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Most plants are photoautotrophic organisms fixing carbon through photosynthesis. Plants are, however, also dependent on water and mineral nutrients which are acquired from the environment (either directly through plant roots or rhizoids or through symbiotic relations with other constituents of ecosystems, principally mycorrhizal fungi). Despite the prevailing autotrophic strategy, several plant lineages have evolved heterotrophic means of resource acquisition, parasitizing fungi (mycoheterotrophy; Leake, 1994; Selosse & Cameron, 2010) or other plants (plant parasitism; Irving & Cameron, 2009) for either all or a subset of resources required to support their vital processes. Among these strategies, hemiparasitism is a plant-specific mechanism of resource acquisition based on both autotrophy and heterotrophy. Hemiparasitic plants are green, performing photosynthesis but at the same time attacking the vascular system of other species and exploiting the host xylem sap which contains both mineral nutrients and organic carbon (Irving & Cameron, 2009). Hemiparasitic species occur in the majority of terrestrial ecosystems, but in temperate Eurasia, grassland communities represent the principal habitats, containing the highest diversity of the hemiparasitic flora. Moreover, parasitic plants are more than botanical curiosities; their ability to suppress host species growth and affect nutrient flows in the ecosystems (Quested et al., 2003; Cameron et al., 2005; Phoenix & Press, 2005) results in the hemiparasites often being considered keystone species or ecosystem engineers, regulating the structure and function of host communities (Cameron et al., 2005; Press & Phoenix, 2005).
The genus Rhinanthus (Orobanchaceae) is a common and widely distributed example of a northern-temperate root-hemiparasitic group. All of its 25–35 species are annual hemiparasites occurring in grasslands of low and medium productivity in Europe and North America (Meusel et al., 1978). As root hemiparasites, Rhinanthus spp. attach to roots of their hosts by a specialized organ called haustorium, providing direct luminal continuity between host and parasite xylem vessels via open conduits called oscula (Cameron et al., 2006). Host-to-parasite solute transfer therefore occurs through mass flow, driven by a water potential gradient between the host and the parasite induced by high transpiration rate of the parasite and accumulation of osmotically active compounds, particularly mannitol, in parasite tissues (Cameron et al., 2006). While direct luminal continuity as a mechanism for solute extraction from a host is not common to all parasitic plants, it is shared by many hemiparasitic species within the family Orobanchaceae, including the noxious hemiparasitic tropical weed Striga spp. (Dörr, 1997). The connection to the host’s xylem and the absence of a phloem connection imply that water and mineral nutrients are the most important resources obtained from the host. Their flows have indeed been quantified in numerous hemiparasitic systems, accounting for a substantial proportion of total host resources (Jiang et al., 2003, 2004, 2010), although the potential for acquisition of organic carbon is often ignored (but see Těšitel et al., 2010a). Despite low organic carbon concentration in xylem, the extensive amount of solutes acquired from the host can also result in significant heterotrophic carbon gain (Těšitel et al., 2010a), which has been observed not only in Rhinanthus but also in numerous representatives of other root-hemiparasitic species, including Euphrasia (Těšitel et al., 2010a), Striga (Press et al., 1987) and Olax (Tennakoon & Pate, 1996) species. The host-derived carbon can account for up to 20–80% of hemiparasite dry mass (see Těšitel et al., 2010b for a review).
The hemiparasitic lifestyle provides notable advantages to Rhinanthus spp., supplying a relatively rich and stable resource of inorganic and organic nutrients without the need for carbon investment in an extensive root network or mycorrhizal associations. The efficient exploitation of host resources is probably closely related to the annual life history (Press et al., 1988; Ehleringer & Marshall, 1995), which is shared with most of the temperate hemiparasitic Rhinanthoid Orobanchaceae and evolved independently in individual genera from perennial ancestors (Těšitel et al., 2010c). The annual life history combined with the absence of a long-living seed-bank (ter Borg, 1985; van Hulst et al., 1987; Kelly, 1989), however, requires successful seed production and seedling establishment in every season, imposing an apparent constraint on the persistence of Rhinanthus spp. populations growing in dense-sward communities dominated by perennial species. Rhinanthus spp. and some other hemiparasites (e.g. Melampyrum species; Těšitel et al., 2010c) compensate for this constraint by production of large, resource-rich seeds, enabling fast growth of seedlings at the start of the growing season and hence allowing them to inhabit sites of medium productivity where no other annuals persist (Kelly, 1989; Strykstra et al., 2002). Highly productive, nutrient-rich sites are, however, generally unfavourable for the persistence of hemiparasite populations, including Rhinanthus spp. (van Hulst et al., 1987; Cameron et al., 2009; Fibich et al., 2010; Hejcman et al., 2011), underpinned by a high intensity of competition for light at such sites (Hautier et al., 2009). In addition, the sensitivity of hemiparasites to light competition was confirmed experimentally (Matthies, 1995; Keith et al., 2004; Hejcman et al., 2011) and is further supported by theoretical mathematical models, suggesting an unstable population dynamics with a high extinction risk (Cameron et al., 2009) or competitive exclusion (Fibich et al., 2010) at highly productive sites.
Limitation of Rhinanthus spp. to grasslands of low and intermediate productivity by interspecific competition might appear to be in conflict with the reported ability of Rhinanthus to withdraw substantial (up to c. 50% of dry biomass for Rhinanthus minor; Těšitel et al., 2010a) amounts of organic carbon from its host, especially given that Hwangbo & Seel (2002) reported no effect of shading on biomass production of R. minor. However, this latter study is based on analyses of adult plants that were shaded immediately before anthesis, limiting their predictive power over the competitive-exclusion hypothesis detailed earlier.
As a facultative hemiparasite, the seeds of Rhinanthus spp. germinate without host induction, produce green leaves and then attach to their host’s roots several days after emergence. They are thus entirely reliant upon their seed reserves and their own photosynthetic activity for carbon and nutrients during the short period before the attachment (Irving & Cameron, 2009). This is in direct contrast to the obligate hemiparasites, which usually require host-borne signalling compounds, such as strigolactones, to induce germination and that attach to their host before the formation and emergence of green shoots (Irving & Cameron, 2009). In Rhinanthus, the seedling stage can therefore be hypothesized as the bottleneck stage of the populations when most mortality occurs as a result of competition for light and nutrients. Competitive exclusion of established Rhinanthus spp. is, on the other hand, hard to imagine since the mature plants can grow relatively tall and their growth can be further supported in moderately productive ecosystems (Fibich et al., 2010; Mudrák & Lepš, 2010).
In the present study, we investigate the effect of light competition on performance of Rhinanthus alectorolophus in the context of its heterotrophic carbon acquisition. Experimental R. alectorolophus plants were cultivated in pots with wheat (Triticum aestivum; C3 photosynthesis) and maize (Zea mays; C4 photosynthesis) as host species. This allowed estimation of the amount of host-derived carbon in hemiparasite biomass using natural abundance of carbon-stable isotopes (method introduced by Press et al., 1987 for Striga spp. and adapted by Těšitel et al., 2010a). The shading treatments were imposed on plants at different stages of development, simulating competitive pressure from co-occurring species. The main aim of this experiment was therefore to test the hypothesis that the highest susceptibility of Rhinanthus to competition for light is at the seedling stage and to uncover a possible role of heterotrophic carbon acquisition regulating the population dynamics of Rhinanthus spp.
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Our study unequivocally demonstrates that the growth of R. alectorolophus is supported by two sources of organic carbon originating from its own photosynthetic activity and host-derived assimilates. Assimilates of autotrophic origin apparently represent the dominant component of carbon balance in adult R. alectorolophus plants in agreement with the high rates of photosynthesis we detected in R. alectorolophus plants under natural field conditions (Fig. 1). The fatal consequences of the total darkness treatment for R. alectorolophus identifies photosynthesis as an absolutely essential physiological process for R. alectorolophus, in contrast to the cases of the obligate hemiparasites such as Striga asiatica, which was shown to grow and reproduce even in absolute darkness (Rogers & Nelson, 1962), or Striga hermonthica, in which albino mutants occasionally occur (Press et al., 1991).
Based on the key role of photosynthesis in the carbon budget of R. alectorolophus, the lower hemiparasite biomass production observed under both shading treatments can be attributed to a reduction of the rate of photosynthesis caused by the shading. This contrasts with the conclusions of Hwangbo & Seel (2002), who recorded little or no effects of shading on hemiparasite growth. This study, however, only imposed limited shading (decreasing PAR intensity to c. 50%) on adult R. minor plants at 4.5 wk post-attachment. Not only did the shading treatments of our experiment affect the rate of assimilation, but they also affected the quantity of host-derived carbon acquired by the hemiparasites. Shaded seedlings had the highest proportion of host-derived carbon in their biomass but we estimated that the total amount was lower than that in shaded young plants and unshaded controls, which furthermore decreased their growth. By contrast, shaded young plants acquired a similar amount of organic carbon from the host as did the control plants. In addition, these plants had a larger photosynthetically active leaf area at the onset of shading, resulting in a higher rate of assimilation per whole plant compared with the shaded seedlings. This in turn allowed production of more leaves, leading to a positive feedback, underpinning the differential performance of shaded seedlings and young plants.
A comparison between the proportion of host-derived carbon, its total amount and the N : C ratio in the biomass of the hemiparasites (Fig. 4a,b) clearly suggests that differences in the assimilation rates of plants under individual shading treatments were the cause of the principal pattern in the proportion of host-derived carbon in the hemiparasite biomass. The low proportion of host-derived carbon and the low N : C ratio in unshaded R. alectorolophus were apparently caused by their high assimilation rates, resulting in ‘dilution’ of host-derived carbon and nitrogen in the hemiparasite biomass dominated by assimilates of autotrophic origin, while in the shaded treatments, this effect was much lower. The detected trend of a higher concentration of host-derived carbon in the lower parts of the plant indicates that host-derived carbon is mostly directed to structural components of the lower stem parts, while the autotrophic carbon is preferentially used for the development of new leaves and vertical growth of the stem, which is producing additional photosynthetically active area. On the other hand, nitrogen is mostly directed to leaves and the upper parts of the plants (particularly in the shaded treatments). These contrasting patterns suggest an intensive retranslocation and metabolic processing of the host-borne resources in the hemiparasite. A very similar pattern was also detected in S. hermonthica; however, the differential allocation of carbon originating from different sources was much more pronounced (Santos-Izquierdo et al., 2008). The elevated proportion of host-derived carbon in the upper parts of shaded seedlings indicates that under extreme deficiency of autotrophic assimilates, host-derived carbon can be also directed to vertical growth.
The host-derived carbon acquired by Rhinanthus is derived from xylem-mobile organic elements (mostly organic acids and amino-acids, e.g. Alvarez et al., 2008) and the inflow of these compounds therefore occurs through mass flow driven by water potential difference maintained by elevated transpiration rate of the hemiparasite and accumulation of osmotically active sugar alcohols in hemiparasite tissues (Jiang et al., 2003, 2008). Playing the key role in determining sink strength of the hemiparasite, the transpiration rate for the whole plant is dependent on its total leaf area, stomatal conductance and difference in water vapour concentration between the leaves and ambient air. Of these, the effect of stomatal conductance can be assumed to be very similar across Rhinanthus plants, which keep their stomata permanently open (Jiang et al., 2003). We did not perform any detailed quantitative analysis of leaf area, but the difference in biomass production between control and shaded young plants appeared to be caused by increased leaf thickness of directly illuminated leaves and their palisade parenchyma (Supporting Information, Fig. S1) rather than by the smaller leaf area of shaded young plants. The pattern whereby the total amount of host-derived carbon in the biomass depends on the shading treatment (Fig. 4b) therefore suggests that the total leaf area could be the key factor underpinning the parasitic resource uptake. The last variable potentially affecting the transpiration rate – the difference in interior water vapour concentration – might have developed between irradiated and shaded leaves as a result of the different temperature increasing the transpiration of leaves under full irradiation. However, leaf temperature data acquired during the field gas-exchange measurement of light-response curves (Fig. 1) indicated a rather low temperature difference (c. 1°C) between leaves irradiated by 500 μmol photons m−2 s−1 and those irradiated by 50 μmol photons m−2 s−1, resulting in a relatively small decrease in the transpiration rate inflicted by shading compared with the effect of leaf area. In addition, a substantial amount of host-borne resources can be acquired during the dark period of the day when host plants close their stomata, while those of Rhinanthus remain open resulting in rather intense night-time transpiration (Press et al., 1988; Jiang et al., 2003). Owing to the resultant difference in sink strength between the host and hemiparasite shoots in the night, this mechanism is hypothesized to play an important role in parasitic resource acquisition by the hemiparasites (Press et al., 1988; Ehleringer & Marshall, 1995), independently of the light conditions during the day.
The lack of a significant difference in height between shaded young plants and controls, and a rather moderate decrease of vertical growth in shaded seedlings achieving > 50% height of control plants (Fig. 2b) indicate that shaded plants mobilize their resources (acquired both autotrophically and heterotrophically) to support their vertical growth in a bid to avoid shading, a fundamental process also observed in nonparasitic plants (Lambers et al., 2008). Shaded young plants were therefore able to grow as tall, and suppress host growth to the same degree, as the control hemiparasites despite the severe shading. This response would potentially allow the parasites to escape shading under natural conditions in grasslands when that shading is caused by competing neighbouring plants. However, if shading is imposed at the seedling stage, parasites remain small and are unable suppress the host growth to a sufficient extent or to compete for light with the surrounding vegetation.
The sensitivity of R. alectorolophus to shading when imposed at a very early stage of development clearly supports the hypothesis of Těšitel et al. (2010a) explaining the conflict between heterotrophic acquisition of a substantial amount of carbon and reported sensitivity to light competition in Rhinanthus (Matthies, 1995; Hejcman et al., 2011) through inefficiency of resource uptake by unattached hemiparasite seedlings. In addition, a moderate increase of grassland productivity, and hence an increase of competition (Hautier et al., 2009), has been reported to increase seedling mortality in hemiparasites, but also the fecundity of the survivors (van Hulst et al., 1987; Mudrák & Lepš, 2010). Similarly, Keith et al. (2004) and Hautier et al. (2010) demonstrated that once they had survived the seedling stage, hemiparasites growing close to the host or attached to a fast-growing host species achieve comparatively high fecundity. All these results suggest that productivity connected with elevated mineral nutrient uptake supports the growth of the hemiparasites after overcoming the seedling stage by increasing the effectiveness of their photosynthetic machinery.
The mortality of hemiparasite seedlings caused by light competition pressure from the surrounding vegetation is also determined by the quality of attachment to the host; that is, seedlings attached by multiple haustoria and to lower-order host roots take up host resources more effectively and their mortality rate is consequently lower (Keith et al., 2004). This can, at least in part, compensate for the effect of shading on the carbon balance of the hemiparasite by providing substantial amounts of host-derived carbon. Indeed, some of the shaded seedlings in our experiment managed to acquire as much host-derived carbon as plants in the other two treatments (Fig. 4b) and these individuals also performed best among the shaded seedlings in terms of biomass production. Supporting this conclusion, Hejcman et al. (2011) demonstrated that R. minor is able to persist even in highly productive grassland plots, although it requires intensive seed rain from surrounding plots of low productivity. These R. minor plants were substantially more vigorous than plants growing in surrounding, less productive, plots (M. Hejcman, pers. comm.) and probably represented a population subset that was able to acquire substantial amounts of carbon heterotrophically. Nonetheless they were still unable to establish a stable population because of the high mortality rate and subsequent low population density.
Persistence of Rhinanthus spp. populations in grassland communities depends on its ability to complete the life cycle. As annual hemiparasites without a substantial long-living seed-bank, the species must germinate, grow, flower and produce seeds during every season. This study identified the seedling stage as the bottleneck of the life cycle, limiting the occurrence of Rhinanthus spp. to grasslands with low to moderate productivity. Such sensitivity of seedlings is in part addressed by a comparatively large seed size and early germination controlled by environmental factors allowing rapid development and overcoming limitation in the critical recruitment stage. However, the ability to acquire substantial amounts of host-derived organic carbon, supporting the growth of young plants, represents a further mechanism enabling Rhinanthus seedlings to escape from competition for light. In this way, the heterotrophic carbon acquisition can broaden the ecological range of the hemiparasites, allowing their occurrence in a moderately productive environment.