Environmental stresses mediate endophyte–grass

interactions in a boreal archipelago

Nora M. Saona1*, Benedicte Riber Albrectsen2, Lars Ericson3 and Dawn R. Bazely1

1Department of Biology, York University, 4700 Keele Street, Toronto ON M3J 1P3, Canada; 2Department of Plant

Physiology, Umea University, SE-901 87 Umea, Sweden; and 3Department of Ecology and Environmental Science,

Umea University, SE-901 87 Umea, Sweden

Summary

1. Both evolutionary theory and empirical evidence from agricultural research support the view

that asexual, vertically transmitted fungal endophytes are typically plant mutualists that develop

high infection frequencies within host grass populations. In contrast, endophyte–grass interactions

in natural ecosystems are more variable, spanning the range from mutualism to antagonism and

comparatively little is known about their range of response to environmental stress.

2. We examined patterns in endophyte prevalence and endophyte–grass interactions across nutri-

ent and grazing (from Greylag and Canada geese) gradients in 15 sites with different soil moisture

levels in 13 island populations of the widespread grass Festuca rubra in a boreal archipelago in

Sweden.

3. In the field, endophyte prevalence levels were generally low (range = 10–53%) compared with

those reported from agricultural systems. Under mesic-moist conditions endophyte prevalence was

constantly low (mean prevalence = 15%) and was not affected by grazing pressure or nutrient

availability. In contrast, under conditions of drought, endophyte prevalence increased from 10% to

53%with increasing nutrient availability and increasing grazing pressure.

4. In the field, we measured the production of flowering culms, as a proxy for host fitness, to deter-

mine how endophyte-infected plants differed from uninfected plants. At dry sites, endophyte infec-

tion did not affect flowering culm production. In contrast, at mesic-moist sites production of

flowering culms in endophyte-infected plants increased with the covarying effects of increasing

nutrient availability and grazing pressure, indicating that the interaction switched from antagonistic

to mutualistic.

5. A concurrent glasshouse experiment showed that in most situations, the host appears to incur

some costs for harbouring endophytes. Uninfected grasses generally outperformed infected grasses

(antagonistic interaction), while infected grasses outperformed uninfected grasses (mutualistic inter-

action) only in dry, nutrient-rich conditions. Nutrient and water addition affected tiller production,

leaf number and leaf length differently, suggesting that tillers responded with different strategies.

This emphasizes that several response variables are needed to evaluate the interaction.

6. Synthesis. This study found complex patterns in endophyte prevalence that were not always cor-

related with culm production. These contrasting patterns suggest that the direction and strength of

selection on infected plants is highly variable and depends upon a suite of interacting environmental

variables thatmay fluctuate in the intensity of their impact, during the course of the host life cycle.

Key-words: antagonist–mutualist continuum, endophyte, Epichloe festucae, Festuca rubra,

grass, herbivory, nutrient stress, Sweden, symbiosis, water stress

Introduction

Endophytes are microorganisms that infect the tissues of

healthy plants but do not cause disease symptoms (Wilson

1995). Vertically transmitted fungal endophytes are obligate

symbionts of pooid grasses (Carroll 1988). These symbionts

spread via their hosts’ seeds, directly coupling the reproduction

of both symbiotic partners. Evolutionary theory predicts that

such symbionts will evolve low virulence, thereby maximizing

the symbionts’ fitness (Ewald 1983, 1987). This pattern has

been widely observed in the agricultural grasses Lolium arundi-

naceum (Tall Fescue) and Lolium perenne (Perennial Ryegrass)*Correspondence author. E-mail: nsaona@yorku.ca

Journal of Ecology 2010, 98, 470–479 doi: 10.1111/j.1365-2745.2009.01613.x

� 2009 The Authors. Journal compilation � 2009 British Ecological Society

infected with fungal endophytes, which has given rise to the

widely accepted idea of a mutualistic relationship (Saikkonen

et al. 2006) existing between them.Here, we use the term ‘endo-

phyte-infected’ to indicate a grass harbouring an endophyte,

without implying anynegative connotation, sensu the use of the

termwith respect tomycorrhizas andnitrogen-fixingRhizobia.

In a mutualistic relationship, the benefits of harbouring the

endophyte should exceed the costs. The cost to the host is that

of providing nutritional support (Ahlholm et al. 2002; Faeth

2002). In turn, the host is protected from herbivores and

pathogens by a range of defensive alkaloid compounds synthe-

sized by endophytes (Cheplick&Clay 1988), and often demon-

strates increased drought and mineral stress tolerance (West

et al. 1993; Ravel et al. 1997a; Monnet et al. 2001) and bio-

mass accumulation (Belesky, Stringer & Hill 1989). Despite

the cost, endophyte infection does not always reduce herbivore

performance (Saikkonen et al. 1999; Faeth 2002) and recent

research has even documented negative effects of endophytes

on the predators of herbivores feeding on infected host plants,

potentially creating multi-trophic feedbacks that will affect

patterns of herbivory (de Sassi, Muller & Krauss 2006; Harri,

Krauss &Muller 2008, 2009).

While the broader significance of endophytes in structuring

plant communities remains unclear, studies show that endo-

phytes can affect plant community dynamics (Rudgers et al.

2007), limit transmission of viruses among plants (Lehtonen

et al. 2006), alter competitive relationships among plants (Mal-

inowski, Belesky&Fedders 1999;Richmond,Grewal&Cardi-

na 2003; Faeth, Helander & Saikkonen 2004) and influence

seed survival and germination (Gundel et al. 2006; Wali et al.

2009). In comparison with numerous studies with agricultural

grass species, there are fewer studies on endophytes in natu-

rally occurring native grass populations. Some of these studies

suggest that the endophyte–grass interaction is not always

mutualistic (e.g. Faeth 2002; Faeth & Fagan 2002; Faeth &

Sullivan 2003; Saikkonen et al. 2004). Furthermore, in con-

trast to agronomic species, where infection rates tend to be

consistently high (Spyreas, Gibson & Basinger 2001; Piano

et al. 2005; Ju et al. 2006), endophyte infection frequencies in

natural systems tend to be lower andmore variable (Saikkonen

et al. 2000; Bazely et al. 1997, 2007;Wali et al. 2007).

The mutualism to antagonism continuum hypothesis sug-

gests that the endophyte–grass interaction varies according to

prevailing abiotic conditions such as nutrient and water avail-

ability (Saikkonen et al.1998;Muller&Krauss 2005), although

the relative importance of these various interacting factors

remains poorly understood (Schulz & Boyle 2005). Schulz &

Boyle (2005) also suggested that the symbiosis spans the entire

continuumfrommutualismtoantagonism,becauseof the shift-

ingbalancebetween fungal virulence andplantdefence.

Biotic stress, such as herbivory, influences endophyte–grass

interactions because both vertebrate and invertebrate herbi-

vores have generally been observed to prefer uninfected grasses

and avoid infected grasses (Clay 1996; Conover & Messmer

1996; Shiba & Sugawara 2005). The ingestion of infected

grasses results in decreased growth and fecundity and

increased mortality compared with animals on endophyte-free

diets (e.g. Bazely et al. 1997; Conover 2003; Meister et al.

2006). This anti-herbivore defence confers a fitness advantage

on grass hosts, resulting in a mutualistic association (Clay

1988). However, the defence function is not always demon-

strated for infected native grasses (Faeth 2009). The outcome

of a plant–herbivore interaction is often far from clear and

recent research documenting the cascading effects of endo-

phytes on the predators of some insect herbivores feeding on

infected plants (e.g. de Sassi, Muller & Krauss 2006; Harri,

Krauss &Muller 2008, 2009) places the defensive nature of en-

dophytes directly within the complex framework of indirect

interactionwebs (Ohgushi 2005).

In the field, increased herbivore pressure has, in some stud-

ies, been associated with increased endophyte prevalence, sug-

gesting that past and current selective foraging may have

created the pattern of prevalence maintaining the defensive

mutualism (Bazely et al. 1997; Koh & Hik 2007, 2008). How-

ever, while high infection may be correlated with high herbiv-

ory pressure, a range of other correlated factors may also

account for these patterns (Gundel et al. 2008).

The state of the symbiosis between the host and the endo-

phyte should, in theory, affect host reproductive success and

therefore, endophyte prevalence in host populations. Under

situations of limited resource availability, endophytes may

interact antagonistically with the grass, perhaps as a result of

the metabolic costs incurred by the host of supporting the

endophyte (Cheplick, Perera&Koulouris 2000; Ahlholm et al.

2002). Here, endophyte-infected plants should have relatively

low fitness, and infection prevalence should decline rapidly

(Ravel, Michalakis & Charmet 1997b). In contrast, in mutual-

istic associations, infection should increase over time (Saikko-

nen et al. 1998). Thus, prevalence has been used as a marker

for a mutualistic interaction, as has reproductive output in the

formof increased seed set and flowering culm production.

We investigated variability in the relationship between the

systemic grass endophyteEpichloe festucae and its hostFestuca

rubraL. in naturally occurring island populations in a Swedish

archipelago, with respect to abiotic and biotic environmental

gradients. We asked if the endophyte–grass interaction

changes (i) across nutrient and herbivory gradients and (ii)

under two contrasting soil moisture regimes namely dry vs.

mesic-moist. We used two response variables: endophyte prev-

alence and number of flowering grass culms as a proxy for host

fitness. We hypothesized that the endophyte–grass interaction

should vary from mutualistic to antagonistic across the gradi-

ents, depending on water availability. We thus expected the

interaction to be more mutualistic in nutrient-rich, moist and

highly grazed populations both when measured as endophyte

prevalence and as flowering culm production.

Materials and methods

STUDY SYSTEM

Epichloe festucae Leuchtmann, Schardl and Siegel (Ascomycota:

Clavicipitaceae) is a fungal endophyte that forms systemic, intercellular

infections in cool-season grasses. The hyphae grow only in the hosts’

Endophytes, grasses and environmental stress 471

� 2009 The Authors. Journal compilation � 2009 British Ecological Society, Journal of Ecology, 98, 470–479

aerial tissues and E. festucae is transmitted vertically through host

seeds (asexual reproduction) and horizontally by meiotic ascospores

(sexual reproduction). The latter has not been observed in the study

area (L. Ericson, pers. obs.) nor has it been reported from northern

Sweden (cf. Eriksson 1992) or northern Finland (Wali et al. 2007).

However, horizontal transmission, while rare (Faeth 2002), cannot

be entirely discounted (Wali et al. 2007). Epichloe festucae produces

alkaloids that deter herbivory by vertebrates and invertebrates

(Wilkinson et al. 2000; Clay & Schardl 2002; Vazquez de Aldana

et al. 2003; Jensen, Mikkelsen & Roulund 2007). Although F. rubra

plants from the study area were not directly screened for alkaloids,

E. festucae in grass from other European populations have been

confirmed to produce alkaloid compounds (Vazquez de Aldana

et al. 2007).

Festuca rubraL. (Poaceae) is a widespread perennial grass native to

Eurasia and with a circumpolar distribution (Hulten & Fries 1986)

and host to E. festucae. Festuca rubra is a naturally occurring, com-

mon and abundant species at our study site in the Skeppsvik archipel-

ago (63�48-51¢ N, 20�35-40¢ E), north of Umea, Sweden, in the Gulf

of Bothnia. Festuca rubra is the dominant characteristic species of

open shores and grows in distinct, rounded tussocks. Here, soil mois-

ture is important for the type of vegetation cover: onmesic-moist sites

F. rubra forms a closed grass sward and an organic soil layer, while on

dry sites it is more scattered. Festuca rubra is a polymorphic taxon

with still unclear intraspecific variation (Markgraff-Dannenberg

1980). The populations in the study area belong to a morphologically

distinct taxon restricted to the sea shores of the northern and eastern

parts of the Baltic area (cf. Vare 2007).

SKEPPSVIK ARCHIPELAGO SURVEY

Characterization of collection sites

From June to August 2005, we collected tillers from 40 individuals

on each of 13 of c. 100 islands in the Skeppsvik archipelago. The

selected islands are all located in the outer half of the archipelago

within an area of 1.7 · 3.5 km. The maximum distance between two

islands was 3.2 km and the shortest distance was 0.3 km. Islands

varied in size from c. 400 to 50 000 m2. Grasses on these islands are

exposed to varying abiotic (soil moisture, nutrient availability) and

biotic (herbivore pressure) conditions. Based on extensive experience

of this archipelago accumulated since the 1970s, we selected islands

so that they would span both abiotic (soil moisture, nutrient avail-

ability) and biotic (herbivory by the native Greylag goose, Anser

anser and the introduced Canada goose, Branta canadensis) gradi-

ents that we hypothesized to be creating significant variation in

stress levels. Sampling was also performed to cover a range of island

ages. On two of the 13 islands, sampling was performed at each of

two nearby sites that differed in nutrient availability (island 3) and

in nutrient and water availability (island 2). Therefore sampling

included 15 sites in total. This was subsequently accounted for in

statistical analyses.

We characterized each island by its overall level of water, nutrient

and herbivore stress. We scored soil moisture on an island as (0) very

dry, (1) dry, (2) mesic and (3) moist. The reason for scoring soil mois-

ture is that the Baltic coast is characterized by its low annual precipi-

tation, in general less than 350 mm (Sjors 1999). This implies that the

climate in spring-early summer is semi-arid which, in combination

with extended drought periods, plays an important role in vegetation

differentiation (cf. Sjors 1999). A mesic-moist site is characterized by

low moisture stress and drought damage does not occur or will occur

only rarely (about once during a 20-year period). A dry site is charac-

terized by highmoisture stress and it suffers during prolonged periods

of drought, about every second year and severely so every fifth year.

We scored nutrient availability on the islands according to a scale

increasing from 0 (low) to 5 (high). To estimate nutrient status, we uti-

lized the species composition of the lichen vegetation that occurred

on boulders and cliffs. We selected seven common species that repre-

sent a decreasing gradient in nutrient availability (Xanthoria candelar-

ia, Physcia caesia, Caloplaca scopularis, Neofuscelia pulla, Lecidea

pantherina, Parmelia saxatilis andUmbilicaria torrefacta) (cf. Ericson

& Wallentinus 1979). Based upon their ranking and abundance at

each island (site), we obtained an index for nutrient availability for

each island (site), ranging from 0 (low) to 5 (high).

We estimated herbivore pressure by scoring, on a scale from 0 to 5,

the damage to grass tussocks from goose herbivory. An island was

scored as 0 if there was little to no visitation by geese to the island and

little to no herbivore damage to plants. An island was scored as 5 if

there was high visitation by geese and high levels of grazing damage

to plants, characterized by most F. rubra tussocks having tillers

grazed to ground level. Geese grazing in the archipelago have been

scored since the early 1980s (L. Ericson, unpubl. data). During this

period, the ranking of the study islands with regard to grazing pres-

sure has remained unchanged because both species prefer to forage

on open islands without any shrubs and trees. Breeding gulls (Larus

spp.) may also forage on grasses, although much less intensely (L.

Ericson, pers. obs.). Since gulls also breed on the small open islands

that are preferred by geese, our grazing index does not discriminate

between the different vertebrate herbivores, although the Greylag

goose is by far the most abundant species. Canada geese are able to

discriminate between endophyte-infected and uninfected grass, and

selectively forage for uninfected grasses while avoiding infected plants

(Conover & Messmer 1996). We are not aware of any similar studies

for Greylag geese but we assumed similar behaviour.

We used island age, range 20–1050 years, as an indicator of grass

population age. Because of isostatic rebound from the retreating gla-

ciers that once covered this area, land is uplifting at a relatively con-

stant rate of c. 0.9 cm year)1(Ekman 1996). Since F. rubra will

regularly colonize newly exposed islands around mean sea level, we

can reliably estimate the oldest age of the F. rubra population on each

island (cf. Carlsson et al. 1990). We assumed that grass populations

on older islands or growing at higher elevations within islands were

older than grass populations on younger islands or growing at lower

elevations. This estimate of grass population age may tend to overes-

timate age because young grass populations may exist at higher eleva-

tions as a result of seed dispersal. However, since the opposite is not

true (old populations do not exist at low elevations or on young

islands), we were confident that on average, older populations occur

on older islands and that island age is a correlate of population age.

Measurements

While tillers were arbitrarily sampled from tussocks, all sampled tus-

socks were at least two metres apart from each other to ensure that

we were collecting from separate genotypic individuals. We estimated

grass performance by counting the number of culms produced by

each individual tussock. To strengthen our estimate of host produc-

tivity, we also measured the area covered by each tussock. Tussock

area was estimated from the length of the major axis (longest diame-

ter) andminor axis (shortest diameter), using the formula

Area ¼ ½pðmajor axis�minor axisÞ�=4

We evaluated endophyte infection status of individual F. rubra

plants by preserving samples in acidified alcohol (1:2 glacial

472 N. M. Saona et al.

� 2009 The Authors. Journal compilation � 2009 British Ecological Society, Journal of Ecology, 98, 470–479

acetic acid : 95% ethanol) and screening the pseudostem of each

tiller for endophyte infection using a Phytoscreen Immunoblot

Assay (Agrinostics Inc., Watkinsville, GA, USA) (Koh et al.

2006). To visually verify endophyte infection in the flattened leaf

sheath, we stained a subsample of these tillers with aniline blue

(Koh et al. 2006) and viewed it with a compound microscope at

400· magnification.

GLASSHOUSE EXPERIMENT

We conducted a glasshouse experiment to determine how water and

nutrient supplementation affect endophyte–grass interactions. We

collected 10 F. rubra tussocks, including endophyte-infected and

uninfected individuals, from a wave-washed morainic seashore,

Brannolandet, located 10 km SSW of the Skeppsvik archipelago

(63�44¢ N, 20�26¢ E). Tussocks were isolated (surrounded by gravel)

at least 2 m from each other; therefore we can safely assume them to

be 10 different genotypes. Our preliminary survey of this site revealed

that infection prevalence was low (less than 5%). We separated indi-

vidual tillers with roots from each tussock and planted 36 tillers per

genotype individually into 4 · 4 cm pots containing low-nutrient

sowing soil and perlite. We grew plants in a glasshouse under natural

light and temperature conditions. We randomly assigned tillers

within the same genotype to one of four treatments: (i) lowwater ⁄ lownutrient, (ii) high water ⁄ low nutrient, (iii) low water ⁄ high nutrient

and (iv) high water ⁄ high nutrient. The experiment began with nine

tillers per treatment per genotype, with 360 tillers in total. Pots were

watered until the soil was thoroughly soaked every day (high) or

twice per week (low). Plants received 1.5 g of Weibulls (N:P:K

= 6:2:4) pulverized slow-release fertilizer pellet (high) or no nutrient

supplementation (low). After 6 weeks, we randomly selected three

pots per treatment per genotype and harvested them by cutting tillers

at soil level. We estimated plant productivity by the length of the lon-

gest leaf, number of leaves and number of daughter tillers produced.

As with the archipelago survey, we used a Phytoscreen Immunoblot

Assay to confirm the infection status of each plant.

STATIST ICAL ANALYSES

To test how endophyte prevalence varied with water, nutrient and

grazing indices, we used logistic regression, with host infection status

(yes or no) as the dependent variable and water index, nutrient

index, grazing index and island age as independent variables. Each

island was sampled once, except for two islands that were each sam-

pled twice. Given that repeated observations on the same island are

not statistically independent, we used a repeated-measures analysis

to account for correlations among observations at the same site. The

test statistic for this analysis is the likelihood ratio (G). To test how

the endophyte–grass interaction varied with environmental stresses,

we used a mixed model with number of culms as the dependent vari-

able and tussock area, host infection status (yes or no), water index,

nutrient index, grazing index and island age as independent variables

and island as a random effect. We included area in the model since

the relation between culm production and infection may be con-

founded by tussock area. To visualize the interaction, we have classi-

fied moisture into two categories: Dry and Moist. The ‘Dry’

category included values less than 2, whereas the ‘Moist’ category

included moisture values from 2 to 3. We square-root transformed

the number of culms to normalize residuals. We assessed the direc-

tion of the endophyte–grass interaction along the antagonism–mutu-

alism continuum by the difference in production of culms between

infected and uninfected individuals, controlling for differences in

tussock area. Within a site (same environmental conditions), if the

number of inflorescences produced by infected plants was greater

than uninfected plants (positive net benefit), then we interpreted this

as a mutualistic interaction between the grass host and its endo-

phyte. If the difference was negative (negative net benefit), then we

interpreted this as an antagonistic interaction. We interpreted a

greater difference between infected and uninfected grasses as stron-

ger mutualisms or antagonisms. We used Spearman’s rank correla-

tion to evaluate the relationship between nutrient and grazing

indices and net culm benefit.

For the glasshouse experiment, to determine how endophyte infec-

tion and experimental treatment affected plant performance, we used

mixed models with tiller production, total leaves produced, number

of leaves per tiller or length of longest leaf after 6 weeks of growth as

a dependent variable and endophyte infection status and soil treat-

ment as independent variables. We tested differences between the

number of tillers, leaves, number of leaves per tiller and length of lon-

gest leaves between endophyte infected and uninfected individuals for

each treatment with linear contrasts. To determine the independent

effects of infection status, nutrient and water supplementation on til-

ler production, we used a mixed model with tiller production, total

leaves produced, number of leaves per tiller or length of longest leaf

as a dependent variable and infection status, nutrient level (high ⁄ low)and water availability (high ⁄ low) as independent variables. In all

cases, tussock identity was the random effect. All analyses were per-

formed using sas version 9.1 (SAS Institute, Cary, NC,USA). In these

analyses, we began with a saturated model and arrived at the final

model using a backwards stepwise elimination process (Sokal &

Rohlf 1995).

Results

ENDOPHYTE PREVALENCE IN THE SKEPPSVIK

ARCHIPELAGO

Although endophyte-infected F. rubra plants were present on

all 13 islands, no E. festucae stromata were observed on F. ru-

bra culms, indicating that the endophytes were not reproduc-

ing sexually. The observed prevalence of F. rubra infected with

E. festucae varied widely among islands, from 10.0% to 52.5%

(mean = 20.8%; Fig. 1) (logistic regression: G = 44.36,

P < 0.001) but was low in general. High levels of endophyte

prevalence were only observed on dry, nutrient-rich islands

that experienced high grazing pressure. The island with the

lowest endophyte prevalence was characterized by mesic areas

having a low nutrient index and high grazing pressure.

Endophyte prevalence among islands reflected variation in

moisture, nutrient availability, grazing pressure and island

age (Table 1). First, endophyte prevalence increased with

increasing nutrient availability at dry sites (moisture · nutri-

ent index: G = 5.44, P = 0.02; Fig. 1a) but did not vary at

moist-mesic sites. Second, endophyte prevalence increased sig-

nificantly with increasing grazing pressure at dry sites, but

prevalence did not vary at mesic-moist sites (moisture · graz-

ing pressure: G = 4.23, P = 0.04; Fig. 1b). Furthermore,

endophyte prevalence also increased with decreasing moisture

availability on older islands (G = 6.34, P = 0.0118), but

there was no relationship between prevalence and moisture

on younger islands.

Endophytes, grasses and environmental stress 473

� 2009 The Authors. Journal compilation � 2009 British Ecological Society, Journal of Ecology, 98, 470–479

REPRODUCTIVE ALLOCATION IN FESTUCA RUBRA

The number of flowering culms produced by F. rubra tussocks

varied with endophyte infection, water and nutrient availabil-

ity, and the intensity of goose grazing (Table 2). Each

explained a significant proportion of the variance in culm pro-

duction. Although there was no difference in the number of

culms produced by infected and uninfected plants across soil

nutrient availability gradients at dry sites (nutrient · infection:

F1,238 = 0.13, P = 0.7170), at moist sites, culm productivity

increased as nutrient availability increased for endophyte

infected plants, but decreased for uninfected plants (nutri-

ent · infection:F1,145 = 8.17,P = 0.0049).

The interaction of the symbiosis along the antagonism–

mutualism continuum varied with the level of nutrient and

water availability. At dry sites, there was no observed rela-

tionship between nutrient index and net culm benefit (Spear-

man rank correlation: r = )0.05, P = 0.9075; Fig. 2a). In

contrast, at moist sites the net benefit of endophyte infection

to the grass host increased with nutrient availability

(Fig. 2a). Uninfected grasses outperformed endophyte-

infected grasses at low nutrient availabilities, but at higher

nutrient availabilities, infected grasses outperformed unin-

fected ones. At moist sites, in conditions of low nutrient

availability, there was a net negative benefit to grass hosts of

having endophytes, indicating an antagonistic interaction.

However, as nutrient availability increased, the net benefit to

hosts increased and endophyte–host interactions became

positive and more mutualistic (Spearman rank correlation:

r = 0.87, P = 0.0103; Fig. 2a). At the majority of the moist

sites, the net benefit of harbouring endophytes was negative,

indicating that most endophyte–grass interactions were

antagonistic.

The interaction of the symbiosis also varied with grazing

pressure (Fig. 2b). At dry sites, there was no relationship

between grazing index and net culm benefit (Spearman rank

correlation: r = )0.30, P = 0.5142; Fig. 2b). In contrast, at

moist sites the net benefit to hosts of endophyte infection

increased with increasing grazing pressure (Spearman rank

correlation: r = 0.92, P = 0.0034; Fig. 2b). Uninfected

grasses outperformed endophyte-infected grasses at low graz-

ing pressures, but at higher grazing pressures, infected grasses

outperformed uninfected ones. As nutrient availability

increased, the net benefit to hosts increased and endophyte–

host interactions changed from negative to positive and the

interaction became more mutualistic at mesic-moist sites

(Fig. 2b).

Fig. 1. Relationships between endophyte prevalence and (a) soil

nutrient availability and (b) intensity of grazing on dry and mesic-

moist islands. Prevalences are the predicted values from a statistical

model that includes water, nutrient and grazing indices, and island

age as explanatory factors.

Table 1. Summary of logistic regression statistics for endophyte

prevalence in Festuca rubra growing at sites with gradients in grazing

intensity, moisture and nutrient availabilities

Source d.f. G P

Age 1 3.93 0.0474

Grazing 1 3.80 0.0514

Moisture 1 2.24 0.1345

Nutrient 1 4.75 0.0293

Age · Moisture 1 5.41 0.0201

Grazing · Moisture 1 4.16 0.0413

Nutrient · Moisture 1 4.93 0.0264

Nutrient, moisture and grazing refer to their respective indices.

Age refers to the age of the island where plants were collected.

Table 2. Summary of mixed model statistics for production of culms

in Festuca rubra growing at sites with gradients in grazing intensity,

moisture and nutrient availabilities

Effect d.f. F P

Area 1,383 117.25 <0.0001

Infected 1,383 0.01 0.9229

Moisture 1,383 0.22 0.6357

Nutrient 1,383 18.03 <0.0001

Grazing 1,383 6.52 0.0110

Nutrient · Infection 1,383 0.24 0.6222

Moisture · Infection 1,383 5.88 0.0157

Nutrient · Grazing 1,383 9.69 0.0020

Moisture · Nutrient 1,383 1.72 0.1906

Moisture · Nutrient · Infection 1,383 6.75 0.0097

Nutrient, moisture and grazing refer to their respective indices.

Area refers to the area covered by a tussock. Numerator and

denominator degrees of freedom are shown for all effects.

474 N. M. Saona et al.

� 2009 The Authors. Journal compilation � 2009 British Ecological Society, Journal of Ecology, 98, 470–479

GLASSHOUSE EXPERIMENT

All individuals produced tillers during the experiment, indicat-

ing that the nutrient and water treatment levels selected for the

experiment were within tolerable limits for the grasses. The

patterns observed in the glasshouse experiment showed

that endophyte-infected (E+) and uninfected (E)) grasses fol-lowed different strategies. E) plants produced more tillers

(Fig. 3a, LSmean±SE throughout; E+: 7.98±0.40, E): 9.97±0.59, F1,103 = 7.87, P = 0.0060) and total number of

leaves (Fig. 3b, E+: 19.80±0.97, E): 23.14±1.45, F1,103 =

3.69, P = 0.0576), whereas E+ plants formed both longer

leaves (Fig. 3d, E+: 18.99±1.40 cm, E): 13.89±2.13 cm,

F1,103 = 3.99, P = 0.0484) and more leaves per tiller (Fig. 3c,

E+: 2.52±0.05, E): 2.31±0.07, F1,103 = 6.45, P = 0.0126).

We found significant within-treatment differences between E+

and E) plants for all response variables. E) plants produced

more tillers than E+ plants in the low water ⁄ low nutrient

(Fig. 3a, F1,100 = 3.96, P = 0.0493) and high water ⁄highnutrient treatments (F1,100 = 6.28, P = 0.0138) and more

leaves than E+plants in the low water ⁄ low nutrient treatment

(Fig. 3b, F1,100 = 4.60, P = 0.0343). E+ plants produced

more leaves per tiller in the high water ⁄high nutrient treatment

(Fig. 3c, F1,100 = 8.80, P = 0.0038) and longer leaves in the

low water ⁄ low nutrient treatment (Fig. 3d, F1,100 = 4.71,

P = 0.0324).

Nutrient and water addition affected tiller production, leaf

number and leaf length differently. Water supplementation

had a positive effect on tiller production (LS mean±SE

throughout; high: 10.17±0.48, low: 7.78±0.49,

F1,104 = 13.36, P = 0.0004) and total leaf production (high:

24.73±1.17, low: 18.21±1.19, F1,104 = 16.54, P < 0.0001),

whereas fertilization negatively affected the production of both

tillers (high: 8.19±0.49, low: 9.76±0.48, F1,111 = 5.73, P =

0.0185) and leaves (high: 19.05±1.19, low: 23.88± 1.17,

F1,104 = 16.54, P < 0.0001). Neither nutrient nor water addi-

tion affected the length of the longest leaf (nutrient:

F1,104 = 0.71,P = 0.4003; water: F1,104 = 1.67P =0.1989).

We did not find significant interactions between endophyte

infection status and soil treatment for any response variable.

Fig. 2. Relationship between (a) soil nutrient availability and (b)

grazing intensity and benefit of endophyte infection. The benefit of

infection is the difference in the mean number of culms produced by

endophyte-infected and uninfected grasses, predicted from a statisti-

cal model that includes grass infection status, individual tussock area,

and water, nutrient and grazing indices as explanatory factors. The

Moisture index was categorized as ‘dry’ and ‘mesic-moist’, as

described inMaterials andmethods.

(a) (b)

(c) (d)Fig. 3. Effects of soil treatments and endo-

phyte infection status of Festuca rubra on (a)

mean number of tillers, (b) mean number of

leaves, (c) number of leaves per tiller and (d)

length of the longest leaf produced by endo-

phyte-infected (E+) and uninfected (E))genotypes. Treatments: O = low nutrients,

low water; N = high nutrients, low water;

W = low nutrients, high water;

NW = high nutrients, high water. Asterisks

indicate significant differences between E+

and E) plants within a treatment group

(*P < 0.05, **P < 0.01).

Endophytes, grasses and environmental stress 475

� 2009 The Authors. Journal compilation � 2009 British Ecological Society, Journal of Ecology, 98, 470–479

Discussion

COMPLEX FACTORS DRIVE ENDOPHYTE FUNCTION IN

GRASSES

Our results clearly illustrate the highly variable and complex

nature of the endophyte–grass symbiosis in the studied system.

First, we found marked variation in endophyte prevalence

across nutrient and herbivore gradients. Second, this variation

differed depending upon soil moisture regime. Third, we found

no relationship between endophyte prevalence and production

of flowering culms, which suggests that the direction and

strength of selection on infected plants is variable. Thus, over-

all, the observed patterns were not as we hypothesized, namely

that the interaction should always be more mutualistic under

(i) increased resource availability (nutrient rich and mesic-

moist sites) and (ii) on grazed sites.

Our results support the view that the endophyte–grass sym-

biosis represents a complex interaction that may vary along an

antagonist–mutualist continuum (Schulz & Boyle 2005).

According to the Metapopulation Theory (Gilpin & Hanski

1991) and the Geographic Mosaic Theory of Coevolution

(Thompson 2005), organisms are patchily distributed in some-

what isolated populations in heterogeneous landscapes where

the direction and strength of selection pressures may change

over space and time. In this framework, the patterns we

observed in this studymay indicate that the symbiosis is at var-

ious stages of interaction on the different islands. Several stud-

ies on symbiotic systems have emphasized the conditional

outcome of these relationships and that resource gradients can

create selection mosaics across habitats (Hochberg et al.

2000). We identified and characterized a number of important

environmental gradients considered to be of importance for

endophyte–grass interactions (Saikkonen et al. 1998) and

detected a number of correlated patterns for the endophyte–

grass interaction. Random effects alone cannot explain the

consistency of these patterns. Instead, we believe that

the observed patterns help to illustrate the complex nature of

the symbiosis.

THE ROLE OF SOIL MOISTURE

We found that endophyte prevalence was generally low under

mesic-moist conditions but that endophyte prevalence on dry

sites increased along the nutrient and herbivory gradients. This

suggests that at mesic-moist sites, the ecological cost of endo-

phyte infection may be high and it may be attributed to stron-

ger competitive interactions in the closed grass sward

characteristic of mesic-moist sites (cf. Study system). In con-

trast, on dry sites, characterized by more sparsely distributed

grass tussocks (cf. Study system), intraspecific competition is

likely to be weaker, resulting in an increased survival of endo-

phyte-infected plants. The observed trend towards increasing

prevalence on such sites may be explained if successful survival

of infected individuals is favoured by increased nutrient avail-

ability. This may reflect either that natural enemies, in general,

depend upon the nutritional status of the host for their success-

ful survival and development (Burdon, Thrall & Ericson

2006), or an increase in endophyte transmission efficiency

(Afkhami & Rudgers 2008) under more benign environmental

conditions. Moreover, because the nutrient availability of an

island is tightly coupled to the presence of herbivores in this

archipelago, it is impossible to separate the positive effect of

nutrients and grazing for this data set.

Thus, we demonstrated a positive effect of nutrient (and

grazing) levels both in moist and dry areas, expressed, how-

ever, in two different ways: as enhanced prevalence on dry sites

and as a reproductive gain on moist sites. This suggests that

synergistic interactions may be expressed in different charac-

ters, in our case prevalence and potential fitness gains. This

confirms the complexity of the interaction in natural systems,

which may be affected by changed intraspecific interactions as

a result of past grazing history (cf. Koh & Hik 2007, 2008). It

further suggests that several responses must be considered

simultaneously when assessing the nature of the interaction.

Our observation that field data on endophyte prevalence

cannot be easily used to evaluate endophyte interaction state

has an interesting parallel in the work on F. rubra in northern

Finland (Wali et al. 2007, 2009). Wali et al. (2007) found that

meadow populations generally showed high prevalence values

(around 50%) whereas river bank populations were consider-

ably lower (10–20%). However, in their study of seedling

establishment, Wali et al. (2009) also found that germination

of endophyte-infected plants was faster and plants showed an

increased survival and growth on the river banks, suggesting a

benefit of the endophyte under the harsher environmental con-

ditions characteristic of the alluvial habitat. Thus, they found

themost positive effects of endophytes at sites characterized by

low prevalences. In this study, seedling establishment may be

linked to a combination of water, nutrient and herbivore stres-

ses, which may change the selective advantage of harbouring

endophytes. The studies by Wali et al. (2007, 2009) together

with our data suggest that to understand the outcome of endo-

phyte–grass interactions in natural systems we must consider

that selection may change during various stages of the host

ontogeny (Ahlholm et al. 2002; Olejniczak&Lembicz 2007).

EVIDENCE OF SELECTION

The significant differences in endophyte prevalence among the

Skeppsvik islands suggest that there is a selective advantage to

endophyte infection at some sites but not at others. We found

that dry sites were often associated with higher endophyte

prevalence, suggesting some selective advantage of the associa-

tion under these conditions, while this was never the case on

mesic-moist sites. This supports the hypothesis that water

availability is one such factor which balances the antagonist–

mutualist continuum. As found by Novas, Collantes & Cabral

(2007), such patterns may differ widely also between species in

the same geographical region. In their field study in Patagonia,

they found that endophyte prevalence in Bromus setifolius

increased with increasing water availability while the reverse

was found for Phleum alpinum, emphasizing the complex nat-

ure of endophyte–grass relationships. Likewise, in a study

476 N. M. Saona et al.

� 2009 The Authors. Journal compilation � 2009 British Ecological Society, Journal of Ecology, 98, 470–479

from the Scandinavianmountains,Granath et al. (2007) found

that with increasing altitude endophyte prevalence decreased

in F. rubra, a species of more nutrient-rich and mesic sites, but

increased in F. ovina, a species of more nutrient-poor and dry

sites. These contrasting patterns highlight the need to unravel

the relative importance of both abiotic and biotic factors for

the antagonist–mutualist continuum.

The pattern of endophyte infection on the Skeppsvik islands

may be due to a combined effect of a selective advantage under

drought conditions, the prevailing climatic conditions in Baltic

archipelagos (Ericson & Wallentinus 1979; Sjors 1999) and

herbivore protection resulting in the selective foraging by geese

(Conover & Messmer 1996). Such patterns are in accordance

with earlier work demonstrating the strong effects of grazing

by herbivores and moisture conditions on endophyte–grass

relationships (Clay 1996; Morse, Day & Faeth 2002).

Although we lack accurate data for Greylag geese, Canada

geese are able to discriminate between endophyte-infected and

uninfected grass and selectively forage for uninfected grass

(Conover & Messmer 1996); therefore endophyte-infected

plants at heavily grazed sites may be at a selective advantage,

resulting in their decreased mortality. However, reduced intra-

specific competition as a result of increased grazing may possi-

bly over-ride the importance of selective foraging. In the study

area, the geese arrive soon after snow melt (in late April) to

their breeding grounds and forage on the sparse green foliage.

Most years, this time and early summer coincide with a pro-

longed drought period, often resulting in extensive mortality of

F. rubra (L. Ericson, unpubl. data).

Our data also show an increase in endophyte prevalence at

drought-stressed sites, especially on old islands, suggesting that

endophyte-infected plants may experience a selective advan-

tage and are selected for under these drought conditions. In the

absence of recurrent and severe drought events and subsequent

drought damages, endophyte-infected plants would not experi-

ence a similar selective advantage. If so, this implies that intra-

specific competitive interactions may play a more important

role in the low prevalence found on mesic-moist sites. This

explanation is supported by the glasshouse experiment, which

shows that the endophyte-infected plants in general showed a

lower tiller production. Our glasshouse experiment found that

E+and E) plants represent two different strategies. E+plants

produced both longer leaves andmore leaves per tiller than E)plants. Furthermore, E+ plants produced both fewer tillers

and total number of leaves than E) plants. However, this pat-

tern changed under dry, nutrient-rich conditions. Under these

conditions, we found a shift that suggested a relative advantage

for E+plants over E) plants, both with regard to number of

tillers as well as total number of leaves. Thus, the experiment

suggests that E+plants in general have a relative disadvantage

compared with E) plants, particularly in response to increased

water availability, which is in accordance with the low preva-

lence of E+ plants on mesic-moist sites in the archipelago,

where competitive interactions are more likely to predominate

in the closed grass sward. Furthermore, the experiment sug-

gests an increased advantage, at least early in life, for E+plants

under the combination of severe drought conditions (cf.

Morse, Day&Faeth 2002; Cheplick 2007) and increased nutri-

ent availability. This may explain the pattern observed in the

studied archipelago, where we found higher prevalences only

on nutrient-rich, dry sites. Interestingly, high E+prevalences,

often close to 100%, seem to characterize areas with Mediter-

ranean climates, with regular, severe drought periods (e.g.

southern France (Lewis et al. 1997) and Spain (Zabalgogeaz-

coa et al. 1999, 2006)). It may be argued that the duration of

our glasshouse experiment was short in comparison with the

natural life span of the grass host and that these results should

be interpreted with caution. However, it is important to note

that this short duration is relevant for testing how selection

operates early in the host life and for understanding why we

found more E+plants on dry, nutrient-rich sites in the archi-

pelago.

This study casts light upon the intricate nature of an endo-

phyte–grass association and demonstrates that this symbiosis

is strongly affected by both abiotic and biotic factors that inter-

act in a complicated manner. Our results suggest that these

interacting factors are likely to obscure the nature of endo-

phyte–grass interactions and that simple single factor

responsesmay be rare.

Acknowledgements

This research was supported byYork International (N.M.S.), YorkUniversity,

NSERC (D.R.B.), International Polar Year (D.R.B.), The Swedish Research

Council (L.E.), Carl Tryggers Foundation (B.A.) and Edlund Brothers Foun-

dation (D.R.B. and B.A.). This manuscript benefited from constructive com-

ments and suggestions fromM.C.Otterstatter and three anonymous referees.

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Received 18 July 2009; accepted 13November 2009

Handling Editor: Jonathan Newman

Endophytes, grasses and environmental stress 479

� 2009 The Authors. Journal compilation � 2009 British Ecological Society, Journal of Ecology, 98, 470–479