The role of heterotrophic carbon acquisition by the hemiparasitic plant Rhinanthus alectorolophus in seedling establishment in natural communities: a physiological perspective

Summary

• •
  Heterotrophic acquisition of substantial amounts of organic carbon by hemiparasitic plants was clearly demonstrated by numerous studies. Many hemiparasites are, however, also limited by competition for light preventing the establishment of their populations on highly productive sites.
• •
  In a growth-chamber experiment, we investigated the effects of competition for light, simulated by shading, on growth and heterotrophic carbon acquisition by the hemiparasite Rhinanthus alectorolophus attached to C₃ and C₄ hosts using analyses of biomass production and stable isotopes of carbon.
• •
  Shading had a detrimental effect on biomass production and vertical growth of the hemiparasites shaded from when they were seedlings, while shading imposed later caused only a moderate decrease of biomass production and had no effect on the height. Moreover, shading increased the proportion of host-derived carbon in hemiparasite biomass (up to 50% in shaded seedlings).
• •
  These results demonstrate that host-derived carbon can play a crucial role in carbon budget of hemiparasites, especially if they grow in a productive environment with intense competition for light. The heterotrophic carbon acquisition can allow hemiparasite establishment in communities of moderate productivity, helping well-attached hemiparasites to escape from the critical seedling stage.

Introduction

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.

Materials and Methods

Experimental species

Rhinanthus alectorolophus (Scop.) Pollich is an annual hemiparasitic species occurring mostly in calcareous grasslands (Karlík & Poschlod, 2009); however, R. alectorolophus was a weed in agro-ecosystems before the industrialization of agriculture, probably infesting cereal crops such as wheat and maize, the species used as hosts in the present study (Skála & Štech, 2000). Together with two closely related species in the genus, R. minor and R. angustifolius, R. alectorolophus is one of the frequently used model species in ecological and ecophysiological studies of root hemiparasites (Joshi et al., 2000; Matthies, 2003; Hautier et al., 2010). Seeds of R. alectorolophus used in this study were collected from fruiting plants of a natural population close to Zechovice near Volyně, Czech Republic (49°09′28″N, 13°52′13″E, 510 m asl).

Resolving the photosynthetic performance of R. alectorolophus in the field

A light-response curve of net photosynthesis over a range of actinic light intensities (0–1500 μmol photons m⁻² s⁻¹) was resolved for R. alectorolophus plants (n = 5) at the site from which the seeds originated in order to capture characteristics of species photosynthesis under natural conditions. Each measured leaf was first allowed to accommodate to the maximal light intensity (1500 μmol photons m⁻² s⁻¹) until a steady-state assimilation rate developed, which was consequently recorded, followed by decrease of the irradiation and steady-state assimilation rate recording in a series of 1500, 1000, 800, 500, 300, 150, 100, 50 and 0 μmol photons m⁻² s⁻¹. Gas-exchange measurements were conducted on leaves of R. alectorolophus plants using an infrared gas analyser coupled to a Li-6400 portable photosynthesis system (Li-Cor Biosciences, Lincoln, NE, USA). Individual curves were fitted by a nonlinear regression models using an equation in Lambers et al. (2008):

• (Eqn 1)

where A_n = rate of assimilation, A_max = light-saturated rate of CO₂ assimilation, φ = apparent quantum yield, θ = curvature factor, and R_d = dark respiration during photosynthesis. A mean light-response curve (Fig. 1) was obtained by averaging the five individual light-response curves, which also allowed calculating its confidence limits.

Figure 1. Light-response curve displaying dependence of the rate of photosynthesis on the intensity of photosynthetically active radiation (PAR) in leaves of five Rhinanthus alectorolophus plants growing under natural conditions. The summary light-response curve (solid line) and the confidence intervals (dashed lines) were calculated as means and 2 × standard errors of values predicted by a light-response model of individual plants. Symbols represent raw data and are classified by plant sample identity. Mean (± SE) parameters of the light-response curve models: A_max = 22.69 (± 1.26) μmol CO₂ m⁻² s⁻¹, R_d = 2.00 (± 0.31) μmol CO₂ m⁻² s⁻¹, φ = 0.085 (± 0.004), θ = 0.467 (± 0.094), compensation point: 24.81 (± 3.7) μmol photons m⁻² s⁻¹, where A_max = light-saturated rate of CO₂ assimilation, φ = apparent quantum yield, θ = curvature factor, and R_d = dark respiration during photosynthesis. Dashed lines represent PAR intensities resulting from the shading treatments: 50 μmol photons m⁻² s⁻¹ under shaded conditions, 450 μmol photons m⁻² s⁻¹ under full irradiation.

Assessment of host-derived carbon in hemiparasite biomass

Experimental R. alectorolophus plants were cultivated in pots containing wheat or maize host plants in order to assess the proportion of host-derived carbon in hemiparasite biomass. R. alectorolophus and wheat display a C₃ photosynthetic pathway while maize performs C₄ photosynthesis. As a result of their photochemical processes, C₄ plants are usually significantly more enriched in ¹³C (expected δ¹³C value ranges between −12 and −18‰; Smith & Epstein, 1971) than C₃ plants (expected δ¹³C value ranges between −23 and −35‰, Smith & Epstein, 1971; the expected δ¹³C of Rhinanthus assimilates is close to the lower limit of the C₃ range as a result of its high stomatal conductance and low water-use efficiency; Press et al., 1988; Lambers et al., 2008). It is hence possible to infer the proportion of host-derived carbon in biomass of a hemiparasitic plant by measuring the relative change in the δ¹³C value of the C₃ parasite attached to a C₄ host compared with when it is attached to a C₃ host using a linear two-source isotope mixing model (an adjusted form of models of Marshall & Ehleringer, 1990 and Gebauer & Meyer, 2003; adopted by Těšitel et al., 2010a), relating the excess of ¹³C in hemiparasites attached to the C₄ host compared with those attached to the C₃ host to the difference in isotope composition between the C₃ and C₄ hosts themselves Eqn 2.

• (Eqn 2)

where %H = the percentage of carbon in parasite biomass that is derived from the host, δ_P(C3) = δ¹³C of the parasite growing on the C₃ wheat host, δ_P(C4) = δ¹³C of the parasite growing on the C₄ maize host, δ_H(C3) = δ¹³C of the infected wheat host, and δ_H(C4) = δ¹³C of the infected maize host. δ¹³C samples were taken of leaves of hosts and a leaf and stem of the upper and lower part of each parasite since we expected variation in isotopic values between these separate parts of individual hemiparasitic plants. Subsequent calculations of the proportion of host-derived carbon in the separate parts of the hemiparasite plants were based on our experimentally derived values for δ_P(C4) of the separate parts of the R. alectorolophus plant and the δ_H(C4) of the leaf to which the parasite was attached. The average values of δ_H(C3) of corresponding treatment and δ_P(C3) of the corresponding separate part of R. alectorolophus plants cultivated under corresponding treatment were entered into the model as the baseline C₃ reference values. The total amount of host-derived organic carbon in above-ground hemiparasite biomass was estimated as a product of biomass dry-weight (DW) and mean proportion of host-derived carbon concentration in the samples of the four separate plant parts.

This approach assumes that ¹³C flows from the host only in a form of xylem-mobile organic compounds and no significant refixation of root-respired CO₂ by the hemiparasite occurs, the effect of which was experimentally excluded by Těšitel et al. (2010a). In addition, the model assumes that no carbon isotope fractionation occurs during the transfer of solutes through haustoria and that the potential difference in δ¹³C between host bulk-leaf and xylem mobile organic compounds is either negligible or identical in both species.

Cultivation and experimental design

Seeds of the hosts were germinated on Petri dishes with moist filter paper. The seedlings were moved to 10 × 10 cm square pots containing a substrate of peat and washed quartz sand (1 : 1, v/v ratio) after successful germination. Each pot contained one host plant. The plants were cultivated in a growth cabinet at the Faculty of Science University of South Bohemia under a light intensity of 450 (400–500) μmol m⁻² s⁻¹ (photosynthetic active radiation, PAR) with a 14 : 10 h light : dark cycle at 18°C. R. alectorolophus seedlings (germinated on moist filter paper at 4°C) were sown at a density of three seedlings per pot, c. 3–4 cm from the host plant after 7 d of host development. After having produced green cotyledons, the parasite seedlings were thinned to one per pot by removing the most and least advanced seedlings to obtain a homogeneous cohort.

Pots were divided into four experimental groups arranged in a completely randomized design. The Rhinanthus plantlets were placed in the shade 8 d after sowing in the first group (referred to as shaded seedlings, n = 18). In the second group (n = 18), young plants were subjected to the same shading on day 24 after sowing (referred to as shaded young plants). The third group (n = 17) consisted of plants that were kept in completely dark conditions, imposed at the same time as the young plant shading. The fourth group (n = 19) was the control, growing under full light. The host plants received full light in all treatments. Shading was adjusted and checked every day to prevent direct light from falling on any part of the shaded plants. Moreover, the position of each of the pots was exchanged randomly every c. 7 d to diminish possible effects of irradiation heterogeneity (although this was rather minimal as indicated by the ranges of irradiation values).

The shading on the experimental plants decreased the PAR intensity from 450 (400–500) to 50 (40–60) μmol photons m⁻² s⁻¹, corresponding to rates of photosynthesis of 14.25 μmol CO₂ m⁻² s⁻¹ (62.8% of A_max in plants growing under natural conditions) and 1.82 μmol CO₂ m⁻² s⁻¹ (8.0% of A_max, Fig. 1), respectively. This PAR intensity ratio of c. 0.1 corresponds well to values typical of light conditions above the grassland canopy and in the understorey (Hautier et al., 2009). The shading was achieved using a square shield made of thick green paper (8 × 8 cm, 218 g m⁻²) blocking the direct light. The shaded plants therefore received indirect irradiation only, similar to a plant growing under natural conditions shaded by nearby competitive species. The shading shields were fixed 20 cm above the pots by a wooden support inserted into the pot substrate. This simple shield design allowed selective shading of Rhinanthus plants only and did not block air circulation, preventing modification of air water potential or vapour pressure by shading. Several Rhinanthus plants grew sufficiently tall to almost interfere with the shading shield at the end of the experiment. The shield was moved upwards by several cm in such cases; however, an additional shield was placed on the pot to keep the irradiation within the desired range of 40–60 μmol photons m⁻² s⁻¹ (confirmed by PAR measurements). The complete darkness treatment was imposed by covering the Rhinanthus plants with a 1-cm-thick isolation polypropylene tube (inner diameter of 5 cm, length 20 cm) which was covered by a small tray and perforated (holes c. 1 cm in diameter, one hole on c. 25 cm² of the tube), pointing downwards to allow as much ventilation as possible while preventing light from coming in.

This experimental design was used to provide sufficient contrast between the light treatments rather than analyse performance or physiology of R. alectorolophus under exact amounts of irradiation used. The shaded plants also performed better than might be expected from the light-response curves of the R. alectorolophus plants (Fig. 1) as a result of the higher the effectiveness of diffuse light photosynthetic utilization (Sinclair & Shiraiwa, 1992; Gu et al., 2002); nonetheless the shading apparently created a contrast of light-deficiency stressed vs nonstressed plants.

Biomass harvesting and stable isotope analysis

Plants were harvested 46 d after sowing of R. alectorolophus. The height of each of the R. alectorolophus plants was measured before the harvest. The above-ground biomass of both host plants and parasites was sampled in addition to the sampling for stable isotope analyses. All samples were dried at 80°C for 48 h and weighed immediately. The DW of samples used for stable isotope analyses was included in the weight the total above-ground dry biomass produced.

The samples for carbon stable isotope analysis were homogenized separately and a 0.4 mg subset of each constituent part was analysed for ¹³C content by a Vario MicroCube elemental analyser (Elementar Analysensysteme, Hanau, Germany) connected to an isotope ratio mass spectrometer (IRMS Delta XL Plus, Finnigan, Germany) at the Faculty of Science, University of South Bohemia. Data were collected as atom%¹³C and re-expressed as delta values relative to the Pee Dee Belemnite standard (d) using Eqn 3:

• (Eqn 3)

where R_Sample is the ¹³C : ¹²C ratio in the sample and R_Standard is the ¹³C : ¹²C ratio in the Pee Dee Belemnite standard. In addition to the δ¹³C data, the atomic N : C ratio was determined in the samples during the mass spectrometry analysis in order to resolve the nitrogen status of the experimental plants. The delta values were related to the internal working standard (cellulose, δ¹³C = −24.389‰) which is related to the international standard saccharose (δ¹³C = −10.40‰) obtained from the International Atomic Agency, Vienna. Quantity calibration for N : C ratio measurements was conducted using an atropine standard.

Data analysis

Factorial analyses of variance (ANOVA) were used to analyse the effects of treatments and host species identity on height and DW of biomass of R. alectorolophus and DW of biomass of the hosts and the estimated amount of carbon present in the hemiparasite biomass. The response variables were log-transformed to improve normality and homoscedasticity of residuals. Tukey honest significant difference post-hoc tests were calculated to test differences among individual levels of statistically significant multilevel terms.

Linear mixed-effect models were used to analyse the carbon isotope composition and N : C ratio of R. alectorolophus plants and the proportion of host-derived carbon in individual parts of its biomass. The analysis of ‘split-plot’ design was used: the host species, shading and their interaction represented the ‘main plot’ fixed factor tested against the variability among individual plants (plant identity was a random factor nested in the interaction of host species × shading). The plant parts represented the split-plot factor tested against the residual variability, similarly to its interactions. A priori defined contrasts were employed for further detailed analyses of model results and their interpretation. The contrasts for the light treatments were defined to compare both shading treatments with the control (treatment contrasts; Crawley, 2007). Custom contrasts were defined for separate R. alectorolophus plant parts from which isotope samples were taken, testing the following: upper vs lower part of the plant; leaf vs stem in the upper part of the plant; and leaf vs stem in the lower part of the plant. All statistical analyses were conducted in R, version 2.12 (R Development Core Team, 2010). Linear mixed-effect models were calculated using the package nlme, version 3.1-97 (Pinheiro et al., 2010).

Results

Survival and growth of the experimental plants

All plants subjected to the total darkness treatment died within 5 d. In the other treatments, Rhinanthus plants survived until the harvest except for three shaded seedlings and one control plant. These plants were excluded from further analyses. Shading had a significant effect on biomass production in R. alectorolophus (Table 1, Fig. 2a). Plants of both shading treatments produced significantly less biomass than unshaded control plants, and shaded seedlings produced significantly less biomass than shaded young plants (Fig. 2a). A different effect of shading treatments on hemiparasite height was observed (Table 1, Fig. 2b), as shaded seedlings grew significantly less tall than control plants, while shading imposed later had no statistically significant effect on plant height (Fig. 2b). Host species identity had no significant effect on either biomass production or height of the parasites (Table 1, Fig. 2a,b).

Table 1.   Summary of factorial ANOVA testing the effects of shading treatments and host species identity on above-ground biomass production and height of Rhinathus alectorolophus and biomass of the hosts

Effect	Rhinathus biomass DW				Rhinanthus height			Host biomass DW
	df	Sum Sq.	F	P	Sum Sq.	F	P	Sum Sq.	F	P
Data were transformed by natural logarithm before the analysis. Sum Sq., Sum of squares. Statistically significant test results (P < 0.05) are indicated in bold.
Host species	1, 41	0.03	0.12	0.73	0.01	0.08	0.77	9.58	97.49	< 10^−10
Shading treatment	2, 41	30.37	58.04	< 10^−9	2.08	10.9	< 0.001	3.59	18.24	< 10^−5
Host species × shading treatment	2, 41	0.32	0.62	0.55	0.05	0.24	0.79	0.078	0.4	0.68
Residuals	41	10.73			3.91			4.03

Figure 2. Above-ground biomass production and height of Rhinanthus alectorolophus, and above-ground biomass production of its hosts under different shading treatments imposed on the hemiparasite. (a) DW of above-ground biomass produced by R. alectorolophus; (b) height of R. alectorolophus; (c) DW of above-ground biomass produced by the hosts to which R. alectorolophus was attached. White bars indicate wheat or R. alectorolophus attached to wheat; grey bars represent maize or R. alectorolophus attached to maize. Note the logarithmic scale of the y-axes. Error bars represent ± SE. Different letters symbolize statistically significant difference inferred from post-hoc pairwise comparisons (P < 0.05).

Host biomass production significantly differed between the host species (wheat produced less biomass than maize) and across the treatments (Table 1, Fig. 2c). The hosts of shaded young plants of R. alectorolophus produced a similar amount of biomass as hosts parasitized by control plants, while the amount of biomass produced by those parasitized by shaded seedlings was significantly higher (Table 1, Fig. 2c)

Carbon stable isotope composition and N : C ratio in biomass of the experimental plants

Carbon stable isotope composition of host species corresponded to values expected for C₃ and C₄ plants (Fig. 3a). δ¹³C values detected in the individual parts of R. alectorolophus biomass displayed substantial variability, which was significantly influenced by host species identity, shading treatment, plant part (from which the sample originated) and the interactions between these factors (Table 2). R. alectorolophus attached to wheat was in general slightly depleted compared with the host, as expected, due the low water-use efficiency of the parasite (Fig. 3a). R. alectorolophus plants attached to maize were significantly enriched in ¹³C compared with those attached to wheat (t₄₁ = 2.45, P = 0.0001). Leaves were significantly depleted in ¹³C compared with stems (t₁₃₀ = −5.04, P < 0.0001 in the upper part of the plants; t₁₃₀ = −4.76, P < 0.0001 in the lower part of the plants), while the difference in δ¹³C between the upper and the lower parts was nonsignificant (t₁₃₀ = 1.64, P = 0.10). The main effects of the shading treatment did not yield statistically significant contrasts (t₄₁ = 0.57, P = 0.57 for shaded seedlings; t₄₁ = 0.84, P = 0.40 for shaded young plants) but there was a significant interaction, indicating ¹³C enrichment in both shaded treatments of R. alectorolophus attached to maize (t₄₁ = 3.40, P = 0.0015 for shaded seedlings; t₄₁ = 2.18, P = 0.035 for shaded young plants). The upper parts of shaded young plants were significantly less enriched in ¹³C than the lower parts (t₁₂₁ = −3.601, P = 0.0005) and their upper leaves were significantly less depleted compared with the upper part of the stem (t₁₂₁ = 2.95, P = 0.0038), as against the situation in control plants. In addition, lower leaves of the parasites attached to maize were significantly depleted in ¹³C compared with the lower part of the stem (t₁₂₁ = −2.54, P = 0.012).

Figure 3. δ¹³C values (a) and N : C atomic ratio (b) in biomass of wheat (C₃ photosynthesis) and maize (C₄ photosynthesis) hosts and individual samples of Rhinanthus alectorolophus plants (C₃ photosynthesis) under individual shading treatments. Black, grey and white boxes indicate shaded seedlings, shaded young plants and control treatments, respectively. The squares in boxes represent medians, boxes represent quantiles (i.e. 25 and 75% quantiles) and the lines extending from the box boundaries (whiskers) represent the range or nonoutlier range of values, whichever is smaller. The nonoutlier range is defined as the interval between (25% quantile − 1.5 × interquartile range) and (75% quantile + 1.5 × interquantile range). Any point outside this interval is considered an outlier and is depicted separately.

Table 2.   Summary of the general linear mixed-effect models testing the effects of host species identity, shading treatment and part of the plant from which the sample was taken on the δ¹³C value and N : C ratio in the biomass of Rhinathus alectorolophus

Effect	δ^13C value			N : C ratio
	df	F	P	df	F	P
Nonsignificant terms were omitted from the final model of N : C ratio distribution. Overall tests of the final models: δ^13C, likelihood ratio = 205.23, df = 17, P < 0.0001; N : C ratio, likelihood ratio = 194.42, df = 12, P < 0.0001 (tested against the null models containing no fixed-effect terms). Statistically significant test results (P < 0.05) are indicated in bold.
Host species	1, 41	89.93	< 0.0001	1, 41	4.07	0.050
Shading treatment	2, 41	14.15	< 0.0001	2, 41	40.89	< 0.0001
Plant part	3, 121	62.74	< 0.0001	3, 121	73.38	< 0.0001
Host species × shading treatment	2, 41	6.86	0.0027	2, 41	1.33	0.28
Host species × plant part	3, 121	3.08	0.0301	3, 121	1.23	0.30
Shading treatment × plant part	6, 121	4.16	0.0008	6, 121	4.75	0.0002

The N : C atomic ratio in the biomass of Rhinanthus was clearly affected by the shading treatments and according which part of the plant the sample originated (Fig. 3b, Table 2), although there was also a marginally significant effect of host species identity (parasites attached to maize had slightly lower N : C ratio, t₄₃ = −2.08, P = 0.043). The N : C ratio in unshaded control plants was close to that of wheat host plants (incl. parasites attached to maize), while shading caused a significant increase of its value in both shaded seedlings (t₄₃ = 8.32, P < 0.0001) and young plants (t₄₃ = 4.39, P = 0.0001). The upper parts of Rhinanthus displayed generally higher N : C ratio (t₁₂₄ = 2.90, P = 0.0044), which also applied to leaves compared with stems in both upper (t₁₂₄ = 2.02, P = 0.045) and lower (t₁₂₄ = 4.93, P < 0.0001) parts of the plants. In addition, there was a pronounced effect of interaction between the shading treatments and parts of the plants. The upper parts of both shaded seedlings and shaded young plants had a higher than additive N : C proportion in their upper part compared with the unshaded control (t₁₂₄ = 2.28, P = 0.024, t₁₂₄ = 3.78, P = 0.0002, respectively). In addition, shaded seedlings had a higher than additive N : C proportion in their upper leaves compared with the stems (t₁₂₄ = 2.85, P = 0.0052) and a similar pattern was present in the lower part of the shaded young plants (t₁₂₄ = 2.04, P = 0.043).

Proportion and total amount estimation of host-derived carbon in hemiparasite biomass

The proportion of host-derived carbon in Rhinanthus biomass was significantly affected by the shading treatments and differed across parts of the plants sampled (Fig. 4a, Table 3). Shaded seedlings had a significantly higher proportion of host-derived carbon in their biomass than did control plants (t₂₃ = 4.30, P = 0.0003). A similar, albeit smaller, difference was detected between shaded young plants and control plants (t₂₃ = 2.62, P = 0.015). In addition, the host-derived carbon proportion was significantly lower in the upper parts of the plants than in the lower parts (t₆₄ = −3.40, P = 0.0012). There were also significant interactions caused by higher than additive proportions of host-derived carbon in the upper parts of shaded seedlings (t₆₄ = 3.31, P = 0.0015) and lower than additive proportions in the lower leaves compared with the corresponding parts of the stem (t₆₄ = −4.64, P < 0.0001) in plants undergoing the same treatment.

Figure 4. (a) Proportion of host-derived carbon in different samples of biomass of Rhinanthus alectorolophus attached to maize and cultivated under different shading treatments. The values were calculated from raw δ¹³C data (Fig. 3a) using an isotope mixing model Eqn 2. (b) Total estimated amount of host-derived carbon in biomass of R. alectorolophus attached to maize and cultivated under different shading treatments. The values were calculated as a product of mean proportion of host-derived carbon in hemiparasite biomass across individual analysed parts of a plant and the DW of its above-ground biomass. Black, grey and white boxes indicate shaded seedlings, shaded young plants and control treatments, respectively. The squares in boxes represent medians, boxes represent quartiles (i.e. 25 and 75% quantiles) and the lines extending from the box boundaries (whiskers) represent the range or nonoutlier range of values, whichever is smaller. The nonoutlier range is defined as the interval between (25% quantile − 1.5 × interquartile range) and (75% quantile + 1.5 × interquantile range). Any point outside this interval is considered an outlier and is depicted separately. Different letters in (b) symbolize statistically significant difference inferred from post-hoc pairwise comparisons (P < 0.05).

Table 3.   Summary of a linear mixed-effect model testing differences in the proportion of host-derived carbon in the biomass of individual parts of Rhinathus alectorolophus plants cultivated under the shading treatments

Effect	df	F	P
Plant identity was used as a random factor nested within the shading treatment. Overall test of the final model: likelihood ratio = 71.86, df = 11, P < 0.0001 (tested against null model containing no fixed-effect terms). Statistically significant test results (P < 0.05) are indicated in bold.
Shading treatment	2, 23	9.91	0.0008
Plant part	3, 64	8.12	0.0001
Shading treatment × plant part	6, 64	8.22	< 0.0001

The shading had a substantial effect not only on the proportion of host-derived carbon in Rhinanthus biomass but also on the estimated total amount of carbon incorporated in the above-ground biomass (Fig. 4b, one-way ANOVA summary: log-transformed data, R² = 0.376, F_(2,23) = 6.93, P = 0.0044). In contrast to the observed positive effect of shading on the proportion of host-derived carbon, its total estimated amount was significantly lower in shaded seedlings than in the other two treatments (Fig. 4b). There was no statistically significant difference between shaded young plants and control plants.

Discussion

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.

Wider perspectives

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.

Acknowledgements

We thank Petra Fialová and Ladislav Marek (University of South Bohemia) for analysing samples for ¹³C content. We are grateful to Renate Wesselingh and two additional anonymous reviewers for their comments, which helped to improve the original version of the manuscript. J.T., J.L. and M.V. are supported by the Grant Agency of the Academy of Sciences of the Czech Republic (grant no. IAA601410805), Grant Agency of the Czech Republic (grant no. 206/08/H044), Grant Agency of the University of South Bohemia (grant nos 138/2010/P and 134/2010/P) and the Ministry of Education of the Czech Republic (institutional grant no. MSM6007665801). D.D.C. was supported by a Royal Society University Research Fellowship (award number: UF090328).