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- Materials and methods
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Parasitic plants are present, often in high abundance, in many ecosystems where they affect not only their hosts, but indirectly impact on many other organisms. They have been shown to have major effects on the structure and function of ecological communities (Press & Phoenix 2005), including changing plant community diversity and structure (Joshi, Matthies & Schmid 2000; Ameloot, Verheyen & Hermy 2005), influencing nutrient cycling (Quested, Press & Callaghan 2003; Fisher et al. 2013), altering soil microbial communities (Bardgett et al. 2006) and affecting the performance of invertebrate herbivores (Marvier 1996; Ewald, John & Hartley 2011).
Rhinanthus minor is a generalist hemiparasitic plant (Gibson & Watkinson 1989). It is a widespread component of grasslands throughout Europe and North America (Westbury 2004) and exerts much of its influence by dramatically reducing host plant biomass (Cameron et al. 2005). Like many other parasitic plants, R. minor establishes cellular continuity with the xylem stream of its hosts via specialized organs (haustoria), thereby extracting nutrients (Kuijt 1969; Riopel & Timko 1995). The severity of the impact of R. minor on its hosts, and its consequent community level effects, depends, at least in part, on host species identity and the ability of the host to tolerate or resist infection (Gibson & Watkinson 1991; Cameron, Coats & Seel 2006; Rowntree, Cameron & Preziosi 2011).
Just as R. minor has differential effects on host species, hosts can be more, or less, beneficial to the parasite. Variation in the performance of R. minor appears to result largely from how well the host plant can defend its xylem stream from the hemiparasite (Cameron, Coats & Seel 2006; Cameron & Seel 2007), and to variation in the types and amounts of solutes that the hemiparasite can remove from its host (Seel, Cooper & Press 1993; Press 1995), although host growth rate is also a determining factor (Hautier et al. 2010). In general, legumes and grasses are thought to be ‘good’ hosts for R. minor while non-leguminous forbs are regarded as ‘poor’ hosts (Seel, Cooper & Press 1993; Seel & Press 1993; Cameron, Coats & Seel 2006), but thus far, only a limited range of potential host species has been tested in terms of impact on parasite performance.
Host and parasitic plants do not interact with each other in isolation. Host plants can indirectly influence parasite herbivores (e.g. Marvier 1996; Adler 2002), while parasitic plants indirectly affect host plant herbivores (Ewald, John & Hartley 2011). The performance of invertebrate herbivores is dependent on the nutritional quality of the plant on which they are feeding (Douglas 1993), as well as the plant's physical (Hanley et al. 2007) and chemical (Bennett & Wallsgrove 1994) defences. As the biomass and nutritional quality of hemiparasites depends on the host species they are attached to (Seel, Cooper & Press 1993; Seel & Press 1993), it follows that the parasite's host plant may also determine the success of parasite-feeding herbivores (Adler 2002).
As a parasite with the ability to infect and utilize numerous host species simultaneously, it is unlikely that R. minor will be attached to a single host species in the field (Gibson & Watkinson 1989). Parasites attached to multiple host species can gain different nutritional components from each host (Govier, Nelson & Pate 1967) and may also receive protection against multiple environmental stressors (Pate et al. 1990), including herbivores (Marvier 1998). Therefore, a mixed host or ‘generalist’ strategy has the potential to benefit the parasite, particularly in the presence of herbivores. However, the extent of such benefits has not been tested, and to our knowledge, this is the first experimental study where the effects of different multiple host combinations on the performance of the parasite and its herbivores have been identified. Investigating the effects of these complex multitrophic interactions on the performance of the organisms concerned will enable us to understand more fully the key role that parasitic plants can play in structuring terrestrial communities and regulating their dynamics.
We used a series of glasshouse experiments to understand the consequences of a generalist host strategy by a keystone parasitic plant, R. minor, for itself and other associated organisms. We used a range of host species alone, and in combination, to test the effects of host identity on the performance of the parasite and an aphid herbivore feeding upon it. First, we investigated the effect of individual host species on the parasitic plant and predicted that host functional group would be more important than host species identity in determining the performance of the parasitic plant. Next, we investigated the effect of single host species on the performance of aphid herbivores feeding on the parasite and predicted that host identity would indirectly influence the success of the parasite's aphid herbivores. Finally, using the results from the first two experiments to inform the choice of host species used, we investigated the effects of the parasite attaching to multiple host species on its aphid herbivores. We predicted that aphid performance would depend on the combination of host species used, with the cumulative effect on aphids reflecting the balance of ‘good’ and ‘bad’ hosts within the mixture.
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- Materials and methods
- Supporting Information
This study is the first assessment of how attachment to different combinations of multiple host species affects both R. minor and its associated aphid herbivores. Our data show that when attached to a single host, R. minor biomass was best explained by the number of haustorial attachments to the host rather than the size, or biomass, of the host plant. While it is known that not all haustoria produce functional attachments (Cameron & Seel 2007), our results demonstrate that the investment in such structures by the parasite reflects the quality of the host. They also support the idea that differential resistance among a variety of host species across functional groups (Cameron, Coats & Seel 2006; Cameron & Seel 2007) is related to the number and effectiveness of haustorial connections in addition to any effects of host growth rate (Hautier et al. 2010). Certainly, haustorial connections were a more important determinant of host quality for the parasitic plant in our study, since host biomass had no influence on R. minor performance.
There was considerable variation in host suitability at the species level, which was not predictable from the species’ functional groups. For example, although when analysed at the level of functional group, legumes and grasses were better hosts for R. minor than non-leguminous forbs (see also Seel, Cooper & Press 1993; Seel & Press 1993); when species level effects were considered, R. minor actually performed no worse on the non-leguminous forbs S. minor and A. millefolium than on the best host the legume L. corniculatus. In fact, the poor performance of P. lanceolata as a host, which has been previously well documented (Cameron, Coats & Seel 2006; Cameron & Seel 2007), is the predominant factor in reducing the performance of the non-leguminous forbs as hosts overall. Similarly, the performance of the functional group legumes as hosts is predominantly influenced by the high biomass attained when R. minor is growing on L. corniculatus, while the other legumes, O. repens and T. pratense, were no better hosts than P. lanceolata. The grasses H. lanatus and D. glomerata were as good hosts as L. corniculatus, but overall, the grasses were no better as hosts than the non-leguminous forbs and no worse than the legumes. In light of this species level variation, we caution against making generalizations of R. minor host performance based only on information about plant functional group.
Aphid abundances feeding on R. minor attached to a single host increased with R. minor biomass, but were not affected by the N : C ratio of the parasite. This suggests that the size of the available resource rather than its quality, at least when expressed in relation to nitrogen, is of greater importance to aphid fitness. Host functional group was not important in determining aphid abundance, but host species was. The aphids used in this experiments had prior exposure to R. minor attached to two of our host species (T. repens and H. lanatus). It is possible, although not necessarily the case (Via 1991), that prior exposure enabled the aphids to better tolerate any negative effects of these hosts. Numbers of aphids were reduced on R. minor attached to H. lanatus and A. millefolium compared with those attached to O. repens, but by far, the greatest effect was seen when the parasite was attached to L. corniculatus. On these plants, numbers of aphids were considerably reduced compared with all other host species, possibly because of antiherbivore secondary metabolites associated with this species. Previous studies on other parasitic plant species have demonstrated that compounds with antiherbivory properties can be transferred from the host to the parasitic plant (Marvier 1996; Adler & Wink 2001). In L. corniculatus, cyanogenic glycosides are the principle toxic metabolites, known to be effective against herbivores (Scriber 1978). These interact with degradation enzymes within the plant to release HCN when plants are under attack. The picrate assay that we used detects the release of HCN as a result of this degradation process, and, while we did detect this compound in L. corniculatus, there was no evidence of HCN production in the parasitic plant. We cannot, however, rule out the transfer of cyanogenic glycosides themselves between the host and parasite as it is possible that the compounds were present in R. minor, but not degraded to release HCN.
Our third experiment investigated the relationship between host species, in particular L. corniculatus, and R. minor aphid herbivores, by testing mixtures of species including L. corniculatus against single host plantings. For all host combinations tested, aphid abundance decreased as the proportion of L. corniculatus plants increased. While this does not reveal the specific mechanisms by which the host plants were influencing the parasite's aphid herbivores, it does demonstrate that L. corniculatus confers resistance to herbivores on R. minor. The most likely mechanism for this is via the transfer of secondary metabolites across the haustoria (Adler & Wink 2001), although we found no evidence of this mechanism here (see above). These results also suggest that the propensity of R. minor to attach to multiple host species in the field is likely to benefit the parasite's herbivores by diluting any negative effects of particular host species.
When attached to multiple host species, the relationship between R. minor biomass and aphid abundance breaks down and depends entirely on host species identity. When O. repens was paired with L. corniculatus, larger R. minor plants supported greater numbers of aphids, but when S. minor and L. corniculatus were paired as hosts, smaller plants supported more aphids. This discrepancy in the impact of host combinations is likely due, in part, to the negative effect of L. corniculatus on the aphids, but also its suitability as a host for R. minor.
Attachment to multiple host species had little discernable effect on R. minor biomass, except when D. glomerata was paired with L. corniculatus. In this case, the parasite was larger when attached to both host species compared with when it was only attached to the grass. Previous work has produced contradictory conclusions on whether attachment to a single host species (Matthies 1996) or multiple host species (Marvier 1998) is the most beneficial for parasitic plants. Our results suggest that the host identity within mixtures is likely to be critical in explaining these apparent contradictions on R. minor performance. Further, the responses of R. minor to host mixtures did not predict the response of the aphids since the relationship between R. minor biomass and aphid abundance is different for each combination of hosts.
In conclusion, host species identity has considerable effects on the performance of both R. minor and its aphid herbivores, and this variation between species cannot be explained solely by plant functional group. Furthermore, this effect holds for interactions with single and multiple host species. Investment in haustorial structures is a good indication of host suitability for the parasite, while parasite biomass is a good predictor of the size of aphid populations feeding on R. minor, but only when the parasite is attached to a single host. When attached to multiple hosts, the relationship between host plant and parasite herbivore is more complex. The ‘generalist’ nature of R. minor is likely to benefit its aphid herbivores by diluting any negative effects of a single host species thereby significantly influencing herbivore population dynamics. With this work, we demonstrate that the interactions between the keystone parasitic plant R. minor and its hosts extend beyond the plants themselves and have significant consequences for the wider ecological community.