Biology of the Root Parasitic Rhinanthoid Orobanchaceae

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1
Phylogeny, life history evolution and biogeography of
the Rhinanthoid Orobanchaceae
Jakub Těšitel*, Pavel Říha, Šárka Svobodová, Tamara Malinová, Milan Štech
Faculty of Science, University of South Bohemia, Branišovská 31, CZ-370 05, České Budějovice,
Czech Republic
* for correspondence: [email protected]
Abstract
The Rhinanthoid Orobanchaceae form a monophyletic lineage including the hemiparasitic genera
Euphrasia, Melampyrum, Tozzia, Bartsia, Nothobartsia, Odontites (s. l.), Rhinanthus,
Rhynchocorys, Parentucellia, Hedbergia and holoparasitic Lathraea. In this study, we aimed to
reconstruct the phylogeny, evolution of life history traits (life cycle and seed size) and explain the
extant biogeographic patterns in this group. For the phylogenetic reconstruction, we used molecular
data obtained by sequencing of the nuclear ITS region and chloroplast trnT-trnL intergenic spacer
and matK+trnK region. The genus Melampyrum was found to occupy the sister position to the rest
of the group. The other genera were assembled in the sister Rhinanthus-Rhynchocorys-Lathraea and
Bartsia-Euphrasia-Odontites subclades. The reconstruction of life cycle evolution yielded
ambiguous results suggesting nonetheless substantially higher likelihood of perenniality compared
to annuality in most ancestor lineages. Seed size varied across two orders of magnitude (average
weight per seed: 0.02-7.22 mg) and displayed a notable pattern characterized by tendency to
reduction in the Bartsia-Euphrasia-Odontites subclade compared to the rest of the group. Seed size
evolution was found to be correlated with life history evolution in the group if the generally smallseeded Bartsia-Euphrasia-Odontites subclade is excluded. We formulated hypotheses relating the
extant biogeographical affinities of individual genera to the geological history of the EuroCaucasian diversity centre of the group. Notable dispersal events in Euphrasia and Bartsia were
hypothesised to be allowed or at least facilitated by a specific combination of the life history traits.
The full version of this paper is protected by copyright. Therefore, it is archived by the Faculty of
Science, University of South Bohemia only in the original print version of the Ph.D. thesis. The
final version of the full paper is available at:
http://www.springerlink.com/content/w02lvq6307346m25/
The final publication is available at www.springerlink.com
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Tšitel et al.: Heterotrophic carbon gain by root hemiparasites
Paper 2
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atom % 13C and re-expressed as delta values relative to the Pee Dee Belemnite standard (G) using
equation 1.
G 13C ( RSample / RStandard 1) 1000[‰]
13
12
where RSample = C: C ratio in the sample and RStandard
standard.
Eqn 1
= C: C ratio in the Pee Dee Belemnite
13
12
Assessment of host derived carbon in hemiparasite biomass
Rhinanthus minor, E. rostkoviana and wheat perform C3 photosynthesis while maize performs C4
photosynthesis. As a result of their photochemical processes, C4 plants are usually significantly
more enriched in 13C than C3 plants. Hence, it is possible to infer the extent of heterotrophic carbon
gain of a hemiparasitic plant by measuring the relative change in the G13C value of the C3 parasite
attached to a C4 host compared to when it is attached to a C3 host. We used an adjusted form of a
linear two-source isotope-mixing model (Marshall & Ehleringer 1990), adapted from Gebauer &
Meyer (2003), to calculate proportion of host-derived carbon in hemiparasite biomass (Equation 2).
§ G 13C P (C4 ) G 13C P (C3 ) ·
¸ 100 [%]
% H ¨ 13
Eqn 2
¨ G C H (C ) G 13C H (C ) ¸
4
3 ¹
©
where %H = the percentage of carbon in parasite biomass that is derived from the host, GCP(C3) =
G13C of the parasite growing on the C3 wheat host, GCP(C4) = G13C of the parasite growing on the C4
maize host, GC+C3) = G13C of the infected wheat host and GC+C4) = G13C of the infected maize host.
For each replicate, GC P(C4) and the GC H(C4) of the corresponding host plant were entered into the
model calculation and the average values of GCH(C3) and GC P(C3) were used as the baseline
reference value. The outcome of this model provides an estimate of proportion of heterotrophic
carbon in hemiparasite biomass produced by an adult host-attached plant. This value integrates
organic carbon flows from the host to the hemiparasite, the actual values of which can be variable in
time. The model assumes that there is no fractionation during the assimilation of host-derived
nutrients by the parasite or that the fractionation has the same effect on G13C in parasites cultivated
with both maize and wheat. Such assumption is reasonable since direct luminal continuity exists in
R. minor haustoria (Cameron et al. 2006). In case of E. rostkoviana, absence of luminal continuity
cannot be excluded; nonetheless, a study on Olax phyllanthi which conducts nutrient transfer via
contact interfacial parenchyma does not report any substantial bias connected to isotope
discrimination during the transfer (Tennakoon & Pate 1996). In addition, xylem G13C is assumed to
be of the same value as the bulk leaf dry matter. Information on isotopic composition of xylem sap
is unfortunately missing from literature probably due to difficulty in measuring this value, caused
by low organic carbon concentration. Variation in G13C across main structures of a normal
autotrophic plant however tends to be fairly restricted (Bowling et al. 2008). Moreover if any
significant fractionation effect occurred, it could be assumed to be equal in both host species
resulting in an equal effect on both terms in the mixing model numerator.
Statistical analysis
Differences between treatment means were analysed by ANOVA followed by Fisher’s multiple
comparison test or using Student’s t test using Minitab version 13 (Minitab Inc., PA, USA). Where
necessary, to satisfy the test assumptions, data were arcsine square root transformed. Welch’s
estimation of degrees of freedom was used for t tests performed on data with unequal samples
sizes. Untransformed means and associated standard errors are presented. The isotope-mixing
model outcomes were tested for significant difference from 0 (i.e. no carbon gain from
heterotrophy) using Student’s t test.
34
Paper 2
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40
3
Interactions between hemiparasitic plants and their
hosts – the importance of organic carbon transfer
Jakub Těšitel1*, Lenka Plavcová2,3, Duncan D. Cameron4
1
Department of Botany, Faculty of Science, University of South Bohemia, Branišovská 31, 370 05
České Budějovice, Czech Republic
2
Department of Plant Physiology, Faculty of Science, University of South Bohemia, Branišovská
31, 370 05 České Budějovice, Czech Republic
3
Department of Renewable Resources, University of Alberta, 4-42 ESB, Edmonton, AB, T6G 2E3,
Canada
4
Department of Animal and Plant Sciences, University of Sheffield, Alfred Denny Building, Western
Bank, Sheffield, S10 2TN.
*for correspondence: [email protected]
Abstract
Hemiparasitic plants display a unique strategy of resource acquisition combining parasitism of other
species and own photosynthetic activity. Despite the active photoassimilation and green habit, they
acquire substantial amount of carbon from their hosts. The organic carbon transfer has a crucial
influence on the nature of the interaction between hemiparasites and their hosts which can oscillate
between parasitism and competition for light. In this minireview, we summarize methodical
approaches and results of various studies dealing with carbon budget of hemiparasites and the
ecological implications of carbon heterotrophy in hemiparasites.
The full version of this paper is protected by copyright. Therefore, it is archived by the Faculty of
Science, University of South Bohemia only in the original print version of the Ph.D. thesis. The
final version of the full paper is available at:
http://www.landesbioscience.com/journals/10/article/12563/
The definitive version is available at www.blackwell-synergy.com.
4
The role of heterotrophic carbon acquisition by the
hemiparasitic plant Rhinanthus alectorolophus in
seedling establishment in natural communities: a
physiologicalperspective.
Jakub T³šitel1*, Jan Lepš1,2, Martina Vráblová3 & Duncan D. Cameron4
1
Department of Botany, Faculty of Science, University of South Bohemia, Branišovská 31, 370 05
Zeské Bud[jovice, Czech Republic
2
Institute of Entomology, Biology Center, Academy of Sciences of the Czech Republic, Branišovská
31, 370 05 Zeské Bud[jovice, Czech Republic
3
Department of Plant Physiology, Faculty of Science, University of South Bohemia, Branišovská
31, 370 05 Zeské Bud[jovice, Czech Republic
4
Department of Animal and Plant Sciences, University of Sheffield, Alfred Denny Building, Western
Bank, Sheffield, S10 2TN.
* for correspondence: [email protected]
Introduction
Most plants are photoautotrophic organisms fixing carbon through photosynthesis. Plants are
however dependent also 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 parasitising fungi
(mycoheterotrophy; Leake 1994, Selosse & Cameron 2010) or other plants (plant-parasitism; e.g.
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 attack vascular system of other species 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 however 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 tropic weed Striga spp. (Dörr 1997). The connection to the host’s xylem and the
absence of a phloem connection implies that water and mineral nutrients are the most important
resources obtained from the host. Their flows have indeed been quantified in numerous
49
Tšitel et al.: The role of heterotrohic carbon in Rhinanthus seedling establishment
Paper 4
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
T³šitel et al. 2010a). Despite low organic carbon concentration in xylem, the extensive amount of
solutes acquired from the host can result also in significant heterotrophic carbon gain (T³šitel et al.
2010a) observed not only in Rhinanthus but also in numerous representatives of other roothemiparasitic species including Euphrasia (T³šitel et al. 2010a), Striga (Press et al. 1987) and Olax
(Tennakoon & Pate 1996) species. The host-derived carbon can account for 20-80% of hemiparasite
dry-mass (see T³šitel et al. 2010b for a review).
The hemiparasitic lifestyle provides notable advantages to Rhinanthus spp. supplying
relatively rich and stable resource of inorganic and organic nutrients without need of carbon
investment in 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 (T³šitel 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 persistence of Rhinanthus spp.
populations growing in dense-sward communities dominated by perennial species. Rhinanthus spp.
and some other hemiparasites (e.g. Melampyrum species; T³šitel 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 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 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 high extinction risk (Cameron et al. 2009) or
competitive exclusion (Fibich et al. 2010) at highly productive sites.
Limitation of Rhinanthus spp. to low and intermediate productivity grasslands by
interspecific competition might appear in conflict with the reported ability of Rhinanthus to
withdraw substantial (up to c. 50% of dry biomass for Rhinanthus minor; T³šitel 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 prior to anthesis, limiting their predictive power over
the competitive exclusion hypothesis detailed above.
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 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 prior the formation and
emergence of green shoots (Irving & Cameron 2009). In Rhinanthus, the seedling stage can be
therefore hypothesized as the bottle-neck stage of the populations when most mortality occurs due
to 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-
50
Paper 4
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 T³šitel 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 hence was 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 agroecosystems prior to the industrialisation of agriculture, infesting cereal crops most likely including
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
frequently used model species in ecological and ecophysiological studies of root-hemiparasites (e.g.
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 Volyn³, Czech
Republic (N 49°09'28'', E 13°52'13'', 510 m a.s.l.).
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 Ámol
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 Ámol
photons m-2 s-1) until steady-state assimilation rate developed, which was consequently recorded
followed by decrease of the irradiation level and steady-state assimilation rate recording in a series
of 1500, 1000, 800, 500, 300, 150, 100, 50 and 0 Ámol photons m-2 s-1. Gas-exchange measurements
were conducted on leaves of R. alectorolophus plants using an infra red gas analyser coupled to a
Li-6400 portable photosynthesis system (Li-Cor Biosciences, Lincoln, USA). Individual curves
were fitted by a non-linear regression models using an equation in Lambers et al. (2008):
II Amax {(II Amax ) 2 4TIIAmax }0.5
Eqn 1
An
Rd
2T
where An = rate of assimilation, Amax = light-saturated rate of CO2 assimilation, I = apparent
quantum yield, = curvature factor, Rd = 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.
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 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
G13C value ranges between -12 and -18‰; Smith & Epstein 1971) than C3 plants (expected G13C
value ranges between -23 and -35‰, Smith & Epstein 1971; expected G13C of Rhinanthus
assimilates is close to the lower limit of the C3 range due to 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 Ä13C value of the C3 parasite attached to a C4 host compared to 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 T³šitel et al. 2010a), relating the excess of
13
C in hemiparasites attached to the C4 host compared to those attached to the C3 host to the
51
Paper 4
difference in isotope composition between the C3 and C4 hosts themselves (Eq. 2).
§ G P ( C 4 ) G P ( C3 ) ·
¸ 100 [%]
%H ¨
Eqn 2
¨ G H (C ) G H (C ) ¸
4
3 ¹
©
Where %H = the percentage of carbon in parasite biomass that is derived from the host, GP(C3) = G13C
of the parasite growing on the C3 wheat host, GP(C4) = G13C of the parasite growing on the C4 maize
host, GH(C3) = G13C of the infected wheat host and GH(C4) = G13C of the infected maize host. G13C
Fig. 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 (full line) and the confidence
intervals (dashed lines) were calculated as means and 2 u standard errors of values predicted by
light-response model of individual plants. Symbols represent raw data and are classified by plant
sample identity. Mean (± standard error) parameters of the light-response curve models: Amax =
22.69 (±1.26) Ámol CO2 m-2 s-1, Rd = 2.00 (±0.31) Ámol CO2 m-2 s-1, I = 0.085 (±0.004), = 0.467
(±0.094), compensation point: 24.81 (±3.7) Ámol photons m-2 s-1. Grey dashed lines represent PAR
intensities resulting from the shading treatments: 50 Ámol photons m-2 s-1 under shaded conditions,
450 Ámol photons m-2 s-1 under full irradiation.
52
Paper 4
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 was based on our experimentally derived values for GP(C4) of
the separate parts of the R. alectorolophus plant and the GH(C4) of leaf of to which the parasite was
attached. The average values of GH(C3) of corresponding treatment and GP(C3) of corresponding
separate part of R. alectorolophus plants cultivated under corresponding treatment were entered into
the model as the baseline C3 reference values. Total amount of host-derived organic carbon in
above-ground hemiparasite biomass was estimated as a product of biomass dry weight 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, effect of
which was experimentally excluded by T³šitel et al. (2010a). In addition, the model assumes that no
carbon isotope fractionation occurs during the transfer of solutes through haustoria and that
potential difference in G13C 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 cm x 10 cm square pots containing 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 of Faculty of Science University of South Bohemia under light intensity of 450
(400-500) Ámol m-2 s-1 (photosynthetic active radiation - PAR) with a cycle of 14h day and 10h dark
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 to 4 cm from the host plant after 7 days of host development.
After having produced green cotyledon, the parasite seedlings were thinned to one per pot by
removing the most and least advanced seedlings to obtain a homogeneous cohort of seedlings.
Pots were divided into four experimental groups arranged in a completely randomised
design. Shading was imposed on Rhinanthus plantlets 8 days after sowing in the first group
(referred to as shaded seedlings, n = 18). In the second group (n = 18), the same shading was
imposed to young plants on day 24 after sowing (referred to as shaded young plants). The third
group (n = 17) consisted of plants that were kept under 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 coming on any part of the shaded plants. Moreover, the position of
each of the pots was exchanged randomly every c. 7 days to diminish possible effects of irradiation
heterogeneity (although this was rather minimal as indicated by the ranges of irradiation values).
The shading imposed on the experimental plants decreased the PAR intensity from 450
(400-500) Ámol photons m-2 s-1 to 50 (40-60) Ámol photons m-2 s-1 corresponding to rates of
photosynthesis 14.25 Ámol CO2 m-2 s-1 (62.8% of Amax in plants growing under natural conditions)
and 1.82 Ámol CO2 m-2 s-1 (8.0% of Amax, 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
understory (Hautier et al. 2009). The shading was imposed by a square shield made of thick green
paper (8 x 8 cm, 218 g m-2) blocking the direct light. The shaded plants therefore received just
indirect irradiation 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 centimetres
in such cases; however an additional shield was placed to the pot to keep the irradiation level within
the desired range of 40-60 Ámol photons m-2 s-1 (confirmed by PAR measurements). The complete
53
Paper 4
darkness treatment was imposed by covering the Rhinanthus plants by a 1 cm thick isolation
polypropylene tube (inner diameter of 5 cm, length 20 cm) which was covered by a small tray and
perforated by holes (c. 1 cm in diameter, 1 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 designed to provide sufficient contrast between the light
treatments rather than analyse performance or physiology of R. alectorolophus under exact levels of
irradiation used. The shaded plants also performed better than might be expected from the lightresponse curves of the R. alectorolophus plants (Fig 1.) due to higher the effectiveness of diffuse
light photosynthetic utilization (e.g. Sinclair & Shiraiwa 1992, Gu et al. 2002); nonetheless the
shading apparently created a contrast of light-deficiency stressed vs. non-stressed plants.
Biomass harvesting and stable isotope analysis
Plants were harvested 46 days after sowing of R. alectorolophus. Height of each of the R.
alectorolophus plants was measured prior to the harvest. 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 hours and weighed immediately. Dry weight 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
analyzer (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 Equation 3:
Ä13 C ( RSample / RStandard 1) 1000[‰]
Eqn 3
13
12
13
12
where RSample = C: C ratio in the sample and RStandard = C: C ratio in the Pee Dee Belemnite
standard. In addition to the Ä13C 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, Ä13C = -24.389‰) which
is related to the international standard saccharose (Ä13C = -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 dry weight of biomass of R. alectorolophus and dry weight 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 u 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
treatment with the control (treatment contrasts; Crawley 2007). Custom contrasts were defined for
separate R. alectorolophus plant parts from which isotope samples were taken, testing i) upper vs.
lower part of the plant ii) leaf vs. stem in the upper part of the plant and iii) leaf vs. stem in the
lower part of the plant. All statistical analyses were conducted in R version 2.12 (R Development
54
Paper 4
Core Team 2010). Linear mixed-effect models were calculated in package nlme version 3.1-97
(Pinheiro et al. 2010).
Results
Survival and growth of the experimental plants
All plant on which the total darkness treatment was imposed died within five days. 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 compared to 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 smaller compared to 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).
Host biomass production significantly differed between the host species (wheat produced
less biomass than maize) and across the treatments (Table 1, Fig 2c). 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)
Table 1 Summary of factorial analyses of variance testing the effects of shading treatments and host
species identity on above-ground biomass production and height of R. alectorolophus and biomass
of the hosts. Data were transformed by natural logarithm prior to the analysis. Statistically
significant test results (P < 0.05) are indicated in bold.
Effect
df
1. Host species
1,41
2. Shading treatment 2,41
2,41
1. u 2.
Residuals
41
Rhinathus biomass
dry weight
Sum Sq.
F
0.03
0.12
30.37 58.04
0.32
0.62
10.73
Rhinanthus height
P
Sum Sq.
0.73
0.01
-9
<10
2.08
0.55
0.05
3.91
host biomass dry weight
F
P
Sum Sq.
0.08
0.77
9.58
10.9 < 0.001
3.59
0.24
0.79
0.078
4.03
F
97.49
18.24
0.4
P
<10-10
<10-5
0.68
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). Ä13C 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 to 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 to those attached to wheat (t41 = 2.45,
P = 0.0001). Leaves were significantly depleted in 13C compared to 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 Ä13C between the upper and the lower part was non-significant (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 leaf was significantly less depleted
compared the upper part of stem (t121 = 2.95, P = 0.0038) if compared to the situation in control
plants. In addition, lower leaves of the parasites attached to maize were significantly depleted in 13C
55
Paper 4
compared to the lower part of the stem (t121 = -2.54, P = 0.012).
The N/C atomic ratio in biomass of Rhinanthus was clearly affected by the shading
treatments and plant part from which 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 that of
wheat host plants (incl. parasites attached to maize) while shading caused 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 to 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 plant parts.
The upper parts of both shaded seedlings and
shaded young plants had higher than additive
N/C proportion in their upper part compared to
unshaded control (t124 = 2.28, P = 0.024, t124 =
3.78, P = 0.0002, respectively). In addition,
shaded seedlings had higher than additive N/C
proportion in their upper leaves compared to
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 hostderived carbon in hemiparasite biomass
Proportion of host-derived carbon in
Rhinanthus biomass was significantly affected
by the shading treatments and differed across
sampled parts of the plants (Fig. 4a, Table 3).
Shaded seedlings had significantly higher
proportion of host-derived carbon in their
biomass compared to 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, host-derived carbon proportion was
significantly lower in the upper parts of the
Fig. 2 Above-ground biomass production and
height of R. alectorolophus and above-ground
biomass production of its hosts under different
shading
treatments
imposed
on
the
hemiparasite. (a) Dry weight of above-ground
biomass produced by R. alectorolophus (b)
Height of R. alectorolophus (c) Dry weight 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 ±1 standard error. Different letters
symbolize statistically significant difference
inferred from post-hoc pairwise comparisons
(P < 0.05).
56
Paper 4
plants compared to 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
to the corresponding parts of the stem (t64 = -4.64, P < 0.0001) in plants of the same treatment.
Not only had the shading substantial effect on the proportion of host-derived carbon in
Rhinanthus biomass but also on the estimated total amount on carbon incorporated in the aboveground 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 hostderived carbon, its total estimated amount was significantly lower in shaded seedlings compared to
the other two treatments (Fig. 4b). There was no statistically significant difference between shaded
young plants and control plants.
Fig. 3 (a) Ä13C values and (b) N/C atomic ratio 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 points in boxes represent
medians, boxes represent quartiles and the lines extending from the box boundaries (whiskers)
represent the range or non-outlier range of values, whichever is smaller. The non-outlier range is
defined as the interval between 25% quantile – 1.5 times interquartile range and 75% quantile + 1.5
interquantile range. Any point outside this interval is considered outlier and depicted separately.
57
Paper 4
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 Ä13C value
and N/C ratio in biomass of R. alectorolophus. Non-significant terms were omitted from the final
model of N/C ratio distribution. Overall tests of the final models: Ä13C: 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).
Ä13C value
N/C ratio
Effect
df
F
P
df
F
P
1. Host species
1,41
89.93
< 0.0001
1,41
4.07
0.050
2,41
14.15
< 0.0001
2,41
40.89
< 0.0001
2. Shading treatment
3. Plant part
3,121
62.74
< 0.0001
3,121
73.38
< 0.0001
1.33
0.28
1. u 2.
2,41
6.86
0.0027
2,41
1.23
0.30
1. u 3.
3,121
3.08
0.0301
3,121
4.75
0.0002
2. u 3.
6, 121
4.16
0.0008
6, 121
Fig. 4 (a) Proportion of host-derived carbon in different samples of biomass of R. alectorolophus
attached to maize and cultivated under different shading treatments. The values were calculated
from raw Ä13C 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 hostderived carbon in hemiparasite biomass across individual analyzed parts of a plant and the dry
weight of its above-ground biomass. Black, grey and white boxes indicate shaded seedlings, shaded
young plants and control treatments respectively. The points in boxes represent medians, boxes
represent quartiles and the lines extending from the box boundaries (whiskers) represent the range
or non-outlier range of values, whichever is smaller. The non-outlier range is defined as the interval
between 25% quantile – 1.5 times interquartile range and 75% quantile + 1.5 interquantile range.
Any point outside this interval is considered outlier and depicted separately. Different letters in (b)
symbolize statistically significant difference inferred from post-hoc pairwise comparisons (P <
0.05).
58
Paper 4
Discussion
Our study unequivocally demonstrates that the Table 3 Summary of a linear mixed-effect model
growth of Rhinanthus alectorolophus is testing differences in proportion of host-derived
supported by two sources of organic carbon carbon in biomass of individual parts of R.
originating from its own photosynthetic alectorolophus plants cultivated under the
activity
and
host-derived
assimilates. shading treatments. Plant identity was used as a
Assimilates of autotrophic origin apparently random factor nested within the shading
represent the dominant component of carbon treatment. Overall test of the final model:
balance in adult R. alectorolophus plants in Likelihood ratio = 71.86, df = 11, P < 0.0001
agreement with the high rates of (tested against null model containing no fixed
photosynthesis
we
detected
in
R. effect terms).
alectorolophus plants under natural field Effect
df
F
P
conditions (Fig. 1). The fatal consequences of 1. Shading treatment
2,23 9.91 0.0008
the total darkness treatment for R. 2. Plant part
3,64 8.12 0.0001
alectorolophus identifies photosynthesis as an 1 u 2
6,64 8.22 < 0.0001
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 S. 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
Rhinanthus minor plants after 4.5 weeks post attachment. Not only did the shading treatments of
our experiment affect the rate of assimilation, but also 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 compared to shaded young plants and
unshaded control, which furthermore decreased their growth. In contrast, shaded young plants
abstracted a similar amount of organic carbon from the host as the control plants. In addition, these
plants had a larger photosynthetically active leaf area at the onset of shading, resulting in higher rate
of assimilation per whole plant compared to shaded seedlings. This in turn allowed production of
more leaves leading to a positive feedback underpinning differential performance of shaded
seedlings and young plants.
A comparison between the proportion of host-derived carbon, its total amount and N/C ratio
in the biomass of the hemiparasites (Fig 4a,b) clearly suggests that differences in assimilation rates
of plants under individual shading treatment caused the principal pattern in proportion of hostderived carbon in hemiparasite biomass. The low proportion of host-derived carbon and 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 higher concentration of host-derived carbon in the lower plant parts indicate hostderived carbon being mostly directed to structural components of the lower stem parts while the
autotrophic carbon is preferentially used for development of new leaves and vertical growth of the
stem, i.e. producing additional photosynthetically active area. On the other hand, nitrogen is mostly
directed to leaves and the upper parts of the plants (particularly in shaded treatments). These
contrasting patterns suggest an intensive retranslocation and metabolic processing of the host-borne
resources in the hemiparasite. A strikingly similar pattern was also detected in Striga 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,
59
Paper 4
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 per whole plant is dependent on its total leaf area,
stomatal conductance and water vapour concentration difference between the leaves and ambient
air. Of these, the effect of stomatal conductance can be assumed closely 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 (Fig. 5) rather than by lower leaf area of shaded young plants.
The pattern whereby the total amount of host-derived carbon in biomass in depends on 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,
differences in interior water vapour concentration, might have developed between irradiated and
shaded leaves due to their different temperature increasing the transpiration of leaves under full
irradiation. Leaf temperature data acquired during the field gas-exchange measurement of lightresponse curves (Fig. 1) however indicated rather low temperature difference (c. 1°C) between
leaves irradiated by 500 and 50 Ámol photons m-2 s-1 resulting in relatively small decrease of
transpiration rate inflicted by shading if compared to the effect of leaf area. In addition, 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). Due to 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, Erhelinger &
Marshall 1995) independently of light conditions during the day.
The lack of a significant difference in height between shaded young plants and control and a
rather moderate decrease of vertical growth in shaded seedlings achieving more than 50% height of
control plants (Fig. 2b) indicate that shaded plants mobilise their resources (acquired both
autotrophically and heterotrophically) to support their vertical growth aiming to avoid shading, a
fundamental process also observed in non-parasitic 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 from shading under natural conditions in grasslands when shading is imposed by competing
neighbouring plants. However, if shading is imposed in 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 in a very early stage of
development clearly supports the hypothesis of T³šitel et al. (2010a) explaining the conflict
between heterotrophic acquisition of substantial amount of carbon and reported sensitivity to light
competition in Rhinanthus (Matthies 1995, Hejcman et al. 2011) by inefficiency of resource uptake
by unattached hemiparasite seedlings. In addition, a moderate increase of grassland productivity and
hence increase of competition pressure (Hautier et al. 2009) has been reported to increase seedling
mortality in hemiparasites but also 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 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 support the growth of the hemiparasites after overcoming the
seedling stage by increasing effectiveness of their photosynthetic machinery.
60
Paper 4
Fig. 5 Light micrographs of
transverse semi-thin sections
of shaded leaves (a) leaves
expose to direct light (b) of
the experimental Rhinanthus
alectorolophus plants. The
shade leaf is represented by a
sample of upper leaf of a
plant of the shaded young
plant treatment and the upper
leaf an unshaded plant was
taken as sample of a sun leaf.
Leaves were fixed in 2.5%
glutaraldehyde, dehydrated
with an acetone series and
embedded in Spurr resin. 400
nm thick sections were cut
using a Leica Ultracut UCT
ultramicrotome
at
the
Laboratory
of
Electron
Microscopy, Institute of
Parasitology, Academy of
Sciences of the Czech
Republic, Ëeské Bud³jovice.
Sections were stained by 1%
toluidine blue prior to
microscopy.
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) have demonstrated that R. minor is able to persist
even in a highly productive grassland plots requiring however 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 (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 due to 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 substantial long-living seed bank, the
species must germinate, grow, flower and produce seeds in 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
61
Paper 4
rapid development and overcoming limitation in the critical recruitment stage. The ability to acquire
substantial amounts of host-derived organic carbon supporting the growth of young plants however
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 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 additional two anonymous reviewers for
their comments helping to improve the original version of the manuscript. JT, JL and MV 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). DDC is supported by a
Royal Society University Research Fellowship (award number: UF090328).
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104

Biology of the Root Parasitic Rhinanthoid Orobanchaceae

Transcription

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