements until two years later to allow trees to recover from the

disturbance of shrub removal (Díaz et al. 2003) and respond to the treatment. In August of 2009,

we collected roots from the study trees and assessed EM fungal communities using the methods

described for hypothesis 1. In August of 2010, we assessed the post-treatment growth of ten trees

in each of the four treatment groups by measuring the shoot length of ten shoots per tree. We

assessed reproduction by counting the number of mature cones per tree.

Data analysis

We visualized data on the community composition of EM fungi using non-metric

multidimensional scaling (NMS) ordinations with a Bray-Curtis distance measure in PC-ORD

5.10 (McCune & Mefford 2006). For hypothesis 1, we tested the influence of insect resistance

category (insect resistant, insect susceptible and susceptible insect removal) and year (1994,

2004 and 2010) on EM fungal community composition with a permutation-based nonparametric

multivariate analysis of variance (PerMANOVA) using relative abundance data (the percentage

of a given EM fungal species relative to all EM fungal root tips in a sample) in PRIMER version

6.1 (Clark & Gorley 2006). We sampled the same trees each year and accounted for this repeated

sampling by including tree identity as a factor nested within insect resistance category. We

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analyzed the main effects of insect resistance class and year as a two-way factorial, followed by

pair-wise tests of insect resistance or year combinations when main effects were significant (P <

0.05). If significant differences were detected, we conducted an indicator species analysis in PC-

ORD to determine if particular species contributed more to community differences. We

considered a species to be a significant indicator if it had an indicator value >25 and a P value

<0.05. We analyzed data on the abundance of ascomycete fungi across insect resistance classes

and years with a two-way ANOVA in IBM SPSS version 20 followed by Tukey’s least

significant difference tests to locate differences.

We conducted similar analyses for hypothesis 2. For pre-treatment data, we compared

growth and EM fungal communities within a tree type (insect resistant and insect susceptible)

only as our goal was to verify that the trees assigned to shrub removal and shrubs intact

treatments within a tree type did not differ prior to the experiment. We used t-tests for shoot

growth and a multi-response permutation procedure in PC-ORD for EM fungal communities.

For the post–treatment comparisons, we constructed NMS ordinations in PC-ORD and

used PerMANOVA to determine if EM community composition differed among treatment

groups after shrub removal. We considered shrub presence (present or removed) and insect

resistance class (insect resistant or insect susceptible) fixed effects. We conducted indicator

species analysis in PC-ORD following a significant PerMANOVA as described above. We

compared EM fungal species richness, stem growth and cone production of insect resistant and

insect susceptible trees with shrubs intact and shrubs removed using a two-way ANOVA in

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SPSS. We used regression analysis in SPSS to identify associations between EM fungal

communities and growth.

Results

Hypothesis 1: Ongoing drought is associated with changes in the EM fungal communities of

insect resistant trees but not insect susceptible trees

We observed 19 species of EM fungi across the three sampling periods, with eight

species appearing in all years (Table 1). Members of the genera Geopora and Rhizopogon were

the most common with species in the genera Tricholoma, Lactarius, Russula, Suillus and Inocybe

occurring less frequently (Table 1). Two species were observed only on a single tree (Clavulina

sp. and Geopora sp. 5) and three others were observed rarely, and in only one year, 1994. Two of

these latter species were not identified beyond division, and one could not be identified beyond

“unknown EM fungus” because of poor quality DNA sequences. All species were included in the

statistical analyses, but the unknown EM fungus was not included in Table 1. We sampled the

same trees for EM fungal communities each year, but tree identity did not explain a significant

portion of the variation in community composition (F22,75 = 6.492, P = 0.077) and there was no

tree identity by year interaction (F44,75 = 5.801, P = 0.086).

Our hypothesis that ongoing drought would be associated with changes in the EM fungal

species composition of insect resistant trees, but not insect susceptible trees, was supported. The

EM fungal communities of insect resistant trees changed substantially, becoming more like the

communities of insect susceptible trees over time as seen in Figure 2 and indicated by the

significant year by insect resistance interaction (F4,75 = 2.36, P = 0.006). The main effects were

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also significant (F2,75 = 7.086, P = 0.001 for year and F2,75 = 6.971, P = 0.05 for insect resistant

category), but year clearly influenced the magnitude of the difference between insect resistant

and susceptible trees. As predicted, communities differed in all years for insect resistant trees (t =

1.882, P = 0.031 for 1994 versus 2004; t = 3.605, P = 0.003 for 1994 versus 2010, and t = 1.853,

P = 0.05 for 2004 versus 2010)(Fig. 2).

The changes in the EM fungal communities of insect resistant trees with time were

associated with the loss of basidiomycete fungi including Rhizopogon roseolus (Indicator Value

(IV) = 42.3, P = 0.006) and Tricholoma terreum (IV = 32.9, P = 0.003), both significant

indicators of 1994 (Fig. 2). The relative abundance of members of the division Ascomycota

increased with time, particularly in insect resistant trees as indicated by a highly significant

insect resistance by time interaction (F4,66 = 8.76, P < 0.001). Although the main effects were

significant (F2,66 = 13.65, P < 0.001 for year, F2,66 = 47.20, P < 0.0001 for insect resistance

category), the differences among insect resistant trees and the two groups of insect susceptible

trees decreased with time (Fig. 3). Tukey’s tests showed that ascomycete abundance on insect

resistant trees differed from that of both insect susceptible and susceptible insect removal trees

while those of the latter groups were similar. Changes with time were substantial (56% increase)

between 1994 and 2004 but not significant between 2004 and 2010 (P > 0.05).

In contrast to insect resistant trees, the EM communities of both groups of insect

susceptible of trees only partially supported our hypothesis. Insect susceptible and susceptible

insect removal trees had similar communities during the mesic (1994) and first drought (2004)

sampling periods (t = 1.5618, P = 0.096 for susceptible trees, t = 0.982, P = 0.367 for susceptible

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insect removal trees), but their communities changed slightly but significantly from previous

years in 2010 (for susceptible trees, t = 2.017, P = 0.021 for 1994 versus 2010 and t = 1.928, P =

0.037 for 2004 versus 2010; for susceptible moth removal trees, t = 2.231, P = 0.005 for 1994

versus 2010 and t = 2.353 , P = 0.001, for 2004 versus 2010)(Fig. 2). Susceptible and susceptible

moth removal communities were dominated by the genus Geopora in all years, with indicator

species analysis indicating that the difference between the two earlier years and 2010 resulted

from shifts in the relative abundance of Geopora spp. 2, 3 and 4.

On average, EM fungal species richness was 20% higher in 1994 than 2004 but it did not

drop further from 2004 to 2010. Mean species richness per tree for insect susceptible trees + 1

SE was 3.44 + 0.580 for 1994, 2.63 + 0.183 for 2004, and 3.12 + 0.350 for 2010, while the

values for susceptible insect removal trees were 3.60 + 0.209 for 1994, 3.25 + 0.365 for 2004

and 3.44 + 0.293 for 2010. Species richness was similar for insect resistant trees with means of

4.13 + 0.398 for 1994, 3.22 + 0.323 for 2004 and 3.48 + 0.218 for 2010. Species richness varied

with time F2,66 = 3.952, P = 0.024, with post-hoc tests showing that 1994 differed from 2004 and

2010, which did not differ from one another. There was no significant difference in species

richness among the three groups of trees (F2,66 = 2.705, P = 0.075) and no significant resistance

by time interaction (F4,66 = 0.628, P = 0.644).

Hypothesis 2: Removal of co-occurring shrubs will have a greater net effect on the EM

fungal community and growth of insect resistant trees than insect susceptible trees.

Herbivore damage by moths was ten-fold lower on insect resistant trees than insect

susceptible trees, corroborating our designations based on architecture (mean shoot mortality +

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1SE = 4.4 + 0.75 for insect resistant trees and 47.0 + 8.26 for insect susceptible trees, t = -6.99, P

< 0.0001). Also, associated with their dramatic differences in architecture, insect resistant and

insect susceptible pinyons differed nearly five-fold in shrub abundance (mean rooting zone shrub

abundance +1 SE = 63.6 + 4.1 for insect resistant trees and 13.9 + 2.2 for insect susceptible trees,

t = 15.69, P < 0.0001). Combined shoot growth during the pre-treatment years of 2006 and 2007

was similar for insect resistant trees designated as shrubs intact and those designated as shrub

removals (mean + 1SE for control = 24.9 + 0.91 and mean = 24.4 + 1.44 for removal, P > 0.80).

Similar patterns were observed for insect susceptible trees (mean + SE for shrubs intact = 29.7 +

2.00 and mean = 31.1 + 2.76 for shrubs removed, P > 0.70). Likewise EM fungal community

composition was similar for insect resistant trees designated as shrub removals versus shrubs

intact (A = 0.0008, P = 0.366) and for susceptible trees designated as shrub removals versus

shrubs intact (A = 0.00013, P = 0.357).

Contrary to the prediction of our second hypothesis, competitive release following shrub

removal was not greater for insect resistant than insect susceptible P. edulis. The shoot growth of

insect susceptible trees exceeded that of insect resistant trees, irrespective of the shrub removal

treatment (F1,36 = 16.727, P < 0.001 for insect resistant versus insect susceptible; F1,36 = 0.679, P

= 0.415 for the insect resistance by shrub removal interaction; Fig. 4). Despite the five-fold

higher abundance of shrubs in the rooting zone of insect resistant trees than insect susceptible

trees, removal of shrubs increased the shoot growth of susceptible trees by 18%, a similar value

to the competitive release seen in resistant trees whose growth was 14% greater than controls

(shrub treatment F1,36 = 5.146, P = 0.029). On average, shoot growth increased 0.2% for each

shrub removed from insect resistant trees and 1.4% for each shrub removed from insect

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susceptible trees. These results indicate that the higher number of shrubs observed in association

with insect resistant trees is only a partial explanation for the overall lower growth of these trees

relative to insect susceptible trees.

Two years of shrub removal did not stimulate female reproduction (F1,36 = 0.018, P =

0.893), but cone production was lower in insect susceptible trees due to the preference of moth

herbivores for cone-producing terminal shoots (F1,36 = 25.79, P < 0.001)(Whitham & Mopper

1985) and there was no significant interaction (F1,36 = 0.037, P = 0.848). Mean + SE cone

production for insect resistant/shrubs intact trees was 90.7 + 18.6 cones per tree while that of

insect resistant/shrub removal trees was 97.0 + 24.9. Insect susceptible/shrubs intact trees

produced a mean of 4.3 + 3.09 cones per tree, while insect susceptible/shrub removal trees

produced a mean of 3.2 + 2.26 cones per tree.

In support of our second hypothesis, shrub removal altered the EM fungal communities

of insect resistant, but not insect susceptible trees. Our analyses showed a significant shrub

removal by insect resistance category interaction (F4,44 = 2.764, P = 0.027) and the ordination

shows a separation in the communities of insect resistant trees with shrubs intact versus shrubs

removed while insect susceptible tree communities with shrubs intact or shrubs removed overlap

(Fig. 5). Shrub removal was associated with a shift in EM fungal communities of insect resistant

trees towards those of insect susceptible trees, though they remained distinct (F1,44 = 3.479, P =

0.012)(Fig. 5). There was no significant effect of shrub removal on EM fungal communities

overall (F1,44 = 1.957, P = 0.108). EM fungal species richness averaged between 2.1 to 2.5

species per tree and was not affected by insect resistance category, shrub removal or their

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interaction (F1,44 = 1.43, P = 0.238 for insect resistance category, F1,44 = 0.029, P = 0.865 for

shrub removal, and F4,44 = 0.729, P = 0.398 for their interaction). We observed ten species of EM

fungi; only Tylospora sp. was missing on trees sampled for hypothesis 1 (Table 1).

Rhizopogon roseolus was an indicator species that differentiated both insect

resistant/shrubs removed and insect resistant/shrubs intact EM fungal communities (IV = 50.9, P

= 0.003), and insect resistant and insect susceptible communities (IV = 48.8, P = 0.004). This

species declined significantly with shrub removal; it was found on all insect resistant/shrubs

intact trees, but only on two of the insect resistant/shrub removal trees (Table 1). The sharp

reduction in R. roseolus with shrub removal in insect resistant trees occurred in concert with a

nearly 20% increase in the abundance of members of the genus Geopora and likely contributed

to the greater growth of insect resistant trees following shrub removal. The relative abundance of

Geopora was significantly positively associated with the shoot growth of insect resistant trees (r2

= 0.465, F1,19 = 13.93, P = 0.0018, slope = 0.15), while the relative abundance of R. roseolus was

significantly negatively associated with shoot growth (r2 = 0.238, F1,19 = 4.99, P = 0.039, slope =

-1.27). The EM fungal communities of insect susceptible trees were dominated by Geopora,

regardless of shrub treatment, so a shift in the abundance of this genus cannot explain the strong

positive growth response of insect susceptible trees to shrub removal.

Discussion

Plant genetics influences EM fungal communities and their temporal stability

Our finding that insect susceptible and susceptible insect removal trees had similar EM

fungal communities even after 27 years of experimental herbivore removal supports earlier

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shorter-term studies suggesting that plant genetics, not herbivory, determines the EM community

of insect susceptible P. edulis (Sthultz et al. 2009b). In addition, the stability of the EM fungal

community was also influenced by plant genotype. The EM communities of insect resistant trees

changed substantially over time while the communities of both groups of susceptible trees

changed very little. Plant genotype also influenced the EM fungal community composition of

Picea alba and Pinus sylvestris (Leski et al. 2010; Velmala et al. 2013), as well as the microbial

communities of some crops (Aira et al. 2010; Weinert et al. 2012), model animal species, and

humans (Spor et al. 2011). Consistent with our studies across time, some Populus angustifolia

genotypes had distinct, temporally stable communities of arthropods, whereas the communities

of other genotypes fluctuated (Keith et al. 2010). Although genetics-based, long-term

experiments on communities like these are rare, they suggest that plant genetic variation

influences not only community composition, but also community stability.

Drought and genetic variation interact to influence EM fungal community stability

Although host plant genetics strongly influenced EM fungal community composition,

environmental variation also appeared important, particularly for insect resistant trees,

suggesting a genotype by environment interaction. Changes in the EM fungal communities of

insect resistant trees towards dominance by ascomycete fungi were associated with ongoing

drought in the region, a result consistent with previous studies of P. edulis. Shifts from

basidiomycete dominance to ascomycete dominance were associated with drought at the stand

scale (Gordon & Gehring 2011), and surviving trees at high mortality sites were colonized by a

low diversity, ascomycete-dominated EM fungal community (Swaty et al. 2004). Importantly,

the communities of insect resistant trees were converging towards those of susceptible trees, yet

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remained distinct. Insect susceptible trees with stable EM fungal communities survived extreme

drought (21% mortality), whereas insect resistant trees with less stable communities suffered

substantially (68% mortality)(Sthultz et al. 2009a), suggesting that the more stable, ascomycete-

dominated community associated with insect susceptible trees provided an adaptive advantage. A

shift toward the EM fungal communities of susceptible trees thus may ultimately be

advantageous for insect resistant trees. However, such community convergence may lead to a

decline in both the alpha and beta diversity of EM fungi in the region, especially give that P.

edulis is frequently the only host for these fungi in pinyon-juniper woodlands.

Competition and plant genotype interact to influence P. edulis growth and EM fungi

Our removal experiment demonstrated that shrubs were competitors for both insect

resistant and insect susceptible P. edulis, a finding that is consistent with the predictions of

Coomes and Grubb (2000) and with a previous study of P. edulis (McHugh & Gehring 2006).

However, the similar magnitude of the competitive release in insect resistant and insect

susceptible trees was surprising given the five-fold higher shrub abundance of resistant trees. If

competitive release per shrub removed is considered, insect susceptible trees showed a seven-

fold higher release effect than insect resistant trees, demonstrating a competition by plant

genotype interaction. We hypothesize that this pattern resulted from the higher performance of

insect susceptible than insect resistant trees during drought, which allowed insect susceptible

trees to respond to even a small reduction in competition. Not only have insect susceptible trees

survived drought better than insect resistant trees (Sthultz et al. 2009a), the shoot growth of

insect susceptible trees now exceeds that of resistant trees by 25%, a reversal of pre-drought

patterns (Gehring et al., unpublished data). However, it is possible that removal of shrubs altered

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soil nutrients due to decomposition of the remaining roots (Díaz et al. 2003), and that this effect

was greater for insect susceptible than insect resistant trees. We believe this is unlikely because

shrub removal should have had a greater effect on the nutrient dynamics of insect resistant trees

because of their higher shrub abundance. Also, decomposition in these ecosystems is slow and

unlikely to have been significant during our short-term experiment (Classen et al. 2005).

Competing shrubs also affected the EM fungal communities of P. edulis, but even these

effects depended on the genetics of the trees. Shrub removal altered the EM communities of

insect resistant but not insect susceptible P. edulis, although this result is complicated by the

greater shrub abundance of insect resistant trees. However, our tests of hypothesis 1 suggested

that the EM fungal communities of insect susceptible trees were remarkably stable. Other studies

found that plant genotype altered interactions among species. In common milkweed (Asclepias

syriaca), plant genotype altered the interaction between aphids and tending ants (Mooney &

Agrawal 2008). Such studies further demonstrate a genetic basis to indirect interactions that

structure communities (Whitham et al. 2012).

The changes in the EM fungal communities of insect resistant trees following shrub

removal likely contributed to their increased growth relative to control trees. The communities of

insect resistant trees responded to shrub removal principally through reductions in Rhizopogon

roseolus, whose abundance was negatively correlated with shoot growth, and increases in the

abundance of members of the genus Geopora, whose abundance was positively associated with

shoot growth. R. roseolus appears to be a poor mutualist in dry conditions for P. edulis as it was

for P. sylvestris (Kipfer et al. 2012). Members of the genus Geopora, and their relatives in the

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order Pezizales, are often abundant in disturbed or early successional systems (Hrynkiewicz et

al. 2010; Gordon & Gehring 2011). Pezizalean EM fungi are thought to be a low cost alternative

for trees growing under stressful conditions (Tedersoo et al. 2006). Our studies suggest that they

may be highly effective mutualists as well. In addition, the relationships between these fungi and

environmental stress appear to be more complex than previously considered. We observed

significant increases in the abundance of Geopora in association with ongoing drought stress

(this study; Gordon & Gehring 2011) but also in association with host stress relief in the form of

competitor removal. These divergent results suggest that understanding the distribution of these

important EM fungi requires consideration of factors other than environmental stress, such as

competition for colonization sites by fungi.

The influence of interactions between soil microbes and their host plants on plant

community dynamics is increasingly recognized (Bever et al. 2010); our study adds to this

emerging field by showing that intraspecific genetic variation in plants, and the linked traits that

result, significantly influence the abundance of interspecific competitors, the magnitude of

competitive release, and the consequences of competition for the surrounding microbial

community. Our studies argue that the above traits are also linked with several others including

drought tolerance and susceptibility to herbivory (Fig. 1). Similarly, plant defenses, herbivore

and pollinator communities were linked in genetically modified plants (Steppuhn et al. 2004;

Kessler & Baldwin 2006) demonstrating that seemingly unrelated traits can be linked and that

changes in one trait can have unexpected consequences on others. Predicting the future

distribution of plant species following global changes will likely require an understanding of

plant genotype, microbial communities, interspecific competition and a suite of interacting traits.

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Acknowledgments

We thank A. Stone and N. Hubert for help establishing the shrub experiment and collecting root

samples, G. Kovacs, C. Myren and M. Howell for help in the field, the U.S. Forest Service and

Sunset Crater National Monument for their cooperation and NSF DEB0816675 and LTREB

DEB0236204 for funding.

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Data Accessibility: DNA sequences of most fungi observed in this study are available in

Genbank (accession numbers KF546489-KF546501). Data used to hypothesis 2 will be available

on Dryad (doi:10.5061/dryad.0157d) within one year of publication. Data used to test hypothesis

1 will be available within one year of publication on Dryad (ascomycete abundance and species

richness; doi:10.5061/dryad.0157d) or as a part of other long-term data on the same study trees at

iplant (http://www.iplantcollaborative.org/).

Author contributions: CG, CS, AW and TW designed the study; CG, CS, DR, and TL

conducted the field work; CS, DR, TL, and LR conducted the molecular analyses; CG analyzed

the data and wrote the first draft of the manuscript; all authors contributed to revisions.

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Table 1. Identification of EM fungi based on ITS sequences. Species indicated in bold were

observed in all years of the test of hypothesis 1. Species that are underlined were observed in

tests of hypothesis 2.

ID Accession #a best BLAST match % identity Divisionb

Clavulina sp. AJ534709 Clavulina sp. 94 B

Cortinarius sp. KF546489 B

Geopora 1c KF546490 A




Geopora 5c DQ822805 Uncultured Ascomycota 94 A

Geopora cooperid DQ974731 Geopora cooperi 99 A

Hebeloma sp. KF546494 B

Inocybe sp. KF546495 B

Lactarius barrowsii KF546496 B

Lactarius deliciosuse B

Rhizopogon roseolus KF546497 B

Russula sp. KF546498 B

Suillus sp. KF546499 B

Tricholoma terreumf KF546500 B

Tylospora sp.g KF546501 B

Unk. Ascomycete AF387653 Phaeangium lefebvrei 89 A

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Unk. Basidiomycete AY969460 Uncultured basidiomycete 91 B

aAccession numbers come from sequences submitted to GenBank for this study unless best

BLAST match and % identity information are provided. If the latter information is provided data

comes from Sthultz et al. 2009, but DNA sequences are no longer available. % identity refers to

the percent similarity of query and GenBank reference sequences.

bA = species belonging to the division Ascomycota and B = species belonging to the division

Basidiomycota.

c These species were listed as Pezizales 1-6 in Sthultz et al. 2009b; two taxa recognized as

separate species by Sthultz et al. 2009b were combined as a result of Gordon & Gehring (2011)

which analyzed five of the six Pezizales species and proposed that four of them were unique

species in the genus Geopora.

dSequences of this species from this study are no longer available due to a data storage failure.

e EM root tips of this species were observed in 1994 only and were identified based on restriction

fragment length polymorphism data matching root tips to L. deliciosus sporocarps. Sequence

data is not available.

f Tricholoma myomyces is synonymous with T. terreum, the name for the species in Index

Fungorum. Sthultz et al. (2009b) listed them as separate species, but they are combined here.

gThis species was observed only in the sampling for hypothesis 2.

Figure 1. A summary illustration of the genetically-based, linked traits associated with insect

resistant and insect susceptible trees. Previous studies have shown that susceptibility to

herbivory by D. albovittella is associated with a shrub-like architecture (Whitham & Mopper

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1985), reduced competitor shrub density (Sthultz et al., unpub data; this study), an

ectomycorrhizal community dominated by fungi in the division Ascomycota (Sthultz et al.

2009b), and tolerance of drought conditions (Sthultz et al. (2009a). Removal of D. albovittella

from susceptible trees (insect removal column) demonstrated that insect herbivory directly

caused the architectural changes (Whitham & Mopper 1985), but that EM fungal community

composition had a genetic basis (Sthultz et al. 2009b). Shrub abundance (N/Aa) has not been

measured on insect removal trees and tree mortality data (N/Ab) are not reported here because

they are not comparable in scope to the results of Sthultz et al. (2009a). However, we designated

insect removal trees as drought tolerant based on the drought tolerance traits of their offspring as

determined by Sthultz et al. (2009a).

Figure 2. An NMDS ordination showing the EM fungal communities of insect resistant, insect

susceptible and susceptible insect removal pinyons at three time periods, one before the onset of

drought (1994), one following an extreme drought year (2004), and one as drought continued

(2010). The tree types are represented by different symbols (insect resistant, upright architecture

and white squares; insect susceptible, shrub-like architecture and black circles; insect removal,

transition from shrub-like to upright architecture, gray diamonds). Each point represents the

centroid of the EM fungal community of eight to nine replicates per treatment with vertical and

horizontal bars depicting +/- 1 SE. The year designations are indicated individually for the

centroids of insect resistant trees, but are combined for insect susceptible and insect removal

trees due to the close proximity of their centroids. Dashed arrows show trajectories of

communities from 1994-2010. The EM fungal communities of insect susceptible and insect

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removal trees shifted slightly from 2004 to 2010 while those of insect resistant trees changed

significantly during all years.

Figure 3. The percentage of EM fungi in the division Ascomycota for insect resistant, insect

susceptible and susceptible insect removal trees at three time periods. Data are means + 1S.E.

Ascomycotan fungi increased with time in insect resistant trees, but remained similar across

years in insect resistant and insect removal trees.

Figure 4. Shoot length of resistant (open bars; tree icon with upright architecture) and

susceptible trees (filled bars; tree icon with shrub-like architecture) is higher when shrubs are

removed (-shrub) than when shrubs are present (+shrub). Data are means + 1SE The number of

shrubs is higher beneath resistant trees, yet shoot growth of both susceptible and resistant trees

increased significantly with shrub removal.

Figure 5. The EM fungal communities of insect resistant (upright tree icon, white squares) and

insect susceptible (shrub-like architecture, black circles) pinyons growing with or without shrub

neighbors. Only the communities of insect resistant trees were altered by shrub removal. Each

point represents the centroid of the EM community of 12 replicates per group with vertical and

horizontal lines depicting +/- 1 SE.

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Plant genetics and interspecific competitive interactions determine ectomycorrhizal fungal community responses to climate change