Contributions of arbuscular mycorrhizal communities to the invasive habit of Bromus
rubens: sources, patterns and mechanisms
Matthew R. O’Neill and Michael F. Allen
Department of Biology, University of California,
Riverside, CA 92521; monei003@ucr.edu; 951-316-7303
Background: Human mediated transport of exotic plant species to novel environments has
increased with the continued globalization of the past few centuries (Vitousek et al. 1997, Mack
et al. 2000). Of those species that become established, an estimated tenth of a percent experience
an explosion in population size, followed by rapid range expansion (Williamson 1996). Species
that express such population dynamics in non-native ranges are by definition invasive
(Richardson et al. 2011). Plant-soil interactions have recently emerged as an important factor
potentially facilitating invasion success (Reinhart and Callaway 2006). However, explicit
consideration of important species-specific interactions and the functional response of
genetically distinct host populations to these interactions has remained lacking, thus limiting the
formation of general principles that underlie observed patterns.
The historical lack of consensus on causal mechanisms in plant invasion can partially be
attributed to two commonly neglected factors: the importance of identifying genetic source
populations and the interaction between plants and soil microbial communities. Identifying
genetic source populations is crucial because it allows for investigations of differential
interactions of the focal species with its environment between source and recipient ranges. For a
specific mechanism to facilitate invasion it must be shown not to function in native ranges
(Hierro et al. 2005). Several recent studies have identified genetic source populations while
simultaneously conducting common gardens to investigate genotypic and/or post introductory
evolutionary dynamics (Lavergne and Molofsky 2007, Dlugosch and Parker 2008, O’Neill et al.
2012). Results of these studies reveal significantly divergent growth patterns, independent of
biotic interactions, among source and introduced populations. Thus post introductory evolution
in novel environments may alter growth habits and confound studies concerned with the
response of focal species to differences in regional biota.
Plant-soil feedbacks (PSFs) describe net differential plant growth responses due to
interactions with antagonistic (negative) and mutualistic (positive) soil organisms (Bever et al.
1997). Negative feedbacks dominate in most ecosystems where coevolved microbial antagonists
accumulate and constrain growth (Mills and Bever 1998, Kulmatiski et al. 2008). These
feedbacks promote diversity by increasing niche stabilizing differences (Chesson 2000) via
density-dependent antagonism (Janzen 1970, Connell 1971), thus limiting population sizes of
otherwise competitively dominant species (Bever 2003, HillesRisLambers et al. 2012). Positive
PSFs result from decreased antagonism, beneficial partnerships with mycorrhizae or N-fixing
bacteria, increased access to resources, or indirect interactions that alter nutrient cycling
(Reinhart and Callaway 2006, van der Putten et al. 2007). These processes increase host
competitive ability, increasing relative fitness differences among co-occurring species (Chesson
2000), ultimately resulting in decreased diversity and dominance of one to a few plant species
(Bever et al. 2012, HillesRisLambers et al. 2012). Such feedbacks are commonly observed
among dominant native and invasive species (Klironomos 2002).
The vast majority of PSF studies in the field of invasion ecology have treated the
reciprocal effects of plant-soil interactions in a most general sense. Often the soil component is
treated as a ‘black box’ whose effects are quantified by differences in aboveground plant
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biomass. While this methodology has revealed the general pattern of invasive species interacting
differently with invaded relative to native range soils, it provides little to no information on why
they differ. To elucidate these mechanisms light must be shed on this ‘black box’ in the form of
species level identification. In addition, studies have primarily focused on the role of
pathogenic/parasitic microbes in limiting host fitness within the native range. Consequently,
authors commonly invoke the enemy release hypothesis – an increase in invader fitness due to
‘release’ from coevolved predators and pathogens (Elton 1958) – to simultaneously explain
negative interactions in the native range and positive interactions in the invaded range. While
this is a parsimonious explanation, it neglects the important role soil mutualists (e.g. arbuscular
mycorrhizal fungi [AMF]) may play in promoting invasive habit. Rather than plants being
released from ‘enemies’ of their native range, divergent growth habits and population dynamics
may be the product of interactions with different soil mutualist communities (Richardson et al.
2000, Reinhart and Callaway 2006, Sun and He 2010). In either case, species of soil microbes
must be different and/or interact differently with hosts among respective regions to conclude that
they promote invasion. To my knowledge no information on differing soil microbial species and
communities among native, genetic source ranges and invaded ranges exist in the plant-soil
literature. To this end there are many critical questions that have yet to be addressed.
Specifically, who are the mutualists and how are they interacting with the invasive host plant?
Research Objectives: Investigations of plant-soil interactions within the context of invasion
have been numerous, yet, due to the static nature of the metrics commonly used and the lack of
species level identification of soil microbes, mechanisms underlying positive growth responses
in introduced relative to native soils remain poorly resolved. The goals of the proposed research
are to (1) quantify genetic and quantitative trait variation among populations of a focal invasive
species from within its native and introduced ranges, (2) identify populations of closest relation
among those ranges, (3) characterize AMF community composition among native and introduced
ranges and (4) evaluate the effects of biogeographical soil community interactions on host
physiology and growth.
Study System: In the Mediterranean climatic regions of California, Bromus rubens L. is one of
the most abundant invasive plant species, often forming near-monospecific stands that decrease
native biodiversity (Minnich 2008). However, in its native Mediterranean range it occurs only
sparsely or patchily in disturbed areas (Jackson and Roy 1989, Allen and Allen personal
communication). In addition, AMF community composition differs within the rhizosphere of B.
rubens and neighboring species in both introduced and native ranges. For example, in the
introduced range Siguenza et al. (2006) showed that B. rubens tended to associate with smallspored Glomus species while native species associated with large-spored species from the family
Gigasporaceae. In the native range (Spain), Torrecillas et al. (2012) found AMF communities of
B. rubens and co-occurring Brachypodium retusum (Pers.) P. Beauv. to differ. Distinct within
habitat differences in soil communities among species in both ranges suggests that speciesspecific host-AMF associations occur in the field. Such specificity, in conjunction with disparate
patterns in distribution and abundance, makes B. rubens a model candidate to elucidate the
mechanisms by which plant-AMF interactions contribute to successful invasion.
Objectives 1 and 2: Quantify genetic and quantitative trait variation, identify populations
of closest relation – Background and rationale: Specific mechanisms that facilitate invasion
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success must be isolated in invaded relative to native ranges to conclude that the mechanism
influences the process of invasion (Hierro et al. 2005). Therefore, before populations of B.
rubens are chosen for microbial community characterization and growth assays, the first
objective of this study is to identify the genetic source population(s) of B. rubens in southern
California from the Mediterranean.
Methods: Seeds of B. rubens were collected from 7 populations within the Mediterranean and 7
populations within California (4/7 were collected in Riverside County) and Mexico. Sixty
individuals from each population were grown for 50 days in a common garden. Photosynthetic
capacity and morphology were quantified to conduct a growth analysis. Shoot tissue was
harvested from twenty individuals and DNA extracted using a DNeasy Plant Mini Kit (Qiagen,
USA). Polymerase chain reaction and sequencing will be carried out using a PTC-100
Programmable Thermal Controller (MJ Research, USA) and an ABI 3730xl (Applied
Biosystems, USA), respectively, following Ridley et al. (2008), with the following primer sets:
trnT-trnL, rpl32-trnL and trnL-trnF. An analysis of molecular variance (AMOVA) will be
performed to partition within and among population haplotype diversity and calculate an index
of genetic differentiation for haploid data, PT. Results of this analysis will allow quantification
of the diversity among regions and determine genetic origins of B. rubens in California.
Objective 3: Characterize AMF community diversity among native and introduced ranges
– Background and rationale: AMF are an ancient group of fungi that are thought to have
facilitated the migration of plants to terrestrial environments 400mya, and currently establish
relationships with approximately 80% of all plant species (Pirozynski and Malloch 1975, Simon
et al. 1993, Smith and Read 2008). The nature of this interaction mainly consists of phosphorus
(P), nitrogen (N) and water transport from AMF to host plants in exchange for photosynthate (C)
(Allen et al. 2003). Due to the obligate biotrophic habit and reduced ability to disperse,
interactions with host plants are imperative to the fitness of AMF species (Allen 1988).
Consequently it is in the best interest of AMF species to exhibit little selectivity among hosts. In
contrast, it behooves plant hosts to preferentially interact with specific species or species groups
of AMF that provide the most benefit (Allen and Boosalis 1983, Smith and Read 2008). This
pattern of symbiont selectivity by potential hosts via increased carbon allocation, prevention or
termination of interaction has direct effects on AMF fitness and community structure (Kiers et al.
2011). Thus, simultaneous bottom-up and top-down dynamics via species-specific host-symbiont
interactions are important factors influencing above- and belowground community composition
(Allen and Allen 1990, Allen 1991, Allen et al. 1995, van der Heidjen 1998, Wardle et al. 2004,
Klironomos et al. 2011).
The ubiquity and importance of AMF have elicited many investigations into the ecology
and evolution of these species (Smith and Read 2008). However, these studies have been
historically complicated by a taxonomy in perpetual flux (Schusler and Walker 2010).
Throughout the first century of research on AMF species were identified based upon
morphological characters (Blaszkowski 2012). Due to the broad scale and the phenotypic
variability of these characters within and among species, the taxonomy of AMF has experienced
repeated expansions and contractions in the numbers of orders, families, genera and species
(Blaszkowski 2012). At present, technological advancements in molecular methods have
increased our ability to identify species at finer scales, yet the lack of coherence between
morphological and molecular species identities has impeded the construction of an unequivocal
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phylogeny. For these reasons, ecological studies of AMF generally rely on multiple techniques to
sufficiently quantify species-specific host-AMF interactions; specifically, quantitative species
identification via morphology, qualitative species identification via molecular methods, and
observations of the degree of colonization via root staining. All three techniques will be utilized
in concert for this study to establish a comprehensive view of important host-AMF community
interactions among genetic source and introduced populations of B. rubens.
Question: Are AMF communities within the rhizosphere of B. rubens significantly different
among native and invaded ranges?
H: Biogeographical differences in AMF community composition occur between native and
introduced ranges.
Methods: Soil samples are to be collected from within the rhizosphere (no more than 15
centimeters from host) of at least 30 B. rubens individuals per region (genetic source and
invasive populations). From each sample 100g of soil will be separated for AMF community
characterization. AMF spores will be isolated via sucrose centrifugation (Allen et al. 1979,
Ianson and Allen 1986) identified by characteristic morphologies (Schenck and Perez 1990,
Blaszkowski 2012) and relative species abundance quantified. This assessment of AMF species
identity, abundance and diversity between regions will provide a baseline measure of the
potential species pool with which hosts can interact.
Molecular analyses on root tips will be used to assess fungal communities among regions
associating with B. rubens at the time of collection. DNA will be extracted as in Objective 1.
Both pathogenic and AMF communities will be assessed simultaneously using high throughput
sequencing technology. The new primer gITS7, coupled with ITS4, will be used to amplify the
entire ITS2 region (following Ihrmark et al. [2012]). Resulting sequence data will allow for
species identification (blast.ncbi.nlm.nih.gov). This will be the first comprehensive survey of
AMF communities among native and introduced ranges of an important invasive species.
Objective 4: Quantification of biogeographical plant-soil interactions
Question: What role do biogeographical AMF communities play in plant-soil interactions
among regions?
H: AMF communities interact differently with B. rubens among ranges, promoting enhanced
physiological activity and growth in the introduced but not the native range soils.
Methods: Seed and soil samples will be collected from the genetic source and invasive
populations following seed maturation and senescence. Seeds will be collected from 50
individuals per site. Soil samples are to be collected as above. Soils will be pooled by region and
thoroughly homogenized. After homogenization, soil will be treated by autoclave for sterilization
(here on referred to as non-mycorrhizal soil treatment, NM). NM soil will be mixed with
sterilized sand (1:1 by volume) and placed in pots. To determine the contribution of total soil
biota to growth, one third of the pots will receive an unaltered 25% by volume soil inocula
addition by respective region (here on referred to as mycorrhizal soil treatment, M). A microbial
wash will be added to one third of the pots of respective soil origin to isolate effects of AMF on
growth (here on referred to as sievate soil treatment, S; Ames et al. 1987, Koide and Li 1989).
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A full factorial design growth study will be utilized to isolate the effects of regional AMF
on growth. A two-factor fully crossed design will consist of seeds (from native [N] and invasive
[I] populations) planted in each of six soils (MN, MI, SN, SI, NMN and NMI). Plants will be
allowed 60 growth days in a growth chamber. Due to expected effects of AMF on N, P and water
availability, maximum photosynthetic rates (Amax, molm-2s-1) and instantaneous water use
efficiencies (WUE, mmol CO2 /mol H2O) will be monitored throughout the growth period (Allen
and Allen 1986, Auge 2001, Allen et al. 2003). Following the allotted growth period plants will
be harvested and the following factors quantified: total, shoot, and root biomass, root:shoot ratio,
shoot mass ratio, root mass ratio, specific leaf area, leaf mass ratio, N% by shoot mass, P% by
shoot mass, and AMF diversity as in Objective 3. All metrics of AMF diversity and interaction
will be used as predictor variables for relative plant response and nutrient content in linear
models. A result of significant predictors, with larger positive slope estimates, in introduced
relative to native range M soil will indicate positive AMF contribution to plant-soil interactions.
Significance: The research outlined in this proposal will: be the first comprehensive survey of
mutualist communities between the genetic source and introduced regions of an important
invasive species; expand the study of invasive plant-soil interactions through the use of dynamic
rather than static metrics of host-symbiont interactions; and move the field beyond the
description of patterns and toward an understanding of mechanistic processes. Beyond the
substantial contribution these data will make to the study of invasive host-symbiont interactions
and invasion ecology, these data will also be used to develop novel projects to manage the spread
of invasive species through exploitation of soil biota variability, a factor that has remained under
appreciated and under utilized.
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Budget:
Item
Graduate student stipend
Quantity
Summer 2013 @ 49% time
Undergraduate assistant stipend
Sample prep kit
Sequencing costs
10 hr per week for 6 months
Cost
$6,075.38
$5,281.56
$3,600
$1,683
Total Cost
$16,640.94
Budget Justification:
A stipend is requested for Matthew O’Neill, a graduate student in the Michael Allen lab. Matthew
O’Neill will oversee all aspects of the research outlined above. The stipend will cover 3 summer months
at 49% time ($5,895) plus benefits ($180.38), when the growth experiments are to be conducted.
To assist in aspects of set up, break down and lab activities associated with the research outlined,
funds for a part-time undergraduate assistant is requested. The undergraduate assistant will work through
the Summer and Fall 2014 quarters ($5,100, plus benefits: $181.56).
The remaining funds requested will be used for sequencing of the fungal ITS2 region for species
level identification. Sequencing will be conducted using the Illumina MiSeq platform at the IIGB
Genomics Core facility at UCR. DNA will be extracted from roots and polymerase chain reactions will
then be carried out using the primers outlined above. A paired-end sample kit will then be used to create
sequence libraries ($3600). Finally, a 600bp V3 kit (2x300bp paired-end) will be used to sequence the
DNA ($1683).
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