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- Materials and Methods
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Niche differentiation is the process by which natural selection drives competing species or individuals into different patterns of resource use (Hutchinson, 1957; MacArthur & Levins, 1964). Most research has focused on niche reduction caused by negative species interactions, but the effects of positive species interactions on niche differentiation have received considerably less attention (Bruno et al., 2003; Warren et al., 2011). By contrast to competition and predation, positive interactions, such as mutualism, can expand the realized ecological niche of a species by conferring benefits such as increased tolerance to abiotic and biotic stresses (Bruno et al., 2003; Afkhami & Strauss, 2011). Partner-generated niche shifts could also lead to niche differentiation within a species, if individuals that associate with partners have different niches from those that do not (Afkhami & Strauss, 2011).
One potential source causing niche shifts in plant populations are microbial symbionts living asymptomatically within tissues of the host plant. These symbionts, called endophytes, are known to alter host phenotypes (Saikkonen et al., 2006, 2010). The variation from microbial symbiosis may arise from different sources. First, differential fitness, imperfect transmission (e.g. sensu Ravel et al., 1997) and migration can create populations with mixed infection frequencies, with part of the population carrying the symbiont while other individuals remain uninfected (Cheplick & Cho, 2003; Cheplick, 2004; Faeth, 2009). Secondly, within the infected part of plant populations, plants may be infected by various genetic strains of the symbionts that differentially alter host phenotypes. For example, symbionts such as asymptomatic, strictly vertically transmitted Neotyphodium grass endophytes may hybridize (Selosse & Schardl, 2007), and infection by these hybrid symbionts may result in different plant phenotypes from those caused as a result of infections by nonhybrid symbionts (Hamilton et al., 2009).
Most grass populations are mixtures of uninfected grasses and grasses infected with endophytes (see, e.g., Lewis et al., 1997; Saikkonen et al., 2000; Wali et al., 2007; Cheplick & Faeth, 2009). In some grass species, such as Arizona fescue (Festuca arizonica), the infecting endophytes are often a mixture of hybrid (H+) and nonhybrid (NH+) endophytes (Sullivan & Faeth, 2008; Hamilton et al., 2009). About two-thirds of infections in cool season grasses are of hybrid origin (Schardl & Craven, 2003). It has been suggested that hybridization provides an infusion of genetic variation that renders the host plant more tolerant of abiotic and biotic stresses (Schardl & Craven, 2003). However, this hypothesis remains largely untested.
Contrary to the general dominance of H+ endophytes in most grass species, NH+ endophytes dominate most of Arizona fescue populations. On average, Arizona fescue populations consist of 55% NH+, 15% H+ and 30% uninfected (E−) grass individuals (Sullivan & Faeth, 2008; Hamilton et al., 2009). A possible explanation for the observed frequencies of endophtye infections in Arizona fescue is that H+, NH+ and E− grasses respond differently to varying environmental factors. There is some observational support of this hypothesis. H+ plants are more common in habitats with low nutrients and moisture, whereas NH+ plants are more prevalent in the areas with higher soil nutrients and moisture (Sullivan & Faeth, 2008; Hamilton et al., 2009).
Sullivan & Faeth (2008) found that H+ hosts produce higher volume : mass ratios than NH+ hosts in moisture- and nutrient-poor habitats, but not in habitats with plentiful resources. They suggested that this change in plant architecture by H+ plants may be a response to plant density, as H+ plants are typically located under dense tree canopy and likely experience greater intra- and interspecific competition for resources than NH+ plants in less stressful environments. In addition, Hamilton et al. (2010) found that hybrid endophytes increase survival of grass hosts in stressful habitats and concluded that infection by H+ endophytes may increase the fitness of the plants in habitats with scarce resources.
We tested the effects of hybridization of Neotyphodium endophytes on the growth and performance of Arizona fescue with and without competition under varying amounts of water and nutrients. To study performance of plant and plant–endophyte combinations found in the natural populations, NH+, H+ and E− plants were compared. To separate the effects of endophyte infections from plant responses, we also compared plants infected with endophytes (H+ and NH+) with those whose endophyte had been experimentally removed (H− and NH−). Based on the hypothesis by Schardl & Craven (2003) and the past research (Sullivan & Faeth, 2008; Hamilton et al., 2010), we expected that H+ plants perform better than H−, NH+ and E− plants when water and nutrients are scarce and the plants are competing, and that NH+ plants perform better than NH−, H+ and E− plants when there is no competition and water and nutrients are abundantly available.
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- Materials and Methods
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Our results support the hypothesis of increased host performance of H+ plants when resources are scarce. There was increased performance of H+ grasses compared with other plant and plant–endophyte combinations found in the natural populations (NH+ and E−) in almost every response variable measured, but only when competing in low-water and low-nutrient treatments. Hybrid endophyte infection was verified to increase above-ground wet biomass and root dry biomass of the host when competing in low-water and low-nutrient treatments by comparing infected plants (H+) with those whose endophyte had been experimentally removed (H−). Our analysis confirmed that the status of the specific competitors did not matter. In other words, H+ plants were equally superior competitors against all other plants (NH+, H− and E−) in low-water and low-nutrient treatments. When not competing, or competing in other treatment combinations (high water and low nutrients, low water and high nutrients, high water and high nutrients), H+ endophyte did not appear to benefit the host grass. In contrast to expectations, the NH+ endophyte did not affect performance of the host compared with other grasses, regardless of treatment. Our results suggest that symbiont-conferred protection against biotic and abiotic stresses may be underlying the observed niche expansion of Arizona fescue infected by H+ endophyte in the environments with low resources (e.g. Hamilton et al., 2009).
The competitive dominance of H+ Arizona fescues in low-resource environments may result from novel or extra genes in hybrid strains (e.g. Schardl & Craven, 2003; Moon et al., 2004). Hybridization has been suggested to be advantageous for the hosts, especially in marginal habitats at the edge of the host range (Rieseberg, 1997; Schardl & Craven, 2003). The advantages may result from higher genetic variation in the H+ endophytes, which, in turn, increase tolerance to biotic and abiotic stresses (Schardl & Craven, 2003). The mechanism by which the novel genes of H+ improve competitive potential of the infected host is unclear. Several hyphotheses explaining improved competitive potential of Neotyphodium-infected grasses have been proposed. These include changes in nutrient metabolism (Lyons et al., 1990), plant hormone–endophyte interactions (De Battista et al., 1990) and osmotic adjustment by the endophyte (Elmi & West, 1995).
Neither NH+ nor H+ endophyte infections improved growth of the host when plants were not competing, contrary to reports in the general literature, which suggest sweeping benefits of Neotyphodium infections (Saikkonen et al., 2010; Faeth & Saari, 2011). However, our findings are in line with previous studies of Arizona fescue where the endophyte does not appear to benefit the host plant, at least in experiments with no competition (Sullivan & Faeth, 2008; Hamilton et al., 2010). In general, endophyte infections have been demonstrated to have variable effects in the growth of the host plant, depending on the plant species, and especially on the plant and endophyte genotypes in question (Cheplick et al., 1989; Cheplick, 1998; Faeth & Sullivan, 2003; Hunt et al., 2005). The combinations of conditions that result in greater growth of endophyte-infected plants are not fully understood.
Because we mainly found NH+ endophytes to have neutral effects on the host, our findings fail to explain the overall high frequencies of NH+ infections in natural Arizona fescue populations (Schulthess & Faeth, 1998; Sullivan & Faeth, 2008; Hamilton et al., 2009). It is possible that NH+ and H+ endophytes affect other characteristics of the host than those measured in this experiment. For example, increased incidence of fungal pathogens has been suggested to limit the distribution of H+ hosts (Hamilton et al., 2010). Also different effects of H+ and NH+ endophytes on reproductive strategies have been reported (Sullivan & Faeth, 2008). In general, Neotyphodium infections have been suggested to increase resistance of the host against herbivores, seed predators, and plant pathogens. However, these benefits, as well as some others (e.g. resistance to fire), are not found in Arizona fescue (e.g. Saikkonen et al., 1999; Tibbets & Faeth, 1999; Faeth & Sullivan, 2003; Neil et al., 2003; Faeth et al., 2004; Hamilton et al., 2010).
Thus, the question of how high frequencies of NH+ infections are maintained in natural Arizona fescue populations remains unanswered. One explanation for the persistence of high NH+ infection rates, and the repeatedly failed attempts to find positive effects of this endophyte on the host, is that NH+ endophyte infections are infrequently mutualistic, and the positive effects only occur at certain times, such as periods of severe and prolonged droughts or rapid population decline (Faeth, 2002; Morse et al., 2002). We also acknowledge that our experiments may have failed to capture long-term selective pressures associated with a long-lived host plant and its symbiont. Furthermore, in more natural settings in the field, the outcome of the interactions between Arizona fescue and NH+ and H+ endophytes may differ.
Until recently, hybridization has been viewed as destructive force, at least in terms of maintaining species diversity in communities (e.g. Rhymer & Simberloff, 1996; Mallet, 2005). However, hybridization can also be a creative force, increasing diversity and allowing species to persist in marginal habitats (Rieseberg, 1997). At least one of the parental species of H+ endophytes in Arizona fescue is Epichloë, which, when horizontally transmitted, is highly pathogenic. Thus, we propose here that the occasional presence and genetic input from the pathogen Epichloë, and subsequent hybridization, may be necessary to maintain the mutualistic interaction of Neotyphodium with it host grass in natural populations, at least in some environments.
In conclusion, our results support the hypothesis (Schardl & Craven, 2003) that hybridization by endophytes may lead to increased survival of the host plant in stressful environments. To fully assess the impact of hybridization of this symbiont and the consequences to expanding its host’s niche, long-term experiments in the field conditions are necessary. Nonetheless, our results suggest that interactions between plants and microbes may have an important role in colonization, metapopulation dynamics and plant community structure.