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- Materials and Methods
- Supporting Information
Symbioses of plants and animals with microorganisms have led to major transitions in the evolution of life (Margulis, 1996). The combination of diverse physiological functions from different species in a symbiosis may aid in dealing with dynamic temporal or spatial fluctuations of the environment (Boyle et al., 2011) and thus allow symbiota to expand into novel habitats, such as the colonization of land by ancient plants associated with mycorrhizas (Humphreys et al., 2010). The present study aims to contribute to a better understanding of the ecological and evolutionary processes necessary for host–symbiont adaptation, an important precondition for symbiota to be successful.
All modern-day plants in natural ecosystems appear to be symbiotic with fungal endophytes, a diverse group of fungi that can have profound effects on plant ecology and evolution (Rodriguez et al., 2009). Among these fungal groups, one of the best known is the clavicipitaceous endophytes, here referred to as the Epichloae (Epichloë and Neotyphodium spp., Ascomycota), which form hereditary symbioses with grasses from the subfamily Pooideae in temperate areas (Schardl et al., 2004). Endophyte infection may increase plant fitness by conferring stress tolerance to drought and poor soil, or resistance to herbivory and fungal diseases (Kuldau & Bacon, 2008). Endophyte effects on host grasses have been studied extensively in grass–endophyte systems of agricultural importance, which are characterized by artificial or anthropogenic selection on the host grass genotypes (Clay et al., 2005; Monnet et al., 2005). In these systems, endophytes mostly provide beneficial effects to their hosts, and symbioses have been characterized as mutualistic (Assuero et al., 2000; Hesse et al., 2003). However, selection acting in a natural environment may be different from that in agricultural settings, and conclusions reported from agronomic grasses may be biased (Saikkonen et al., 1998). Documented effects of endophytes on wild host grass species in non-agricultural settings are less homogeneous (e.g. Brem & Leuchtmann, 2001; Gonthier et al., 2008; Kannadan & Rudgers, 2008; Craig et al., 2011; Iannone et al., 2012) and have been found to vary from positive to negative (Morse et al., 2002; Faeth et al., 2004; Rudgers & Swafford, 2009; Kane, 2011). Infection frequencies observed in wild grasses are often very high and have been interpreted to reflect a mutualistic interaction between the host grasses and their endophytes (Clay & Schardl, 2002). However, the infection rate may also be influenced by the potential of the endophyte for contagious spread and may be modulated by the endophyte transmission rate (Gundel et al., 2008, 2011; Gibert et al., 2012). Moreover, the outcome of a symbiosis appears to depend on the particular combination of host genotype, endophyte genotype and environmental factors (Meijer & Leuchtmann, 2000; Faeth & Fagan, 2002; Cheplick & Faeth, 2009). Understanding the interactions among genotypes and environment requires experiments that combine variations in all three factors, but, to our knowledge, such a comprehensive study in wild grass species is still lacking.
Asexual Epichloae (genus Neotyphodium) include hybrid and non-hybrid taxa. Non-hybrid endophytes are haploid and genetically resemble sexual Epichloë species, from which they are derived (Moon et al., 2004). Hybrid endophytes are heteroploid (incompletely polyploid) and presumably result from somatic fusion of distinct Epichloë and Neotyphodium species infecting the same host individual (Schardl & Craven, 2003). Hybridization may remedy the accumulation of deleterious mutations of asexual strains by adding genes derived from sexual recombination in different species (Muller, 1964; Schardl et al., 1994; Tsai et al., 1994). The fusion of genomes may also allow hybrids to occupy a broader ecological niche by expressing traits from both ancestral species (Clay & Schardl, 2002). Thus, hybrid endophytes may have a selective advantage relative to non-hybrid endophytes, and are indeed often prevalent among infected host species and populations (Moon et al., 2004; Iannone et al., 2009; Oberhofer & Leuchtmann, 2012). However, the mechanisms driving hybrid predominance in natural host grasses remain unclear.
Selection favoring hybrid over non-hybrid endophytes may vary under different environmental conditions. Several studies on the grass species Festuca arizonica, which hosts one hybrid and one non-hybrid taxon, have indicated that the hybrid endophyte mainly occurs in stressful environments (Morse et al., 2007; Sullivan & Faeth, 2008; Hamilton et al., 2009). An experiment comparing naturally infected grass individuals with their disinfected counterparts revealed that hybrids particularly benefit their hosts under dry/stressful conditions (Saari & Faeth, 2012). Although the authors concluded that hybrids may extend the niche of F. arizonica, hybrid-infected grasses appear to be limited to marginal habitats. These results seem inconsistent with the presumed broader ecological niche of symbiota with hybrid endophytes relative to symbiota with non-hybrid endophytes. Festuca arizonica represents an exception among wild grass species by hosting predominantly non-hybrid endophytes, so that the restricted niche of plants hosting hybrids might be unique to this host species. It is also conceivable that F. arizonica populations hosting hybrids differ genetically from those hosting non-hybrids, so that any stress-specific effects attributed to hybrid endophytes in previous studies are actually related to the host's genotype. To distinguish between host and endophyte genotype effects, the same host accessions need to be inoculated with both endophyte types, and grown under different environmental conditions (Cheplick & Faeth, 2009).
In a survey of 28 natural populations of Hordelymus europaeus across Europe, hybrid endophytes were found in 20 populations, non-hybrids in five populations and one was endophyte free (two populations hosted both types) (Oberhofer & Leuchtmann, 2012). In Switzerland, populations containing a non-hybrid endophyte were only found at a single location on dry, shallow calcareous soil, whereas hybrid endophytes were found at all other locations, with varying water availability (A. Leuchtmann, unpublished).
These observations suggest that, in contrast with F. arizonica, H. europaeus plants hosting the non-hybrid may be more drought tolerant than hybrid-hosting plants. Furthermore, the predominance of hybrid-hosting populations may be a consequence of greater fitness, greater transmission rate or a broader ecological niche of hybrids, as proposed previously (Schardl & Craven, 2003; Moon et al., 2004; Selosse & Schardl, 2007).
To test the niche expansion hypothesis, we exposed H. europaeus plants that had been artificially infected by hybrid or non-hybrid endophytes to drought stress, and examined plant growth performance. We expected plants with hybrid endophytes to thrive in both treatments and plants with non-hybrids to perform well in the drought treatment. Source material originated from four distant European populations, including two populations with hybrid and two with non-hybrid endophytes. Each host accession was recombined with its resident endophyte, the same endophyte type (different genotype), a different endophyte type or mock inoculated with sterile water, which allowed us to differentiate between host and endophyte effects on plant growth and drought tolerance (Cheplick & Faeth, 2009). We specifically asked the following questions: (1) do endophyte-infected plants perform better than disinfected plants?; (2) do plants infected by hybrid and non-hybrid endophytes perform differently?; (3) does plant performance depend on the endophyte–host type combination, that is, are grass populations naturally hosting hybrid or non-hybrid endophytes influenced differently by either endophyte type?; and (4) is there evidence for local co-adaptation, that is, does a particular endophyte genotype provide the greatest benefit to plants from the population in which it naturally occurs? With the results of this experiment, we expect to gain more detailed insight into the nature of the symbiosis between H. europaeus and its different endophytes and to explain the distribution and abundance of hybrid and non-hybrid endophytes. Moreover, we describe the method and results of re-inoculations in detail as these may have relevance for future research.
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- Materials and Methods
- Supporting Information
The experiment started with 6450 seeds from endophyte-free mother plants of four accessions. The germination rate was, on average, 46%, so that 2983 seedlings could be re-inoculated. Between 33% and 98% of the seedlings survived up to the start of the actual experiment, and 2–50% of these (260 plants) proved to be successfully infected by the endophyte (Table 1). Seedling survival and infection success differed among plant accessions and fungal strains (Table 1); in addition, infection success was, on average, three times higher for seedlings re-inoculated with their resident endophyte strain than for seedlings re-inoculated with non-resident strains (binomial generalized linear model (GLM), X2 = 44.8, P < 0.001). Only plant accession E2 had a very low re-inoculation success even with its resident endophyte.
Drought stress strongly reduced the growth and reproduction of H. europaeus plants (Table 2; Fig. 1). At harvest 1 (H1, drought harvest), shoot DW of plants subjected to drought was decreased, on average, by 66.1% (Fig. 1a). At harvest 2 (H2, recovery harvest), the growth of previously drought-stressed plants was still reduced significantly compared with well-watered plants: tiller number was reduced, on average, by 34% (Fig. 1c), and total DW by 41.4% (P < 0.0001, Fig. 1d). Drought increased significantly the DMC (DW : FW) of shoot tissues at H1 (Fig. 1b), but decreased it at H2 (Fig. 1e), and had no influence on the root : shoot ratio (Fig. 1f). A small proportion of plants that had been subjected to drought formed spikes (15.8% vs 49.2%). Seed number per plant was not affected by previous drought, but the average weight per seed was reduced (Table 3).
Table 2. Analysis of variance (ANOVA) results for the effects of Epichloë endophyte infection (uninfected, hybrid or non-hybrid endophyte), drought stress and plant accession type (naturally hybrid or non-hybrid hosting) on vegetative traits of Hordelymus europaeus
| ||df||Harvest 1||Harvest 2|
|Shoot weight||DW : FW||Tiller no.||Plant weight||DW : FW||Root : shoot|
|Plant accession type||1||1.06||5.53*||3.34||4.55*||3.16||9.15**|
|Endophyte × drought||2||0.79||0.39||0.35||1.38||1.28||0.06|
|Endophyte × plant type||2||0.35||0.53||1.82||0.39||1.21||0.37|
|Plant type × drought||1||0.25||5.18*||2.00||0.02||0.27||1.05|
|Endophyte × plant type × drought||2||2.34||1.24||2.16||0.91||0.02||0.72|
|Residuals||219|| || || || || || |
|Contrasts for endophyte effects|
|Uninfected vs infected||1|| || ||3.76***||2.94**|| || |
|Hybrid vs non-hybrid||1|| || ||0.36||−0.59|| || |
Table 3. Analysis of variance results for the effects of re-inoculation type (resident vs same type or different type), drought stress and plant accession on vegetative traits of Hordelymus europaeus
| ||df||Harvest 1||Harvest 2|
|Shoot weight||DW : FW||Tiller no.||Plant weight||DW : FW||Root : shoot|
|Re-inoculation × drought||2||2.79||2.61||2.24||1.56||0.38||0.49|
|Re-inoculation × accession||4||0.90||2.05||3.06*||2.63*||10.15***||3.46*|
|Accession × drought||2||3.00||5.57*||1.65||0.12||4.45*||0.69|
|Inoculation × accession × drought||4||2.06||1.05||0.62||0.83||1.85||0.33|
|Residuals||136|| || || || || || |
Endophyte infection had no significant effect on plant growth during the first phase of the experiment (Table 2, Fig. 1a,b), but influenced significantly tiller production and biomass production during the second phase of the experiment (Table 2, Fig. 1c,d). The effect was related to endophyte presence (Table 2): at H2, the tiller number was 18.2% higher and the total DW was 6.6% higher in plants with endophytes than in endophyte-free plants (Fig. 2). Conversely, there was no significant difference between hybrid- and non-hybrid-hosting plants (Table 2). The effect of endophytes was similar in both water treatments, that is, endophyte–water interactions were not significant (Table 2, Fig. 2). The DMC of shoots and the root : shoot ratio were not influenced significantly by endophyte treatments (Table 2, Fig. 1e,f).
Plant accession types (naturally hosting hybrid or non-hybrid endophytes) differed in DMC at H1 (especially after drought), and in plant weight and root : shoot ratio at H2 (Table 2, Fig. 1b,d,f). However, the effect of endophyte treatments on plant growth did not differ between the two plant accession types: there was no significant endophyte–plant type interaction (Table 2).
When comparing plants re-inoculated with their resident endophyte with those re-inoculated with endophytes from other populations (different or alternate), no significant differences in growth were apparent, that is, plants did not generally produce more tillers or biomass when hosting an endophyte with which they had previously co-existed (Fig. 3). Plants did, on average, have a higher DMC at H2 when hosting their resident endophyte (Table 3, Fig. 3e). However, this effect was dependent on plant accession and was mainly found for accession A2 (Table 3, Supporting Information Fig. S1). Plants from accession A2 also had a 29% greater plant weight at H2 after drought stress when hosting their resident endophyte than when hosting other endophytes (Fig. S1).
Both the proportion of plants forming spikes and the average weight per seed were reduced by drought, whereas seed number was not affected (Tables 4, 5). Spike formation was influenced significantly by endophyte treatments: the proportion of plants producing reproductive tillers was higher among plants hosting non-hybrid endophytes than among plants hosting hybrid endophytes (Tables 4, 5). This difference was slightly more pronounced after drought stress (3.7-fold) than in the control treatment (2.8-fold), although the interaction was not significant. Spike formation was also dependent on plant type, with a significant endophyte–plant type interaction (Table 5): the positive effect of non-hybrid endophytes was stronger in plant accessions A2 and B, which naturally host non-hybrid endophytes (45% of plants produced spikes), than in accession C1, which naturally hosts a hybrid endophyte (only 13.6% of the plants produced spikes). Seed number per plant and average seed weight did not differ significantly among endophyte treatments (Table 5). However, it is worth noting that none of the hybrid-hosting plants produced any ripe seeds after drought stress (Table 4).
Table 4. Reproductive traits of Hordelymus europaeus plants grown with or without drought stress and with different Epichloë endophyte infection treatments
|Water treatment||Endophyte treatment|| n ||Plants with spikes (%)||No. of seeds per plant with spikes||Mean weight per seed if spikes present (mg)|
|Control||No endophyte||48||45.8||15.2 (+ 5.06)||7.0 (± 0.44)|
|Hybrid||19||21.1||17.7 (+ 15.2)||7.1 (± 0.76)|
|Non-hybrid||62||60.6||13.1 (+ 2.9)||6.8 (± 0.21)|
|Drought||No endophyte||48||14.6||8.7 (+ 3.4)||6.3 (± 0.77)|
|Hybrid||19||5.3||0.0 (n = 1)||na|
|Non-hybrid||66||19.7||14.2 (+ 5.9)||5.4 (± 0.51)|
Table 5. Analysis of deviance or analysis of variance results for the effect of Epichloë endophyte infection (uninfected, hybrid or non-hybrid endophyte), drought stress and plant accession type (hybrid or non-hybrid hosting) on reproductive traits of Hordelymus europaeus
| ||df||Formation of spikes||No. of seeds if spikes present||Average seed weight if seed present|
|Plant accession type||1||24.8***||0.00||2.53|
|Endophyte × drought||2||0.3||1.33||0.37|
|Endophyte × plant type||2||7.4*||0.49||0.14|
|Plant type × drought||1||0.9||na||na|
|Endophyte × plant type × drought||2||1.8||na||na|
|Residuals||181/69|| || || |