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- Materials and Methods
- Supporting Information
Nearly all plants have developed symbiotic associations with endophytic or mycorrhizal fungi (Petrini, 1986). The fossil record suggests that some of these interactions are older than 400 million yr (Redecker et al., 2000), suggesting that fungal associations have played a long and important role in the evolution of life on land. These symbionts have been important in plant evolution because they alter the ecology of their hosts, often by enhancing nutrient uptake, increasing stress tolerance, or providing protection from host enemies (Smith & Read, 1997; Clay & Schardl, 2002; Hartley & Gange, 2009). Furthermore, both mycorrhizal fungi, which occur belowground, and aboveground fungal endophytes can have strong impacts on community composition (Clay & Holah, 1999; Hartnett & Wilson, 1999), succession (Janos, 1980; Rudgers et al., 2007), and nutrient cycling (Franzluebbers et al., 1999; van der Heijden et al., 2008). Given the ecological and evolutionary importance of plant–fungal symbioses, there is great interest in understanding mechanisms through which these symbioses are maintained at high frequencies in plant populations (Rudgers et al., 2009). Isolating these mechanisms necessitates identifying the ecological factors that generate variation in the relative costs and benefits of symbiosis.
Positive, negative and neutral effects of symbionts on plant fitness are expected to arise from variation in the relative costs and benefits of the interaction under different ecological contexts (Bronstein, 1994). In some cases, identifying factors causing this variation is straightforward. For example, in plant–mycorrhizal fungi associations, plant hosts and fungal symbionts benefit from the exchange of mineral and organic resources (Smith & Read, 1997; Hoeksema et al., 2010). Consequently, variation in nutrient availability in the soil can influence the net outcome of the interaction (Johnson et al., 1997; Allen et al., 2003). Although increased access to immobilized soil nutrients has traditionally been recognized as the major benefit of mycorrhizal symbiosis, evidence suggests alternative benefits beyond resource limitation, including greater host resistance to soil-borne pathogens and root parasites (Azcón-Aguilar & Barea, 1996), increased host water uptake (Auge, 2001), improved host tolerance to heat (Kytoviita & Ruotsalainen, 2007), and indirect effects on herbivores (Koricheva et al., 2009). These results demonstrate the potential for microbial symbionts to provide a diverse set of benefits to host plants, and highlight the importance of measuring both changes in plant performance and symbiont-induced alterations of host phenotypes under a variety of ecological contexts.
In the aerial tissues of plants, endophytic and epiphytic fungal symbionts are incredibly abundant and diverse (Rodriguez et al., 2009). Here we focus on the symbiosis between grass hosts and vertically transmitted, systemic fungal endophytes (class 1 endophytes, Clavicipitaceae; Rodriguez et al., 2009). Relative to belowground symbioses, far less is known about the costs and benefits of endophytes. Class 1 endophytes inhabit aboveground plant tissues and are estimated to occur in c. 20–30% of grass species (Leuchtmann, 1992). Historically, endophyte symbioses have primarily been recognized for benefiting host plants through increased resistance to herbivores (Clay, 1996; Bush et al., 1997). However, research has also shown that endophytic fungi can increase competitive ability (Clay et al., 1993), drought tolerance (Malinowski & Belesky, 2000; Kannadan & Rudgers, 2008), pathogen resistance (Gwinn & Gavin, 1992; Mahmood et al., 1993), and the accumulation of nutrients (Malinowski et al., 2000; Rahman & Saiga, 2005), suggesting that endophytes may ameliorate the effects of a wide variety of environmental stressors. Most research has focused on a few species of agronomically important grasses (Saikkonen et al., 2006; Cheplick & Faeth, 2009), and far less is known about the nature of plant benefits in wild grass species. Several studies have highlighted the continuum from mutualism to parasitism in grass–endophyte interactions (Saikkonen et al., 2004; Schardl et al., 2004; Muller & Krauss, 2005), but the breadth of ecological factors that produce variation in the outcome of the interaction remains poorly characterized.
In this study, we examined whether endophytes modulate plant responses to shade stress across six grass species that differed in the breadth of light habitats they typically inhabit. Our interest in shade stems from two observations. First, class 1 endophytic fungi associate primarily with C3 grasses (Clay & Schardl, 2002), which are more common in shaded habitats than C4 grasses (Klink & Joly, 1989). Secondly, grass–endophyte symbioses can occur across a range of habitats, from sunny, open fields to shaded, forest understories. However, to our knowledge, there have been no experimental investigations manipulating both endophyte symbiosis and light availability. Recent work on tall fescue grass (Lolium arundinaceum) found that symbiotic hosts in shaded microsites produced more alkaloids and phenolics than symbiotic hosts in open sites (Belesky et al., 2008, 2009). However, sites differed in attributes other than shade, and little is known about the potential for endophyte symbioses to modulate plant growth and trait responses to shade stress alone.
Decreased irradiance generally reduces biomass production in grasses (Eriksen & Whitney, 1981). However, reduced biomass may be mitigated through plastic changes in plant traits, such as reduced root : shoot ratios or increased specific leaf areas (SLAs) (Chapin et al., 1987; Sultan & Bazzaz, 1993), both of which are associated with the ability of plants to optimize light capture. Functionally adaptive plasticity can contribute to environmental tolerance, and ultimately, interspecific differences in plasticity may underlie species’ ecological amplitudes and abilities to persist in novel environments (Sultan, 2000). Widely distributed species are expected to cope with broader environmental gradients and show larger plastic responses than species with restricted ecological amplitudes. The degree to which symbionts, such as endophytes, may influence a host plant’s level of plasticity remains unclear. Here, we hypothesized that, if symbiota are adapted to high-shade environments, then hosts would gain some benefit from the endophyte at high levels of shade, and endophyte symbioses would be more common in hosts from shaded habitats. In addition, we hypothesized that this benefit could occur through increases in plant productivity or changes in plant traits typically associated with adaptive plant responses to shade. Alternatively, endophytes could become more costly to host plants under shaded conditions because they acquire carbon directly from the host (Thrower & Lewis, 1973). By examining hosts that differed in the breadth of light habitats they occupy, we evaluated whether plastic responses to increased shade and to endophyte symbioses differed between species that are restricted to shaded habitats and those that are not. First, we conducted a literature review to ask: Are endophyte frequencies higher in shaded habitats? Secondly, we assessed the effects of four levels of shade and the presence of fungal endophytes on the performance of six grass species to address the question: Do endophyte symbioses affect plant growth and plant traits in response to shade? Thirdly, we assessed the performance of the endophyte within leaf tissues to ask: Does shade alter endophyte density?
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- Materials and Methods
- Supporting Information
To our knowledge, this is the first study to experimentally investigate whether endophyte symbioses can alter plant responses to light availability. Despite the significantly higher frequency of endophyte symbioses in grasses occupying shady habitats, endophyte symbioses do not appear to mediate plant growth in response to light alone, at least across the range of host species we tested. Compared with plants growing in shade, those in full sunlight typically have greater structural defenses against herbivores (Roberts & Paul, 2006), and are often more prone to drought as a result of stronger winds, higher temperatures, and lower air humidity (Larcher, 1975). Our experiment decoupled the effect of light from other microclimate (and biotic) variation by controlling water availability, and pests were minimal in the glasshouse. In the future, it would be useful to explore these factors in combination as they are often correlated in natural settings, and endophytes are known to mediate both drought stress and herbivory (e.g. Clay et al., 2005; Kannadan & Rudgers, 2008).
Although the endophyte did not alter plant biomass production in response to shade for any species, in the only host that reproduced during our short-term glasshouse experiment, endophyte symbiosis increased plant reproductive fitness when light was limiting. Under high shade, symbiotic A. perennans produced 53% more inflorescences than endophyte-free plants, but endophyte presence had no effect under high light. If shading reduces the long-term survival of this perennial grass, this symbiont-mediated shift toward reproductive investment could increase host fitness. This would also be an adaptive strategy for the endophyte if vertical transmission rates to seeds are high. However, for both host and symbiont, an assessment of symbiont effects throughout host ontogeny would be required to fully characterize the fitness consequences of symbiosis, as there could be trade-offs between reproduction and survival (Rudgers et al., 2010). In addition, the generality of this result remains unclear as only a single species flowered during the experiment.
Independently, both shade and endophyte symbiosis influenced biomass accumulation in some grass species. As predicted, shade reduced biomass (up to 75%) in five of the six species examined. Most strikingly, in P. alsodes, loss of the endophyte reduced biomass by 55%, highlighting the importance of the symbiont for host growth in this species (see also Kannadan & Rudgers, 2008). In fact, the magnitude of the effect of endophyte symbiosis was comparable to the magnitude of the shade effect in P. alsodes, for which the 90% shade treatment resulted in a 47% reduction in biomass.
Phenotypically plastic allocation patterns can influence a plant’s ability to capture resources (Poorter et al., 1990), produce offspring (Sultan, 2000), and compete with neighbors (Tilman, 1988). As predicted, greater shading generally increased SLA and reduced the root : shoot ratio. For SLA, grass species from broad habitats were more sensitive to increasing levels of shade than shade-restricted species. Few other studies have examined class 1 endophyte-mediated changes in plant biomass allocation, and ours is the first to examine a suite of native host species. Previous results have been variable, with some studies documenting decreases in root : shoot ratios (Lewis et al., 1996; Lehtonen et al., 2005; Cheplick, 2007), and others finding the opposite (or no) effects (Hesse et al., 2003; Kannadan & Rudgers, 2008). Our study suggests that species’ habitat breadths may help to explain these previous idiosyncrasies, because our shade-restricted and unrestricted host species responded differently to the symbiosis. In species from shade-restricted habitats, symbiosis had no effect on the root : shoot ratio, perhaps indicative of a relatively ‘fixed’ allocation strategy. However, in species from broad light habitats, the presence of symbiosis increased the root : shoot ratio, with symbiotic plants investing relatively more resources belowground. This result does not support the hypothesis that endophyte symbiosis mediates plant response to shade stress, but does highlight the ability of symbiosis to alter the plasticity of some host species. Symbiosis has the potential to alter host plasticity in two ways. First, the presence vs absence of the symbiont may alter host phenotype, allowing the host to adjust phenotype through the gain or loss of symbiosis. Secondly, symbiont presence may increase the degree of plasticity of the host response to altered ecological conditions. This effect occurred in P. autumnalis, for which symbiotic plants showed greater plasticity in SLA in response to shade than did symbiont-free plants. Neither of these types of host plasticity led to endophyte-mediated increases in plant growth in our experiment, and it remains unclear whether such plastic responses could alter other aspects of host demography, such as survival or reproduction.
Given the fundamental importance of examining the responses of both partners for understanding the context dependence of symbioses, surprisingly few studies have examined how variation in biotic or abiotic environments influences the performance of the endophyte (Rasmussen et al., 2007; Mack & Rudgers, 2008). Previous studies suggest that host and endophyte genotype (Rasmussen et al., 2007), abiotic factors such as nitrogen concentration (Rasmussen et al., 2007; Mack & Rudgers, 2008), and biotic interactions with mycorrhizal fungi (Mack & Rudgers, 2008) may influence fungal concentration in plant tissues. Here we showed that shade had a consistently positive influence on endophyte density across the six host species. Much remains to be elucidated regarding the mechanisms that regulate endophyte growth and fitness, but several hypotheses have been proposed. Changes in density could occur through a dilution effect (Lane et al., 1997), which can occur if an environmental factor stimulates growth of the grass host more than growth of the fungus. In the present study, we are not able to rule out this explanation as we did not measure plant or endophyte growth rates, and our shade treatment significantly altered host aboveground biomass. However, inclusion of aboveground biomass as a covariate in the statistical model did not eliminate the significant effect of shade on endophyte density (Table 2), suggesting that this dilution effect may not be strong. Alternatively, changes in host metabolic profiles in response to the environmental context could also play a role in altering endophyte density (Rasmussen et al., 2008). Variation in hyphal density could additionally alter the costs and benefits of host–symbiont resource exchanges (e.g. carbon and nitrogen) or indirectly alter biotic interactions (e.g. endophyte abundance has been positively correlated with anti-herbivore alkaloid concentrations (Spiering et al., 2005; Rasmussen et al., 2007)).
The consistent effect of shade on endophyte density across the six grass species suggests alternative explanations for the higher frequency of symbiosis in shade-restricted host species. First, given that shade-grown plants may be more vulnerable to herbivores and pathogens because of reduced structural defenses (Roberts & Paul, 2006), increased alkaloid concentrations associated with higher hyphal densities could be advantageous for hosts growing in shady habitats and thereby contribute to the persistence of higher symbiont frequencies in shade-restricted species. Our glasshouse experiments, conducted in the absence of herbivores, were not designed to test this mechanism. Secondly, endophyte density could constitute an important component of endophyte fitness that may be independent of host fitness if increases in hyphal density increase rates of vertical transmission of the endophyte to seeds. With the exception of conditions of high heat and humidity, which kill the endophyte, data on the ecological factors that influence rates of vertical transmission are lacking, but high frequencies of endophyte symbioses in grasses restricted to shady habitats could ultimately reflect changes in transmission rates and be unrelated to host fitness (see Gundel et al., 2008).
Understanding the breadth of factors that generate variation in the costs and benefits of interactions between plants and their microbial symbionts is of fundamental importance to elucidating the mechanisms of symbiont persistence. In this study, we found a strong association between endophyte symbiosis and plant species restricted to shaded habitats. However, our glasshouse experiment detected few endophyte-mediated effects under shade, and no enhancement of host plant growth. Notable exceptions included the findings that endophyte symbioses increased plant reproduction in the one species that flowered and altered the plasticity of plant traits associated with light capture in response to shade in another species. This study highlights the importance of examining symbioses across multiple host species and in novel environments for understanding the factors that alter costs and benefits of symbioses and that, ultimately, influence the persistence of symbioses in host populations.