Biol Invasions (2015) 17:1239–1251
DOI 10.1007/s10530-014-0791-1
ORIGINAL PAPER
Mixed fitness effects of grass endophytes modulate impact
of enemy release and rapid evolution in an invasive grass
Roo Vandegrift • Wilma Blaser •
Felipe Campos-Cerda • Allison F. Heneghan
George C. Carroll • Bitty A. Roy
•
Received: 7 March 2014 / Accepted: 23 September 2014 / Published online: 16 October 2014
Ó Springer International Publishing Switzerland 2014
Abstract Endophytic fungi in grasses are often
considered to be mutualistic because they can increase
host resistance to herbivory and drought. However, not
all endophytes are beneficial to their hosts, but may
instead be specialist enemies. Brachypodium sylvaticum is an invasive grass in the USA. In its European
native range, it is nearly always infected by the hostspecific endophyte Epichloë sylvatica. While this
fungus decreases herbivory, it also decreases the
growth rate and size of infected plants, making them
less competitive. After showing that B. sylvaticum has
lost its endophyte in the invaded range, we use
greenhouse assays to deconfound the effects of
endophyte infection and range origin to test assumption of the evolution of increased competitive abilities
Electronic supplementary material The online version of
this article (doi:10.1007/s10530-014-0791-1) contains supplementary material, which is available to authorized users.
R. Vandegrift (&) F. Campos-Cerda
G. C. Carroll B. A. Roy
Institute of Ecology and Evolution, University of Oregon,
Eugene, OR 97403-5289, USA
e-mail: awv@uoregon.edu
W. Blaser
Institute of Integrative Biology, Swiss Federal Institute of
Technology, Universitätstrasse 16, ETH Zentrum, CHN,
8092 Zurich, Switzerland
A. F. Heneghan
New Mexico State University, Las Cruces, NM, USA
(EICA) hypothesis. Brachypodium in its invaded
range appears to have lost tolerance mechanisms
present in the native range, allowing Epichloë to
greatly increase seedling mortality and reduce growth
rates. Additionally, there is some evidence for
increased competitive abilities in the form of increased
seedling growth rates in the invasive range. Together,
these results provide strong support of the EICA
hypothesis.
Keywords Enemy release hypothesis (ERH)
Evolution of increased competitive abilities (EICA)
hypothesis Endophyte Brachypodium sylvaticum
Epichloë sylvatica Mutualist–pathogen relationship
Introduction
The enemy release hypothesis (ERH) postulates that
one major factor facilitating invasion is the relative
lack of specialized enemies in the invaded range,
allowing for faster growth and spread (Keane and
Crawley 2002). An alternative, the evolution of
increased competitive ability hypothesis (EICA),
assumes that the success of invasive species is
evolutionary in nature, driven by the change in
selective pressures of the new environment. Under
the EICA framework, enemy release provides selective pressure to reallocate resources from defense to
growth and reproduction (Blossey and Notzold 1995),
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though there are many complicating factors (Colautti
et al. 2004). One complication is the distinct difference
in defensive strategies used to protect against generalist versus specialist enemies (van der Meijden 1996;
Müller-Schärer et al. 2004). Specialist enemies are
theorized to be more important to plant invasions
(Keane and Crawley 2002) because of the disproportionate effect they have on controlling populations in
their native ranges, and the relative metabolic costliness of specific defenses. Thus, evolved increased
competitive abilities may be due to reallocation of
resources from specialist defenses to generalist
defenses (Joshi and Vrieling 2005).
There has been much debate and intensive research
surrounding the EICA hypothesis (reviewed in:
Atwood and Meyerson 2011; Felker-Quinn et al.
2013), much of it inconclusive or contradictory (e.g.,
Willis et al. 2000; van Kleunen and Schmid 2003;
Bossdorf et al. 2004; but see also Vilà et al. 2003;
Meyer et al. 2005). Founder effects, novel hybridization opportunities, and strong abiotic environmental
selective pressures can drive rapid evolutionary
change in invasion. This complicates EICA research,
and many studies have not tested competitive abilities
and defense in the same organisms (see Bossdorf et al.
2005; Atwood and Meyerson 2011 and citations
therein). It is impossible to make inferences about
energetic tradeoff without a measure of both competitive abilities (usually growth) and defense. Here, we
test the EICA hypothesis using Brachypodium sylvaticum (Huds.) P. Beauv., an aggressive invasive species
in the northwest of the USA (Roy 2010), with the ERH
(phenotypic plasticity in the face of specialist enemy
loss) as an explicit alternative hypothesis. Previous
work indicates that pathogens and herbivores of B.
sylvaticum show some, but not all, of the characteristics predicted by the ERH (Roy et al. 2011).
Insecticide and fungicide sprays were used to remove
herbivorous insects and pathogenic fungi from the
plants in multiple populations in both the native and
invaded ranges. In accordance with the ERH, population growth rates were higher in the native range in
the sprayed plots, where enemies were fewer than in
the control plots. There was no statistically significant
effect of enemy removal in the invaded range.
Contrary to the ERH, all the common enemies were
generalists and there was more herbivory in the
invaded range relative to the native range (Roy et al.
2011; Halbritter et al. 2012).
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Increased herbivory in the invaded range and
reduced seed germination in the native range suggested that there might be differences in endophyte
infection, since endophytes would not have been killed
by the non-systemic fungicides used in previous
studies (Roy et al. 2011; Halbritter et al. 2012).
Endophytes are fungi that live between the cell walls
of plants and cause no visible disease symptoms on the
surface of the plant; they are common in grasses (Clay
1990; Rudgers et al. 2009). While it is not obvious that
a plant is infected when endophytes are present, they
may nonetheless have a range of consequences for
their hosts, from true mutualism that increases insect
or drought resistance, through commensalism, to
antagonist pathogenicity that decreases survival and
reproduction (Carroll 1988; Faeth and Sullivan 2003;
Saikkonen et al. 2006). The same species of endophyte
can either be a mutualist or pathogen depending upon
its lifecycle stage, genotype, or environmental conditions. All symbioses exist on a continuum from
pathogen to mutualist: if the benefit to the host (e.g.,
from reduced herbivory) is greater than the cost (e.g.,
reduced growth and seed-set) the fungus is a mutualist.
The environment within which a host is embedded will
impact the position of a symbiont along this continuum because it will alter the balance between costs
and benefits.
Brachypodium sylvaticum in its native Eurasian
range appears to be almost ubiquitously infected with
a host-specific fungal endophyte, Epichloë sylvatica
Leuchtm and Schardl (Eckblad and Torkelsen 1989;
Raynal 1994; Väre and Itämies 1995; Bucheli and
Leuchtmann 1996; Enomoto et al. 1998; Zabalgogeazcoa et al. 2000; Roy et al. 2011; Leuchtmann,
pers. com.), which may act as a pathogen rather than a
mutualist, despite common assumptions about Epichloë endophytes of grasses (Schardl 1996). Smallscale studies done with infected and uninfected plants
in Switzerland, by Brem and Leuchtmann (2002),
indicate that while plants infected with an asexual
strain of E. sylvatica have less herbivory, they also
have decreased growth rates and competitive abilities.
Recent research in our lab (Roy et al. 2011; Halbritter
et al. 2012) suggests that Epichloë infection may
decrease germination rates in B. sylvaticum by seed
infection, as the fungus is spread vertically from
mother plant to daughter (Brem and Leuchtmann
1999). Thus, E. sylvatica appears to be a specialist
enemy of B. sylvaticum.
Impact of enemy release and rapid evolution
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b Fig. 1 Comparison of predictions of the ERH (a), the EICA
hypothesis (b), and founder effects or other evolutionary forces
unrelated to enemy release (c). Relative sizes of the cartoon
grasses and the green bars indicate relative differences in fitness
for the hosts. Presence (E?) or absence (E-) of E. sylvatica is
shown on the X-axis, and range of origin on the Y-axis of each
panel. ERH (a): If the observed increase in fitness in B.
sylvaticum is due only to escape from E. sylvatica, reintroduction of the fungus to invasive range plants should
recapitulate native range fitness levels, while removal of the
fungus from native range grasses should release them from
control, increasing fitness to levels to those observed in invasive
range plants. EICA (b): If populations of B. sylvaticum have
evolved in response to enemy release in the invasive range, we
expect invasive range plants to experience a disproportionate
fitness loss when infected by E. sylvatica compared to their
native range conspecifics, as well as having increased fitness in
the absence of the fungus. Founder effects (c): If founder effects,
or evolution not related to enemy release, is responsible for the
increase in fitness observed in the invasive range, we expect
invasive range plants to be more fit than native range plants
regardless of infection with E. sylvatica, though infection will
likely still negatively affect the host, likely in a manner
proportionate to the effect on native range plants
Here, we document the near total absence of E.
sylvatica infection in the invaded range. If E. sylvatica
is generally pathogenic, the near lack of fungal
endophyte infection in B. sylvaticum within the
invaded range may constitute strong support for some
form of the ERH or the EICA hypothesis.
In addition to documenting the virtual absence of the
endophyte in the invaded range, we compare germination
and growth rates of seedlings, a common proxy for fitness
(Poorter and Garnier 1999; Matzek 2012), from the
native and invaded ranges to explicitly test the EICA
hypothesis. We utilized a greenhouse experiment using
seeds collected during the same season in both ranges,
clearing the seeds of Epichloë infection and then
selectively re-inoculating half of each group. This
bifactorial design permits us to effectively compare the
effect of E. sylvatica and plant origin independent of each
other, allowing us to distinguish between ERH, EICA,
and potential founder effects (Fig. 1). If release of B.
sylvaticum from control by E. sylvatica is sufficient to
explain observed increases in fitness in the invasive range
(Holmes et al. 2010; Roy et al. 2011; Halbritter et al.
2012), we expect that removing it from native range
plants should increase their performance to be on par with
those from the invasive range. Additionally, we would
expect invasive range plants to be affected similarly to
those from the native range. In short, under the hypothesis
that E. sylvatica is directly impacting fitness in B.
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sylvaticum, such that it is a controlling specialist enemy in
the native range, we expect the fungus to impact native
and invasive range plants similarly (Fig. 1a).
Alternatively, if release from the specialist enemy
E. sylvatica has provided selective pressure for B.
sylvaticum to have evolved some reallocation of
resources from defense to growth and reproduction,
we expect to be able to observe the reduction in
defensive capabilities by invasive range plants in the
form of strongly reduced fitness when infected as
compared to infected native range plants. In other
words, we expect the invasive range plants to be
disproportionately negatively affected by infection
with E. sylvatica. Additionally, if such evolution has
occurred, we would also predict that even in the
absence of the controlling enemy, invasive range
plants will out-perform native range plants (Fig. 1b).
Factors independent of enemy release may be
driving the evolution of invasive range populations of
B. sylvaticum, such as founder effects and drift, or
selection unrelated to enemy release. If the invasive
range plants show increased fitness relative to their
native range counterparts in both infected and uninfected states, such other evolutionary forces may be the
best explanation for the observed increases in fitness in
the invasive range (Holmes et al. 2010; Roy et al. 2011;
Halbritter et al. 2012), though more work will be
needed to determine the extent to which founder effects,
genetic drift, or selective pressures not related to enemy
release are responsible for such evolution (Fig. 1c).
This experiment also allowed us to test explicitly for
effects of infection by E. sylvatica on germination rates.
All germination rates observed to date of uninfected
seeds of European origin are from naturally infected
seeds that were treated to kill the endophyte (Roy et al.
2011; Halbritter et al. 2012). It is necessary to compare
germination rates of seeds from the same population
produced with and without the endophyte to accurately
determine the effect of Epichloë on germination rate,
because infection of seeds at any time may negatively
impact germination, including prior to heat treatment.
R. Vandegrift et al.
Fig. 2 Wild endophyte screen results. We only found evidence
of E. sylvatica in one wild population in the invaded range, the
Fisherman site near Mill City, Oregon. All individuals from all
populations tested from the European native range were
infected, however. *Native range data from Bucheli and
Leuchtmann (1996)
2010), introduced in the early 1900s by the US
Department of Agriculture (USDA) for agronomic
research. Records from the Office of Foreign Plant
Introduction dating back to 1912 indicate that B.
sylvaticum was being imported from India, Sweden,
Russia, and probably other localities (Rosenthal et al.
2008). The grass was first collected in the wild in
Oregon in 1939 (Chambers 1966), and has become
increasingly common during the last 15 years (Rosenthal et al. 2008). This grass is of particular concern
because it is shade-tolerant (Holmes et al. 2010) and
forms vast, virtually monospecific carpets in the forest,
which crowd out other vegetation (Kaye and BlakeleySmith 2006) and, similar to other grasses, may reduce
conifer seed germination (Powell et al. 2006). It is
found commonly and in high densities in the central
Willamette valley, particularly from Eugene to Corvallis, and appears to be in the midst of rapid range
expansion (Rosenthal et al. 2008; Roy 2010).
Materials and methods
Study sites
Focal species
We are working with the grass B. sylvaticum (Huds.) P.
Beauv., an aggressive invasive species in the USA (Roy
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We sourced seed and tested for endophyte infection in
Switzerland (center of the native range) and Oregon
(USA, epicenter of the invaded range) at 21 field sites
Impact of enemy release and rapid evolution
(Supplementary Table S1; Fig. 2), a subset of which
were used for germination, growth rate, and mortality
assays. Climate in the two areas are similar: Zürich
(Switzerland) has an annual precipitation of 1,086 mm
and Eugene (Oregon, USA) has 1,254 mm. Mean
temperatures for Zürich and Eugene are 8.5 and
11.9 °C, respectively (climate information from www.
meteoschweiz.admin.ch and the Western Regional
Climate Center www.wrcc.dri.edu). There are, however, seasonal differences between the two sites:
summer is much drier in Oregon (mean precipitation
for July and August in Zürich is 124.5 mm, but it is
only 20 mm for Eugene).
Infection rates
To determine whether the fungus was present in the
invaded range we used the Agrinostics Field Tiller
immunoblot kit (Agrinostics Ltd. Co., Watkinsville,
GA, USA). Because the kit was developed for a
different species, we verified its efficacy by isolating
E. sylvatica from B. sylvaticum seeds collected in
Switzerland where infection rates are at or near 100 %
(Fig. 2; Bucheli and Leuchtmann 1996; Leuchtmann
and Schardl 1998). For additional positive controls we
used leaf tissue from plants grown from Swiss seeds
and Dactylis glomerata showing choke symptoms
caused by Epichloë typhina (Pers.:Fr.) Tul. Finally, we
verified a subset of immunoblot results with an E.
sylvatica-specific PCR screen; these indicated that the
immunoblot results were valid (see Supplemental
Methods for details).
Genetic data and historical records suggest that B.
sylvaticum was likely initially introduced from two
Bureau of Plant Introduction experimental plots, one
near Eugene, Oregon, and one near Corvallis, Oregon
(Rosenthal et al. 2008). We therefore screened three
populations near Eugene (Mount Pisgah, Jasper, and
Jasper State Park) and two near Corvallis (Bald Hill
and Sweet Home; see Table S1). The vegetation and
other site characteristics have been described elsewhere (Roy et al. 2011).
We tested for E. sylvatica in the invaded range at
three times: peak growing season (20 June 2010),
7 weeks later (4 August 2010), and at the end of the
summer (26 August 2010). All tillers were collected at
ground level within 24 h of analysis, wrapped in a paper
towel and placed on ice. Thirty tillers per population
were randomly sampled by taking the nearest tiller to a
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meter mark along two parallel transects each 15 m long
and approximately 10 m apart.
Initial germination and growth rate assays
For the initial assay, seed material of B. sylvaticum
was collected in late August 2007 from three wild
populations in the native range and six in the invaded
range (Table S1). Seeds were stored at 4 °C until
needed. Prior to germination, seeds were deglumed
and winnowed to remove aborted seeds. To reduce
fungal attack, seeds were surface sterilized in 5 %
bleach solution for 30 s and then rinsed twice with tap
water. On May 22nd 2008 seeds were placed between
four sheets of filter paper in a sterile Petri dish and
dampened with a solution of gibberelic acid (50 mg
GA3/500 ml tap water). Petri dishes were kept at room
temperature and checked daily to ensure correct
moisture level. As they germinated the seedlings from
each of the US populations were transplanted into
200 cm3 Containers (D-40 cells, Steuwe and Sons,
Corvallis, Oregon) filled with Rexius, Patio Potting
SoilTM (one seedling per tube). The Swiss seeds had
extremely low germination rates, and after day 11 we
transferred the remaining seeds to trays filled with
potting soil and transplanted them into containers
upon germination.
We measured the aboveground height of seedlings
9 days after transplanting (with a few exceptions of
8–11 days). Seedling growth rates, a proxy for fitness
(Poorter and Garnier 1999; Matzek 2012), were
calculated by dividing the height at the time of
measurement by the number of days since emergence.
To test differences between ranges we used a mixedmodel analysis of variance (ANOVA) with restricted
maximum likelihood (REML) estimation of variance
components. Range was designated as a fixed effect
and population as a random effect.
EICA greenhouse experiment
A large number of seeds were collected from two
native (Flaach:654, Rafz:395) and two invaded (Pisgah:293, Jasper:291; see Table S1) range populations
of B. sylvaticum at the end of summer 2011. These
were deglumed by hand, and then treated to remove
the endophyte, following Nott and Latch (1993). The
seeds were surface sterilized by immersion in 95 %
EtOH for 1 min, full-strength bleach (6.15 %
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NaHClO) for 3 min, 30 s in 95 % EtOH, then triple
rinsed in autoclaved deionized water. The seeds were
allowed to dry on sterile filter paper, then were placed
in sterile petri dishes and placed in 100 % humidity at
37 °C for 3 weeks in a sealed incubator. The seeds
were then germinated on sterile water agar, with any
seeds showing fungal infection being discarded.
Half of the germinants from each population were
inoculated with E. sylvatica, for a total of 65 plants per
population. Inoculation of B. sylvaticum with the
endophyte was accomplished following Leuchtmann
and Clay (1988). Working under a dissecting microscope, a 27-gauge sterile hypodermic needle was used
to make a small incision just above the apical
meristem of the seedlings at the two- or three-leaf
stage of development (typically 5 days post germination). The needle was then used to collect a small
sample of cultured fungal hyphae (isolated from Swiss
seeds as described above), which was then inserted
into the incision. Control plants were injected with a
small drop of sterile deionized water (Leuchtmann and
Clay 1988). Plants were grown for 5 days on agar
before transplantation into soil (Black Gold, Sun Gro
Horticulture, Agawam, MA, USA) in 10 cm pots.
The plants were randomly distributed in racks in the
greenhouse, and were re-randomized every week.
Height (length from longest leaf tip to soil surface)
was measured every other day, from initial transplant
into soil (19 December 2011) to harvest (29 February
2012). Daily growth rates were calculated as the
difference in height between two subsequent measurements, divided by two. The data were analyzed
using a repeated measures, mixed model analysis of
variance (ANOVA), including site, nested in range,
and inoculation as fixed effects, population included as
a random effect, and daily growth rate as the response
variable. Number of tillers at harvest and oven-dried
biomass were analyzed by mixed model analysis of
variance (ANOVA) as well, though without repeated
measures. Tukey’s HSD was used to compare means.
Mortality was analyzed using the log-rank Mantel–
Haenszel test (Harrington and Fleming 1982).
A subset of both treatment groups for all populations (ten plants per treatment per population) were
repotted into gallon pots and allowed to set seed,
which was collected for second-generation germination assays. A subset of 100 seeds from each parent
plant were deglumed by hand, surface sterilized as
above, plated onto water agar to germinate, and
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R. Vandegrift et al.
observed for germination for 30 days. Ten seeds from
each plant were also tested using the Agrinostics Seed
Immunoblot kit (Agrinostics Ltd. Co., Watkinsville,
GA, USA) to confirm infection status of the parent.
Because seed infection by Epichloë was not entirely
all-or-nothing, germination rates were analyzed by
linear regression to examine trends in germination in
response to rate of seed infection. Additionally, a
mixed model ANOVA and Tukey’s test were used to
examine treatment and range differences. Student’s
t tests were used to examine pairwise differences.
All analysis was performed in R (version 2.15.1),
using the packages vegan (Oksanen et al. 2013) and
survival (Therneau 2013).
Results
Endophyte screening
Endophyte infection in Oregon, epicenter of the
invasion, appears to be limited to a single population
of the eight we sampled. Using the immunoblot test we
found E. sylvatica in 41 of the 455 wild collected
tillers from the invaded range (Fig. 2; Supplementary
Table S1). The only infected plants from the invaded
range were collected from Fisherman, near the northern limit of the invaded range. There was no effect of
time sampled; within a population, all samples were
either infected, or not infected. Our positive controls
were consistently positive (see ‘‘Methods’’). Immunoblot results were validated by screening a subset of
samples (24 negative, 3 positive) with an Epichloëspecific PCR assay, which gave identical results to the
immunoblot.
Germination rates
In our initial assay, germination was significantly
higher in the invaded range (F = 13.101,7.33,
P = 0.0079), and all populations in the invaded range
had higher germination than the native range (Fig. 3a).
In our second germination assay with seeds originating from the common greenhouse study, endophyte infection and seed origin were decoupled
(Fig. 4). We still found significantly higher germination in invasive populations (t = 5.0820.788,
P \ 0.0001), but there was also a significant interaction between range and infection status (F = 8.231,22,
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60
40
20
Seed Germination (%)
80
100
Impact of enemy release and rapid evolution
0
European (Native)
American (Invasive)
0
20
40
60
80
100
Seeds Infected (%)
Fig. 4 Relationship between seed infection by E. sylvatica and
germination. Each point represents a single maternal genotype
originating in the native range (red) or invaded range (blue),
with seed produced being open-pollenated in a common
greenhouse. Two native and two invasive range populations
were included, and there were no statistically significant
differences between populations within a given range (invasive:
P = 0.2311;
native:
t = -0.4811.251,
t = 1.289.966,
P = 0.6402). For linear regressions, the American range has
r2 = 0.20 with P = 0.080; the European range has r2 = 0.24
with P = 0.043. Percentages were transformed using the
standard arcsine square root transformation
Fig. 3 Results from our initial germination rate (a) and growth
rate assays (b), May–June 2008. There is a significant difference
in germination rates for the two ranges (t = 3.975.588,
P = 0.0085), which we initially attributed to the fact that all
native range populations are ubiquitously infected with E.
sylvatica, and all invasive range populations tested lack the
endophyte. There is also a significant difference in growth rates
for the two ranges (t = 16.48159.539, P \ 0.0001), in addition to
differences by individual populations (letters represent differences at P \ 0.05 by pairwise t test)
P = 0.0087), such that there is no difference in
germination rates for infected seeds by range
(t = 1.5911.001, P = 0.1397), while differences in
germination rates of uninfected seed drove the entire
trend (t = 9.318.736, P \ 0.0001). In invasive populations there was a marginally significant trend towards
reduced germination with endophyte infection
(F = 3.791,10, P = 0.0801, r2 = 0.20), but in native
range seeds germination rates significantly increased
with increasing rates of endophyte infection
(F = 5.111,12, P = 0.0432, r2 = 0.24). These results
are in line with Brem and Leuchtmann (2002): they
cite unpublished germination data showing higher
germination rates in endophyte infected seeds in the
native range.
Growth rates
In our initial seedling growth rate assay, we found that
growth rates were significantly higher for plants from
the invaded range (F = 20.921,7.22, P = 0.0024;
Fig. 3b).
Our second growth rate assay was designed to decouple endophyte infection from range of origin. We
found that growth rates were not significantly reduced
in the inoculated treatment for those seedlings from
the native range (Fig. 5a; F = 0.0071,19, P = 0.933),
but were significantly reduced for seedlings from the
invaded range (Fig. 5b; F = 26.041,19, P \ 0.0001).
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R. Vandegrift et al.
A
A
B
B
Fig. 5 Daily growth rate (ratio of change/day) of B. sylvaticum
seedlings in the greenhouse. In seedlings originating in the
plant’s native range (Europe; a), there is no significant
difference (F = 0.0071,19, P = 0.933) between seedlings inoculated with E. sylvatica (red) and those receiving the control
treatment (blue). In seedlings originating in the invaded range
(United States; b), however, there is significant effect of
inoculation (F = 26.041,19, P \ 0.0001)
Interestingly, there was no significant effect of range
origin in this greenhouse experiment once the negatively affected invasive range inoculated plants were
removed from the analysis (F = 0.0661,19,
P = 0.797). There was no significant effect of inoculation on final tiller number of surviving plants for
either range (native: t = 1.894, P = 0.2326; invasive:
t = 1.895, P = 0.2321), nor was there a significant
effect of inoculation on the biomass of surviving
plants for either range (native: t = 1.834, P = 0.2586;
invasive: t = 0.103, P = 0.9996).
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Fig. 6 Survival function estimates for B. sylvaticum seedlings.
In seedlings originating in the plant’s native range (Europe; a),
there is no significant difference (v2 = 1.1, P = 0.299) between
seedlings inoculated with E. sylvatica (red) and those receiving
the control treatment (blue). In seedlings originating in the
invaded range (United States; b), however, there is a significant
effect of inoculation, leading to significantly reduced survivorship (v2 = 34, P \ 0.0001) within the first 2 weeks, and
continuing to drop through time
Seedling mortality
Our second greenhouse experiment tracked mortality
through time, in addition to growth rates. We observed a
significant treatment by range interaction here, with
inoculation not significantly changing mortality for
Brachypodium originating from the native range
(Fig. 6a; v2 = 1.1, P = 0.299), but significantly
increasing mortality for those seedlings originating
from the invaded range (Fig. 6b; v2 = 34, P \ 0.0001).
Impact of enemy release and rapid evolution
Discussion
Pathogen or mutualist?
Harboring this endophyte has fitness costs for B.
sylvaticum, but whether or not an endophyte is a
pathogen or a mutualist depends on the specific
context of host, symbiont, and environment (Carroll
1988; Scholthof 2007). Theoretically, if herbivores are
present that significantly decrease fitness, then
infected plants will have an advantage, provided the
herbivores are deterred by the fungal alkaloids
produced (Richardson et al. 2000; Brem and Leuchtmann 2001). Our results indicate that the endophyte E.
sylvatica has low incidence in the invaded range (9 %
overall; Fig. 2), while literature indicates that it is
nearly ubiquitous in the native range (Fig. 2). In
addition to the Swiss infections reported in Fig. 2
(Bucheli and Leuchtmann 1996; Roy et al. 2011;
Leuchtmann, pers. com.), there are also reports of
infection in Scandinavia, Finland, France, Spain, and
Japan (Eckblad and Torkelsen 1989; Raynal 1994;
Enomoto et al. 1998; Zabalgogeazcoa et al. 2000; Väre
and Itämies 1995). Additionally, Adrian Leuchtmann
reports having seen 100 % infection levels in populations of B. sylvaticum from Holland, Sweden,
England, and Italy. He does, however, note that in
one population from Sardinia, only two out of three
plants were infected (A. Leuchtmann, pers. com.).
Data from the native range indicates that the
endophyte increases resistance to insect herbivory
(Brem and Leuchtmann 2001), but also decreases
competitiveness (Brem and Leuchtmann 2002). We
found that in the invaded range it can be detrimental to
growth rates (Fig. 5) and seed germination (Fig. 4),
but increases seed germination in the native range
(Fig. 4). While the loss of anti-herbivore properties
conferred by the endophyte could have made it more
susceptible to being eaten in the invaded range, data
show that vegetative insect herbivory has little effect
on fitness in either range (Brem and Leuchtmann
2001; Halbritter et al. 2012). However, the observed
elevation in herbivory in the invaded range may be
evidence that loss of the endophyte does mean a loss of
protection (Halbritter et al. 2012).
Brachypodium sylvaticum may be controlled by the
host-specific endophytic fungus E. sylvatica in its
native range, given the effects of the fungus on its
host’s competitive abilities (Brem and Leuchtmann
1247
2002), and the differences in performance between
ranges (Halbritter et al. 2012). This may contribute to
the grass’s success as an invader in the Pacific
Northwest, where Epichloë is largely absent. Given
preliminary germination data in both ranges (Fig. 3a),
we expected to see control by E. sylvatica acting
through reduced germination; however, when infection status and range are deconfounded, it appears that
lower germination in the native range is not caused by
E. sylvatica (Fig. 4). In the absence of the endophyte,
the European seeds germinate at significantly lower
rates than the American seeds, indicating that differences in germination may be determined by genetic
factors. The increase in germination with Epichloë
infection in the native range, and the trend towards
decrease in germination rates with infection in the
invaded range (Fig. 4), lends support to our hypothesis
that B. sylvaticum in the USA has lost defense and/or
tolerance mechanisms (the ability to survive and
reproduce despite being infected; see Roy and Kirchner 2000) through evolution. Demonstrating that the
loss of tolerance is in direct exchange for increased
fitness will be interesting future work.
We show a near total absence of E. sylvatica
infection in the invaded range, as well as a clear loss of
tolerance of such infection by invasive-range B.
sylvaticum from multiple populations throughout the
invaded range (including the only population found to
host the endophyte within that range). It is likely that
ubiquitous infection of B. sylvaticum in Europe is
maintained by a strong selection that is largely absent
in the Pacific Northwest. This selective pressure may
be acting through the seeds: while protection from
folivores proved not to be important in previous
studies (Halbritter et al. 2012), Epichloë may provide
protection from seed-damaging insects or pathogens in
the native range. There is, indeed, higher incidence of
seed-associated insects and pathogens in the native
range than the invasive (Halbritter et al. 2012). Further
studies will be necessary to clarify the role of Epichloë
endophytes in protection of seed.
EICA versus ERH
With regards to enemy release, EICA can be construed
as a sub-case of ERH (Joshi and Vrieling 2005),
though the mechanisms are distinct. Enemy Release
Hypothesis can be explanatory in the absence of
evolution where populations of an organism are
123
1248
directly controlled by co-evolved enemies (Keane and
Crawley 2002; Liu and Stiling 2006), for example as
in Ambrosia artemisiifolia, which seems to not have
lost any defensive capabilities despite herbivore
release upon invasion in France (Genton et al. 2005).
Evolution of increased competitive abilities (EICA),
however, is important when the release from those
enemies provides selective pressures to re-allocate
resources from defense to competitive traits, such as
increased growth and reproduction (Blossey and
Notzold 1995), or production of allelopathic chemicals (Uesugi and Kessler 2013). These two hypotheses
lead to different predictions in our study system
(Fig. 1).
Our experimental design allowed us to assess
evolutionary change in the invaded range, such that
we can effectively distinguish between the ERH and
the EICA hypothesis. We found significantly
increased mortality of inoculated B. sylvaticum originating from the invaded range as compared to their
native range equivalents (Fig. 6). We also found
reduced seedling growth rates in inoculated invasiverange plants (Fig. 5). Both of these results show a loss
of tolerance for the host-specific fungal enemy in the
invasive range populations tested, consistent with
evolutionary loss of defensive mechanisms against
this specific enemy, as predicted by the EICA
hypothesis. The difference in germination rates seems
to point to genetic mechanisms for increased germination in the invaded range, as well as loss of other
factors controlling germination in the native range,
such as seed-damaging pathogens and herbivores.
These facts, taken together, are strong support for the
EICA hypothesis, which predicts such a loss of
defensive mechanisms to specific enemies in
exchange for increased fitness in the invasive range.
Enemy release and invasion history
The story of any invasive species is unique, and while
there may be unifying trends, each species has a
particular history of introduction and a particular
biology that influences its success. B. sylvaticum is no
different; the success of this grass as an invader in the
Pacific Northwest is no doubt influenced by the way in
which it was introduced. During introduction, seed
stock from all over the native range was planted in
USDA test plots near Corvallis and Eugene (Rosenthal
et al. 2008), promoting novel genetic combinations.
123
R. Vandegrift et al.
Rapid range expansion may also have contributed to
evolutionary changes (Rosenthal et al. 2008), independent of selective effects of enemy release. Such
evolutionary drivers are theorized to be more important generally (Felker-Quinn et al. 2013), but it is
necessary to keep in mind the individual nature of
species invasions (Mitchell et al. 2006). This confluence of genotypes and brisk range expansion may have
led to the rapid spread and fixation of resistance genes
in the population, likely before subsequent dispersal,
which is theorized to have been facilitated by logging
in the region of the abandoned USDA test plots
(Rosenthal et al. 2008). This argument supposes that
there is selection for endophyte infection in the native
range that is absent in the invaded range. If this is so,
then it is unlikely to be leaf attacking insect herbivores, as these do not reduce fitness in B. sylvaticum,
and insect herbivory is conspicuously elevated in the
invaded range (Roy et al. 2011). Similarly, it is
unlikely to be a large herbivore, as B. sylvaticum is
unpalatable to most macroherbivores due to high silica
content: rabbit, deer, and other macroherbivore
browsing makes up an extremely small portion of
total plant herbivory for this grass (Brem and Leuchtmann 2001). This appears to be true in both ranges
(Roy et al. 2011). It is more likely to be a seed eating
insect or seed pathogen, or an enemy affecting young
seedlings, as these have stronger effects on fitness
(Roy et al. 2011; Halbritter et al. 2012), and are in line
with our germination results (Figs. 3a, 4).
Alternatively, the endophyte may have been lost
during introduction: seeds could have been treated,
either accidentally or purposefully, in ways that would
have killed seed endophytes. During slow shipment or
uncooled storage in the early twentieth century, seeds
were likely subjected to conditions of heat (37 °C) and
high humidity (*100 %) that would have led to loss
of infection. Storage duration has also been shown to
result in endophyte loss, with endophyte viability
decreasing before seed viability (Gundel et al. 2009).
Endophytes and invasion
Vertically transmitted endophytes are commonly
assumed to be mutualists, and are expected to have a
positive effect on invasiveness (Richardson et al.
2000). For example, Rudgers et al. (2004) said
‘‘Specifically, vertically transmitted fungal endophytes may confer predictable advantages to invading
Impact of enemy release and rapid evolution
grasses when they accompany their host to new
environments (pp. 47)’’. However, there is no reason
to believe that all endophyte infections lead to more fit
plants. In our first assay, we found greatly reduced
growth rates of plants whose seeds originated in the
native range (Fig. 3b), where infection is 100 %, and
in our second assay, we found that endophyte infection
significantly reduced growth rates of plants originating
in the invaded range (Fig. 5). Given the trade-off
between the costs of hosting a given endophyte and the
benefits that such a symbiont can provide in a given
context, it is no surprise that endophyte effects in
invasion ecology are context-dependent. Our results
are consistent with the published results of Brem and
Leuchtmann (2002), who found that when they
removed the endophyte from seeds, the resultant
uninfected plants were faster growing, larger, and
more competitive than infected plants.
Enemy release, in this case, is more complicated
than loss of a single controlling organism upon
invasion. In its native range, E. sylvatica seems to
control B. sylvaticum in some ways (reducing growth
rates and competitive abilities), but those detriments
seem to be off-set by increased germination rates and
potential protection from seed herbivores and pathogens. In the invaded range, the grass is released from
control on growth and competitive abilities imposed
by the fungus, likely because it is not necessary to
harbor such a costly endophyte to maintain high seed
viabilities in the invaded range. Whether that is
through the additional release of control by a seeddamaging organism, or through novel genetic recombination that allows for high germination rates in the
absence of the fungus is still to be determined.
Acknowledgments Adrian Leuchtmann introduced B. Roy to
Brachypodium sylvaticum and Epichloë sylvatica in
Switzerland. Several individuals or agencies kindly allowed us
to work on their land. In Switzerland we thank: Forstämtern,
Grün Stadt Zürich, and Kreisförstern. In the US we thank: S.
DeGhetto and Corvallis Parks for Bald Hill, B. Marshall and
Cascade Timber for Sweet Home, T. Winters and Lane County
Parks for Mt. Pisgah (HBRA), J. Reed and G. de Grassi for
Jasper, D. Johnson for MacForest within the MacDonald-Dunn
Forest, and J. Leroux for Owl; the remaining US sites were
located in the Willamette National Forest. We are most grateful
to K. Blaisdell, A. Clark, M. Cruzan, M. Davis, B. Dentinger, K.
McCulloch, A. Miller, and L. Reynolds for assistance with lab
work and discussions. Funding was provided by NSF DEB0515777 and DEB-0841613, granted to B. A. Roy; R.
Vandegrift was supported by a National Science Foundation
Graduate Research Fellowship, DGE-0829517.
1249
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