Journal of Ecology 2014, 102, 1183–1194
doi: 10.1111/1365-2745.12274
Photosynthesis in perennial mixotrophic Epipactis spp.
(Orchidaceae) contributes more to shoot and fruit
biomass than to hypogeous survival
dric Gonneau1,2,3†, Jana Jersa
kova
4†, Elo€ıse de Tredern1, Ire
ne Till-Bottraud5,6, Kimmo
Ce
lanie Roy8, Toma
s
Ha
jek4 and Marc-Andre
Selosse1,9†*
Saarinen7, Mathieu Sauve1, Me
1
Centre d’Ecologie Fonctionnelle et Evolutive (UMR 5175), 1919 Route de Mende, 34 293 Montpellier Cedex 5,
de Lorraine, LSE, UMR1120, 54518 Vandœuvre-le
s-Nancy, France; 3INRA, LSE, UMR1120,
France; 2Universite
4
s-Nancy, France; Faculty of Science, University of South Bohemia, Branisovska
31, 370 05
54518 Vandœuvre-le
Bude
jovice, Czech Republic; 5Laboratoire d’Ecologie Alpine (LECA), Univ. Grenoble Alpes, 38000 Grenoble,
Cesk
e
€a
€ ka
€ritie
France; 6CNRS, LECA, CNRS, 38000 Grenoble, France; 7South Karelia Allergy and Environment Institute, La
Biologique (UMR 5174), Universite
Paul Sabatier 15, 55330 Tiuruniemi, Finland; 8Laboratoire Evolution et Diversite
^t. 4R1, 118, route de Narbonne, 31062 Toulouse Cedex 4, France; and 9De
partement Syste
matique et
CNRS, Ba
um national d’Histoire naturelle, CP 50, 45 rue Buffon, 75005 Paris, France
Evolution (UMR 7205 ISYEB), Muse
Summary
1. Some forest understorey plants recover carbon (C) not only from their own photosynthesis, but
also from mycorrhizal fungi colonizing their roots. How these mixotrophic plants use the resources
obtained from mycorrhizal and photosynthetic sources remains unknown.
2. We investigated C sources and allocation in mixotrophic perennial orchids from the genus Epipactis. Based on the assumption that fungal biomass has high d13C and N content, while photosynthetic biomass has lower d13C and N content, we indirectly estimated the respective contributions of
these two resources to various organs, at various times over the growth season. Fully heterotrophic
and fully autotrophic plants from the same sites were used as references for d13C and N content of
biomass purely issuing from fungi and photosynthesis, respectively.
3. In four investigated populations, the biomass shifted from fully heterotrophic in young spring
shoots to 80–100% autotrophic in leaves and fruits at fruiting time, suggesting that photosynthesis
supported mostly fruiting costs. In addition, fungal colonization decreased in roots over this period.
4. Based on d13C and N content, below-ground organs and young spring shoots from green (mixotrophic) individuals and spontaneous achlorophyllous variants (fully heterotrophic) displayed similar
fungal C contributions. Similar fungal contributions were also found in shoots of individuals that were
either sprouting (and thus partially photosynthetic) or dormant (and thus fully heterotrophic) in the
previous years. Therefore, fungal C supported mostly young spring shoots and below-ground organs.
5. Although experimentally shaded plants had decreased contributions of photosynthetic C in shoots,
experimentally defoliated plants showed no increase in fungal C contribution as compared with nondefoliated controls. Strikingly, these defoliated plants maintained the same seed production: they
likely compensated defoliation by increasing stem and fruit photosynthesis.
6. Synthesis. We propose a falsifiable model of C resource allocation in mixotrophic orchids, where
mycorrhizal fungi mostly support below-ground organs and survival, while photosynthesis mostly
supports above-ground sexual reproduction, but not below-ground reserves. We discuss how this
allocation pattern, where seed production depends on photosynthesis, complicates the evolutionary
route to full heterotrophy in mixotrophic orchids.
Key-words: 13C, albino plants, dormancy, mycoheterotrophy, mycorrhizas, N content, photosynthesis in the shade, plant–soil (below-ground) interactions, stable isotopes
*Correspondence author. E-mail: [email protected]
†These three authors equally contributed to this work.
© 2014 The Authors. Journal of Ecology © 2014 British Ecological Society
1184 C. Gonneau et al.
Introduction
In the last decade, some green forest plants have been discovered to combine heterotrophic nutrition with photosynthesis
(Gebauer & Meyer 2003; Selosse et al. 2004; Julou et al.
2005). These mixotrophic (MX) plants recover part of their
carbon (C) from their mycorrhizal partners, the soil fungi
symbiotically associated with their roots (see Selosse & Roy
2009 and Hynson et al. 2013 for reviews), and reverse the
usual mycorrhizal exchange where plants provide the fungi
with sugars as a reward for mineral nutrients. Indeed, some
fully heterotrophic plants, the so-called mycoheterotrophic
(MH) plants, are known to use mycorrhizal fungi as an exclusive C source (Leake 2004; Hynson et al. 2013). For this reason, MX plants are also called partial mycoheterotrophs
(Gebauer & Meyer 2003; Selosse & Roy 2009).
MX nutrition was found in perennial orchids and Monotropoideae, an Ericaceae subfamily (Hynson et al. 2013). Molecular barcoding allowed identification of their (often
uncultivable) mycorrhizal associates, which also form ectomycorrhizas with surrounding trees (e.g. Selosse et al. 2004;
Julou et al. 2005; Abadie et al. 2006; Tedersoo et al. 2007;
Zimmer et al. 2007; Hashimoto et al. 2012; Tesitelova et al.
2012; Yagame et al. 2012). This points towards surrounding
trees as ultimate C source and provides an isotopic fingerprint
of MH nutrition in MX plants: since ectomycorrhizal fungi
are enriched in 13C as compared with host trees (Mayor,
Schuur & Henkel 2009), MH biomass is enriched in 13C as
compared with autotrophic biomass (Trudell, Rygiewicz &
Edmonds 2003). Thus, MX orchids and Montropoideae display a 13C abundance intermediate between autotrophic and
fully MH plants (see review in Hynson et al. 2013). MH and
MX plants also have higher 15N and N concentration than
autotrophic plants (Gebauer & Meyer 2003; Trudell,
Rygiewicz & Edmonds 2003), reflecting a position in the
trophic chain above ectomycorrhizal fungi; however, 15N and
13
C abundances do not always correlate (Selosse & Roy
2009; Girlanda et al. 2011).
Little is known about C nutrition and C allocation in MX
plants. Investigations on photosynthesis (CO2 exchange, chlorophyll fluorescence, pigment concentrations) showed
impaired photosynthetic functions in the MX orchids Limodorum abortivum L. Swartz (Girlanda et al. 2006) and Corallorhiza trifida Chatel. (Cameron et al. 2009). Together with
reduced leaves, misshaped stomata and/or low light conditions
of their forest habitats, this often maintains photosynthesis
around or under the light compensation point (Julou et al.
2005; Girlanda et al. 2006; St€ockel, Meyer & Gebauer 2011).
Moreover, MX nutrition responds to the light level. Autotrophic plants normally exhibit lower 13C abundance in more
shaded conditions since CO2 is assimilated at a slower rate,
allowing a higher isotopic fractionation when compared with
high light conditions (Farquhar, Ehleringer & Hubick 1989).
In contrast, higher 13C abundances in the shade were recorded
in the MX Cephalanthera damasonium (Mill.) Druce (Preiss,
Adam & Gebauer 2010) and in MX Ericaceae (Tedersoo
et al. 2007; Zimmer et al. 2007; Matsuda et al. 2012). This
is because MX plants either obtain more 13C-enriched fungal
C in the shade than in full light, or simply obtain the same
amount of 13C-enriched fungal C, mixed in a lower amount
of 13C-depleted photosynthetic C (Hynson et al. 2012; Matsuda
et al. 2012; Roy et al. 2013).
In some Cephalanthera and Epipactis MX orchid species,
fully achlorophyllous MH variants (called albinos) survive up
to 12 years (Salmia 1989; Julou et al. 2005; Abadie et al.
2006), indicating that these species can use fungal C. However, compared with green individuals, albinos of the MX
C. damasonium display various maladaptations to MH life
that reduce their fitness (Roy et al. 2013): albinos have more
frequent dormancy (a below-ground survival over one or several years, without sprouting or flowering; Shefferson, Kull &
Tali 2005; Light & MacConaill 2006); their shoots dry more
frequently before fruit ripening, and surviving shoots bear
fewer seeds with lower germination abilities. Among explaining factors, C limitation is supported by two observations
(Roy et al. 2013): first, albinos have 39 lower basal metabolism than green individuals, as estimated by CO2 production
in the dark; secondly, fungal C proportion in green shoots
shifts from 80% heterotrophic at emergence to 20% at the fruiting stage; at the latter stage, photosynthesis is more efficient,
but mycorrhizal colonization is reduced or even absent. Albinos also have reduced mycorrhizal colonization at fruiting,
which may limit their ability to compensate for photosynthesis loss through fungal C. This supports the hypothesis that
photosynthates are required for late shoot nutrition and fruiting, while individual survival and early shoot development
rely more on fungal C through MH nutrition (Roy et al.
2013).
Here, we investigated the allocation of fungal versus photosynthetic C in MX orchids and tested response to C deprivation in above-ground organs in situ. To challenge the data
hitherto obtained from C. damasonium, we tested predictions
from Roy et al. (2013) on the related MX Epipactis species
E. helleborine (L.) Crantz and E. fibri Scappatici and Robatsch.
We used 13C abundance to estimate fungal (heterotrophic)
versus photosynthetic (autotrophic) C contributions and also
measured 15N and N concentrations. First, we monitored
shoot photosynthetic C proportion and root fungal colonization over the growth season: based on C. damasonium data,
we predicted (i) a decrease of fungal C proportion in shoots,
and thus of 13C abundance, and (ii) a decrease of fungal colonization in roots during the growth season (prediction #1).
Secondly, we investigated the impact of experimental shading
on C sources: we predicted that the fungal C proportion
would increase and buffer the expected decrease of 13C abundance over the growth season (prediction #2). Thirdly, we
investigated the effect of experimental defoliation on photosynthesis and fruiting: we predicted (i) a decrease of photosynthetic C proportion in shoots and fruits, that is, an
increase in 13C abundance, and (ii) a decrease in seed production (prediction #3). Fourthly, we tested the hypothesis that
photosynthesis makes little or no contribution to belowground reserves. We focused on dormancy, which is likely
supported by fungal C in MX species (Shefferson 2009; Roy
© 2014 The Authors. Journal of Ecology © 2014 British Ecological Society, Journal of Ecology, 102, 1183–1194
Resource allocation in mixotrophic orchids 1185
et al. 2013). If the previous year’s photosynthesis contributes
to below-ground reserves, shoots should contain relatively
more photosynthetic C when no dormancy occurred the year
(s) before sprouting. Conversely, if our hypothesis applies,
fungal C proportion and thus 13C abundance in emerging
shoots that reach the soil surface are independent of the number of dormant years (prediction #4). Correspondingly, nonphotosynthetic albinos and green individuals should have
same 13C abundance in their below-ground parts and emerging shoots (prediction #5). We tested these five predictions in
four European sites (Table 1).
Materials and methods
STUDIED SPECIES AND POPULATIONS
Several populations were used to account for the limited number of
individuals and local protection rules (Table 1). Epipactis helleborine
was studied in the Finnish M€antyl€anm€aki (M€a) nature reserve
(67°690 913 N, 35°630 960 E, elevation 90 m), at Kasperske Hory in
the Czech Republic (KH; 49°80 37″ N, 13°350 41″ E, 860 m) and at
Chauriat in France (Ch; 3°170 23″ E, 45°460 25″ N; 500 m). Previously, Julou et al. (2005) investigated C. damasonium at Ch, and
Salmia (1989) investigated E. helleborine albinos at M€a. Epipactis
fibri was studied in the French ^Ile du Beurre (IB) nature reserve
(45°280 25″ N, 4°460 52″ W, elevation 155 m). In a preliminary fungal
barcoding at IB (as in Selosse et al. 2002), E. fibri symbionts proved
Table 1. The five hypotheses (#1–#5) on mixotrophic orchids tested in
this study at four sites, encompassing three Epipactis helleborine populations (at M€antyl€anm€aki, M€a; Kasperske Hory, KH; and Chauriat, Ch)
and one Epipactis fibri population at ^Ile du Beurre (IB)
Four populations
E. helleborine
E. fibri
Five predictions tested in this study
M€a
KH*
Ch*
IB
#1 Proportion of fungal C† in shoots and
root fungal colonization decrease during the
growth season
#2 Shading increases the proportion of
fungal C† that buffers the expected
decrease of 13C abundance in shoots
#3 Defoliation decreases photosynthetic C
proportion† in shoots and fruits, as well as
seed production
#4 Fungal C proportion† in emerging
shoots‡ is independent of the number of
previous dormant years
#5 Albinos and green individuals have same
high proportion of fungal C† in belowground parts and emerging shoots‡
+
+
+
+
+
+
+
+
+
*Experimental manipulations were performed at KH (shading) and
Ch (defoliation) only; the two other populations were simply monitored.
†
Proportions of photosynthetic versus fungal C are indirectly estimated from their, respectively, low versus high d13C, and the fact that
these C sources are accompanied by low versus high N content.
‡
Emerging shoots are shoots sampled few time after they reached the
soil surface, early in the growth season.
to be ectomycorrhizal fungi (GenBank Accession numbers
KF414685–KF414693) dominated by Tuber spp. (KF414687, a Tuber
ITS, was recovered seven times), as expected for MX orchids.
DYNAMICS OF ISOTOPIC ABUNDANCES OVER THE
€
GROWTH SEASON AT MA
To test predictions #1 and #5 (Table 1), E. helleborine tissues were
harvested during the 2011 growth season at the stage of bud emergence with leaves stacked together (1 June), developed shoots with
leaves expanded (23 June), flowering (15 July) and fruiting (capsules
mature, but closed; 28 July). We collected mycorrhizas (i.e. roots
checked for fungal colonization), stems, leaves, and fruits or flowers
when available. For each stage, up to five green E. helleborine and
four albinos were sampled, together with leaves of autotrophic
Convallaria majalis L. and Solidago virgaurea L. (five replicates per
sampling date). Stems of the MH Hypopitys monotropa Crantz
(= Monotropa hypopitys L.) that appeared at the fourth sampling date
were harvested (five replicates).
SHADING EXPERIMENT AT KH
To test predictions #1 and #2 (Table 1), E. helleborine plant tissues
were harvested in 2010 at the same stages as in M€a (respective dates:
7 June, 20 June, 1 August, 26 August). During the first sampling, we
enclosed 12 plants in wire cages covered with a double layer of garden
green shade cloth on a 10 9 10 m experimental area. For each shaded
individual, an unshaded one was selected within a distance of 1 m, so
that both treatment and control plants were intermingled. The amount
of photosynthetically active radiation (PAR) inside and outside the
cages was measured in situ with an LI-190 Quantum sensor (LI-COR,
Lincoln, Nebraska, USA) four times for four different cages at midday.
For each stage, we harvested four shaded and four unshaded E. helleborine plants. We sampled leaves and fruits or flowers when available, as
well as mycorrhizas and non-mycorrhizal roots at the second sampling
date (mycorrhizal status was assessed by viewing fine washed root sections under a stereoscopic microscope). Leaves of autotrophic Alchemilla
vulgaris L. and Fragaria vesca L. (four replicates for each sampling
date) were sampled. The few F. vesca growing in two cages were sampled on 1 August (n = 4). The impact of shading on CO2 assimilation
was estimated using a portable gas-exchange system LI-6400 (LI-COR)
in situ on 1 August 2010, during a sunny day (between 12:00 and
16:00, solar time). The youngest, fully developed E. helleborine leaf
was acclimated in the standard 2 9 3 cm gas-exchange chamber at
PAR irradiance of 2000 (1000 for the shaded plants) lmol m2 s1 for
about 5 min until steady-state photosynthesis. Then the irradiance was
gradually reduced through 1000, 500, 250, 120, 60, 30, 15 and 8
(shaded only) down to 0 lmol m2 s1 (temperature was maintained at
23 2 °C and CO2 concentration at 400 ppm; n = 3 plants per treatment). No limitation of stomatal opening due to drought stress was
observed.
DYNAMICS OF MYCORRHIZAL COLONIZATION AT CH
To test prediction #1 (Table 1), roots of five individuals were harvested on 18 April 2010 (bud emergence) and 15 July of same year
(time of fruiting). Thin hand-cut sections were investigated in all roots,
every 5 mm, starting from the rhizome, under a magnification light
microscope. Sections were attributed to four colonization categories as
in Roy et al. (2013), C0 to C3 (see Fig. 3). Mean colonization was
estimated from the formula: 0.15 9 C1 + 0.45 9 C2 + 0.8 9 C3.
© 2014 The Authors. Journal of Ecology © 2014 British Ecological Society, Journal of Ecology, 102, 1183–1194
1186 C. Gonneau et al.
DEFOLIATION EXPERIMENT AT CH
Prediction #3 (Table 1) was tested on eight pairs of plants of similar
size, situated 40 cm from each other (i.e. not from the same rhizome)
and at the same light level. One shoot per pair was defoliated (bracts
included) on 1 June 2009 (flower buds still closed). Stems, ripening
fruits and leaves when available were collected from surviving shoots
at fruiting on 25 July 2009, before seed dispersal. Fruit maximum
width and length were measured with a vernier calliper. Fruits were
then weighed after 3 days of drying at 70 °C. Dried seeds were
spread on Petri dishes and observed under a 609 dissecting microscope. Since leaves were unavailable for comparison, isotopic abundances were assessed on stems and fruits. To evaluate the expected
differences between stems and other organs, a separate comparison of
isotopic abundances in mycorrhizas, rhizome, stems, leaves and fruits
was made in six other nearby plants harvested on 25 July, together
with leaves of surrounding autotrophic species (Hedera helix L.,
Cornus mas L. and Vincetoxicum officinale Moench; n = 4 replicates
each). Leaves from the plants defoliated in June (n = 8) and control
plants harvested in July (n = 5) were also compared for isotopic
abundances to test prediction #1.
SAMPLING OF PLANTS WITH DIVERGING DORMANCY
HISTORIES AT IB
Using three-year monitoring of individuals of E. fibri, which displays
frequent dormancy (80–90% of individuals; Scappaticci & Till-Bottraud 2010), we tested prediction #4 on the impact of dormancy on C
nutrition (Table 1). We sampled two subpopulations, one from a sunny
site (grassland) and one in the shade of a riparian forest where Alnus
glutinosa (L.) Gaertn., Fraxinus angustifolia Vahl, Populus sp. and
climbing Vitis vinifera L. cover 80% of the surface. Light levels at these
sites were compared as in Matsuda et al. (2012) during sunny days of
October 2009 and July 2010. We sampled one leaf per individual at fruiting (2 October 2009) and shoot emergence (5 July 2010), from individuals in each of the three types of life history described in Fig. 4,
together with leaves of autotrophic species (H. helix and Trifolium aureum Pollich for the sunny site, and F. angustifolia, Pimpinella saxifraga L. and V. vinifera for the shaded site; n = 4 for each species; no
MH species was available). Thus, involving early and late sampling
dates of plants from different light environments, our sampling also
allowed us to test predictions #1 and #2 (Table 1).
CARBON AND NITROGEN ISOTOPE COMPOSITIONS
All samples were dried and kept in silica gel until processing as in
Tedersoo et al. (2007) at the Technical Platform of Functional Ecology (OC081; INRA, Nancy, France) to measure abundances of 13C
and 15N and total N. The same significant differences were obtained
when using C/N instead of N content (not shown). Isotopic abundances were expressed in conventional d notation in &. The standard
deviation (SD) of the replicated standard samples (n = 18) was
0.043& for 13C and 0.162& for 15N. At all sites, all leaves were harvested at the same light level and same distance from the ground as
Epipactis individuals, with a maximal distance between samples of
5 m, to avoid any bias.
STATISTICS
All statistical tests were conducted using R software v 2.14.1 (http://
www.r-project.org/) with the a type I error fixed at 5% (thus, all non-
significant differences have P > 0.05). Normality of variables was
first tested using a Kolmogorov–Smirnov test. In the text, unless
otherwise stated, means are followed by standard deviation (SD).
d13C, d15N and N contents were compared over the growth season by
phenotype or by organ using Kruskal–Wallis (KW) tests and pairwise
comparisons using Mann–Whitney (MW) bilateral tests. Figures present means with SD as bars and, unless otherwise stated, different letters denote significant differences according MW bilateral tests for
each sampling date (using a post hoc Bonferroni correction to the
MW tests).
Results
DYNAMICS OF ISOTOPIC ABUNDANCES OVER
€
THE GROWTH SEASON AT MA
We monitored the heterotrophy level of albino and green
E. helleborine individuals from the M€a population over the
growth season, testing for predictions #1 (decrease of fungal
C proportion in shoots) and #5 (MH nutrition of emerging
shoots; Table 1). Albinos significantly increased in d13C
between the first two sampling dates (MW, U = 72,
P < 0.05; Fig. 1), without significant changes in d15N (see
Fig. S1) and N content (not shown), but these albino shoots
disappeared before the third sampling date. Epipactis helleborine albinos did not differ from the MH Hypopitys monotropa
for d13C and N content (MW; Fig. 1 and not shown), but had
significantly higher d15N (MW, U = 16, P < 0.001; Fig. S1).
As expected, at each sampling date where they were available, albinos and MH H. monotropa always had significantly
higher d13C (Fig. 1), d15N (Fig. S1) and N content (not
shown) than autotrophic Convallaria majalis and Solidago
virgaurea. Over the growth season, d13C of green E. helleborine leaves significantly decreased from 26.6 0.6 to
29.1 0.6& (KW, K = 16, P < 0.001), while autotrophic
leaves of C. majalis and S. virgaurea significantly increased
in d13C (KW, K = 15, P < 0.05; Fig. 1). Between shoot
emergence and fruiting, no significant variation occurred for
d15N and N content in autotrophs, or for d15N in green
E. helleborine (Fig. S1); N content significantly decreased in
green E. helleborine (KW, K = 26.5, P < 0.001; not shown).
Thus, green E. helleborine did not differ in d13C and N content from albinos at the first sampling date (suggesting a similar MH nutrition of emerging shoots for both phenotypes;
prediction #5), or from the autotrophic C. majalis at the last
two samplings (yet S. virgaurea remained significantly lower in
d13C and N content). We estimated the fungal C contribution
to MX green leaves by applying a linear mixing source model
(Hynson et al. 2013), using MH and autotrophic plants from
the same sampling date as references. Based on albinos, fungal
C contributed ca. 100% of green leaf biomass at shoot emergence and 35% at the second sampling date; based on H.
monotropa at the final sampling date, fungal C contribution fell
to 20% (likely an overestimation, since H. monotropa was
significantly more 13C-depleted than albinos). In all, this supported prediction #1 (decrease of fungal C proportion in
shoots over the growth season).
© 2014 The Authors. Journal of Ecology © 2014 British Ecological Society, Journal of Ecology, 102, 1183–1194
Resource allocation in mixotrophic orchids 1187
–21
–23
–25
–27
–29
–31
Shoot emergence
Leaves expanded
Flowering
Hypotitys monotropa
Solidago virgaurea
Convallaria majalis
E. helleborine green
fruits
E. helleborine green
Solidago virgaurea
Convallaria majalis
E. helleborine green
flowers
E. helleborine green
Solidago virgaurea
Convallaria majalis
E. helleborine albinos
E. helleborine green
Solidago virgaurea
E. helleborine albinos
E. helleborine green
–35
Convallaria majalis
–33
Fruiting
Fig. 1. d13C of leaves from green (n = 5; black columns) and albino (n = 4 when available; white columns) Epipactis helleborine over the 2011
growth season at M€antyl€anm€aki (M€a), compared with leaves of autotrophic Convallaria majalis and Solidago virgaurea (n = 5 each; grey and
striped grey columns) or stems of Hypopitys monotropa (n = 5; dotted column), with values for fruits and flowers wherever available (black dotted columns). For each sampling date, values with different letters differ significantly according to Mann–Whitney pairwise tests.
Flowers and fruits did not significantly differ from leaves
in d13C (Fig. 1), d15N (Fig. S1) and N content (not shown),
suggesting similar, mainly photosynthetic C sources. d13C
decreased (significantly at the last sampling) in the order:
mycorrhizas > stems > leaves (see Fig. S2a); d15N increased
in the order: mycorrhizas < stems < leaves (significantly at
the last two samplings only; see Fig. S2b); and N content
decreased in the same order (significantly at the last three
samplings; not shown). Thus, d13C and N content, but not
d15N, indicated decreasing fungal C proportion in these
organs. Mycorrhizas never differed significantly between albinos and green individuals in isotopic abundances (see Fig.
S2) and N content (not shown). Over the growth season, d13C
and N content did not vary significantly in mycorrhizas of
green individuals (KW, K = 5; Fig. S2a and not shown),
while d15N increased significantly (KW, K = 10, P < 0.05;
Fig. S2b). Thus, d13C and N content suggested a similar and
high proportion of fungal C in mycorrhizal biomass of both
phenotypes over the growth season (supporting prediction
#5), while variations of d15N suggested incongruent trends.
SHADING EXPERIMENT AT KH
We tested predictions #1 (decrease of fungal C proportion in
shoots) and #2 (increase of fungal C proportion in shaded conditions; Table 1) by monitoring experimentally shaded and
unshaded green E. helleborine individuals in the KH population. Compared with unshaded conditions (grassland at forest
edge), shading cages excluded 95.1 0.3% of outside PAR.
Shaded E. helleborine leaves displayed typical shade acclimation,
with lower dark respiration rates and thus a lower light compensation point than unshaded ones (see Fig. S3), and their net
CO2 assimilation rate was at least 2.5 times lower. As
expected, shaded leaves of autotrophic Fragaria vesca in
cages had induced d13C (d13C = 31.7 1.0& vs. 28.1
0.6& out of cages; Fig. 2), whereas shaded and unshaded
E. helleborine leaves never differed significantly (Fig. 2). For
each species, d15N abundance and N content also never differed significantly between treatments (not shown). The
E. helleborine d13C response thus suggested that a relative
increase of 13C-enriched fungal C buffered the expected 13C
depletion in the shade, in line with prediction #2.
Over the growth season, E. helleborine leaf d13C decreased
significantly from 27.3 1.5 to 30.0 1.0& (KW,
K = 9, P < 0.01), while leaves d13C in autotrophic F. vesca
and Alchemilla vulgaris did not change significantly, so that
the d13C difference between E. helleborine and the two autotrophs shifted from significant to non-significant (Fig. 2).
Epipactis helleborine d15N and N contents (not shown) were
always significantly above values for autotrophs and increased
significantly over the growth season. Thus, d13C and N content, but not d15N, supported prediction #1.
Flowers and fruits (unshaded only, because pollination
failed in cages) did not significantly differ in d13C, d15N and
N contents from corresponding leaves (n = 4 per treatment;
not shown), suggesting similar autotrophic C sources. At the
second sampling date, we investigated below-ground organs
(n = 4 each): mycorrhizal and non-mycorrhizal roots did not
differ significantly between shaded and unshaded E. helleborine in d13C (see Fig. S4), d15N or N content (not shown);
© 2014 The Authors. Journal of Ecology © 2014 British Ecological Society, Journal of Ecology, 102, 1183–1194
1188 C. Gonneau et al.
–21
13C
( )
–23
c
c
–25
a
b
a
b
a
a,b
a
a,b
a
Fragaria vesca
a,b
E.h. shaded
b
–27
b
–29
a
a
c
–31
–33
Shoot emergence
Leaves expanded
Flowering
E.h. shaded
E.h. non shaded
Alchemilla vulgaris
E.h. non shaded
Alchemilla vulgaris
Fragaria vesca shaded
Fragaria vesca
E.h. shaded
E.h. non shaded
Alchemilla vulgaris
Fragaria vesca
E.h. shaded
E.h. non shaded
Alchemilla vulgaris
Fragaria vesca
–35
Fruiting
Fig. 2. d13C of Epipactis helleborine leaves from individuals shaded (95% PAR reduction; black columns) or not (white columns) over the
growth season (n = 4 at each sampling; the shading began after the first sampling date) at Kasperske Hory (KH). As baselines, leaves of
trophic Alchemilla vulgaris and Fragaria vesca were sampled in full light (n = 4; grey and white-striped grey columns respectively), and
tional F. vesca leaves were sampled in shaded conditions on the third sampling (n = 4; black-striped grey column). For each sampling
values with different letters differ significantly according to Mann–Whitney pairwise tests.
2010
autoaddidate,
within each treatment, d13C decreased significantly in the
order: mycorrhizas > non-mycorrhizal roots > leaves (KW,
K = 16, P < 0.001; Fig. S4). Although d15N and N content
did not differ significantly (not shown), this suggested
decreasing proportions of 13C-enriched fungal C in these
organs.
DYNAMICS OF MYCORRHIZAL COLONIZATION AT CH
We tested prediction #1 on variation of fungal colonization
(Table 1) between bud emergence and fruiting by excavating
five green E. helleborine individuals per stage at Ch. We
observed a significant increase in frequency of non-colonized
root sections (C0), which reached 45.4%, and a significant
decrease of ≥ 60% of colonized sections (C3; Fig. 3). The
mean percentage of cortical root cell colonization decreased
from 51.1% to 17.2%, in line with prediction #1.
DEFOLIATION EXPERIMENT AT CH
We tested prediction #3 (defoliation should decrease photosynthetic C proportion in shoots and fruits, as well as seed
production; Table 1) at Ch, by defoliating green E. helleborine individuals before flowering, and analysing them after
54 days at the fruiting stage. For comparison, we also investigated isotopic abundances and N content of different organs
from six intact fruiting plants. All E. helleborine organs
Fig. 3. Mycorrhizal colonization of Epipactis helleborine roots at
Chauriat (Ch) in 18 April (bud emergence) and 15 July 2010 (fruiting; white and grey columns, respectively), expressed as the mean
percentage of all investigated root sections (from five individuals;
n = 45–85 sections per individual) falling into four colonization categories: C0, no fungal peloton; C1, pelotons in 1–30% of the cortical
cells; C2 pelotons in 31–60% of the cortical cells; C3, pelotons in
≥60% of the cortical cells (***, P < 0.001; ns, not significant according to a Mann–Whitney U-test).
showed significantly higher d13C than leaves of autotrophs, as
expected for MX species (see Fig. S5), and d13C values, and
thus fungal C proportion, decreased in the order: mycorrhizas ≥ rhizomes ≥ stems > leaves = fruits (see Fig. S5 for statistical supports). d13C was 2& lower in leaves and fruits
than in stems, with no significant difference in N content and
d15N (not shown).
© 2014 The Authors. Journal of Ecology © 2014 British Ecological Society, Journal of Ecology, 102, 1183–1194
Resource allocation in mixotrophic orchids 1189
Table 2. Comparison of d13C, shoot survival and fruiting (means SD; n, number of repetitions) for 54-day defoliated and control Epipactis
helleborine plants at Ch in July 2009. Values followed by different letter differ significantly according to Bonferroni-corrected Mann–Whitney Utests (P < 0.05), and there is no significant difference otherwise
Shoot survival
d13C in stems
d13C in fruits
% pollinated flowers
% fruits reaching maturity
Fruit length (cm)
Fruit width (cm)
Fruit dry weight (g)
Seed dry weight (mg)
Seed number
Seed number with embryo
Defoliated plants (n)
Control plants (n)
5 out of 8
25.91 0.71 (5)
26.38 0.74 (13)
0.94 0.12 (37)
0.59 0.46 (18)
1.98 0.23 (18)
0.71 0.06 (18)
0.11 0.02a (18)
17.46 7.31 (18)
4031 950 (18)
2786 723 (18)
5 out of 8
26.33 0.48 (5)
27.12 1.10 (13)
0.99 0.03 (33)
0.57 0.36 (12)
1.80 0.17 (12)
0.67 0.07 (12)
0.07 0.01b (12)
14.94 2.62 (12)
3608 509 (12)
2507 432 (12)
At defoliation, leaves had higher d13C (25.8 0.3&;
n = 8; MW, U = 42, P < 0.001) than 54 days later
(27.4 0.3&; n = 5), with significantly higher N content
and non-significantly higher d15N (not shown), so that d13C
and N content again followed prediction #1 (temporal
decrease of fungal C proportion in shoots). Defoliation did
not detectably affect survival, pollination or fruiting success
(Table 2; seed viability was, however, not tested). d13C was
non-significantly higher in defoliated stems and fruits than in
controls (Table 2), with no significant variation in N content
or d15N (not shown). In each treatment, stems had higher
d13C than fruits, but the difference was not significant
(Table 2), as expected from Fig. S5. Fruit parameters as well
as seed quantity and quality were always higher in defoliated
plants, but this was only significant for fruit dry weight (1.6fold higher). Thus, defoliation did not entail a higher proportion of fungal C in stems and fruits, or reduced fitness, and
this invalidated prediction #3.
ISOTOPIC ABUNDANCES AND DORMANCY HISTORIES
AT IB
Prediction #4 on the impact of dormancy on C nutrition
(Table 1) was tested in an E. fibri population at IB, where
individual plants are monitored annually (Scappaticci & TillBottraud 2010). Two subpopulations were monitored, respectively, from a shaded and a sunny site where PAR irradiance
was 11-fold (July) to 13-fold (October) higher, allowing further testing of prediction #2. Samplings were replicated in
October 2009 and July 2010, allowing further testing of prediction #1. Three dormancy histories were available (Fig. 4):
individuals sprouting in the previous year but dormant two
years before ([011]); individuals dormant in the previous two
years ([001]); and individuals dormant in the previous year
but sprouting two years before ([101]).
At both dates, all E. fibri leaves had higher d13C and d15N
than autotrophic leaves (Fig. 4; see Fig. S6; significantly
except for d13C in the shaded site in October), and higher N
content (significant only in the sunny site; not shown), as
expected for MX orchids. Autotrophs had lower d13C in the
shaded than in the sunny site (significant only in October;
Fig. 4) but did not differ in d15N (Fig. S6) and N content
(not shown). Conversely, E. fibri in the shaded site had
higher d13C (significant only for individuals [011] at both
sampling dates and [001] in October; Fig. 4) and higher N
content (often significantly; not shown) than in the sunny site.
d15N sometimes differed among sites, with incongruent trends
(Fig. S6). Thus, d13C and N content supported prediction #2
(shading increases fungal C proportion). d13C of E. fibri was
higher in July than in October, at shaded (27.2 1.1 vs.
30.4 0.9&, respectively) and sunny (28.6 1.5& vs.
31.6 0.3) sites (MW, P < 0.001 for both sites); similarly,
d15N was higher (significant in the sunny site only; Fig. S6)
and N content was significantly lower (not shown) in July.
All parameters indicated higher fungal C proportion in July
than in October, in line with prediction #1.
Individuals with different dormancy histories ([011], [001]
and [101]) never differed in d13C (Fig. 4) and N content (not
shown). Individuals with a shoot in a previous year tended to
have lower d13C in July 2010 (Fig. 4b), but this was not significant. d15N sometimes differed (Fig. S6), but with incongruent trends among sites and sampling dates. Thus, d13C and
N content, but not always d15N, indicated identical fungal
contributions in shoots regardless of dormancy history, in line
with prediction #4.
Discussion
Epipactis autotrophy increased in above-ground organs over
the growth season in all species and sites, paralleling a
decrease in mycorrhizal fungal colonization at Ch (prediction
#1; Table 1). Autotrophic C did not (or marginally) contribute
to below-ground reserves, so that (i) shoots had a similarly
high proportion of fungal C whatever the number of dormant
years (prediction #4), and (ii) below-ground parts and emerging shoots of albinos and green individuals did not differ in
d13C (prediction #5). Experimental approaches revealed
decreased autotrophic nutrition in shaded shoots (prediction
#2), but strikingly not in defoliated plants, which maintained
fruit and seed production (invalidating prediction #3). N
© 2014 The Authors. Journal of Ecology © 2014 British Ecological Society, Journal of Ecology, 102, 1183–1194
1190 C. Gonneau et al.
(A)
(B)
Fig. 4. d13C of Epipactis fibri individuals with different life histories
at the end (A, October 2009) or beginning (B, July 2010) of the
growth season at the ^Ile du Beurre (IB; replicates number within columns). Grey and white bars represent shaded and sunny IB sites,
respectively. The three life-history types are encoded by Booleans
representing the status (0, dormant or protocorm; 1, sprouting) over
three successive years (all individuals sprout on the sampling year):
individuals [011] sprouted in the previous year but were dormant two
years before, [001] sprouted in the year of sampling only, and [101]
were dormant the year before sampling, but sprouted two years
before. Autotrophs are Hedera helix and Trifolium aureum for the
sunny site, and Fraxinus excelsior, Pimpinella saxifraga and Vitis
vinifera for the shaded site (n = 4 per species and date). Values with
different letters differ significantly according to Mann–Whitney pairwise tests.
content (which usually increases with MH level) showed similar trends to d13C, while d15N (which often increases with
MH level) was often inconsistent with d13C trends, either
between organs or over the growth season. Indeed, N and C
sources and uptakes are only indirectly related, even in MX
plants (Girlanda et al. 2011; Hynson et al. 2013), and the
existing N pool may dilute the 15N signal of newly acquired
N, making d15N values difficult to interpret in our data.
SHOOT AUTOTROPHY INCREASES OVER THE GROWTH
SEASON (PREDICTION #1)
An increasing autotrophy over the growth season was supported at all sites by decreasing d13C and N content of leaves
(and stems at M€a), which sometimes even reached the values
for autotrophs at fruiting time. Increasing water availability
can also decrease d13C in photosynthetic plants (Farquhar,
Ehleringer & Hubick 1989), but this is unlikely to act here
since the d13C of nearby autotrophic plants did not vary over
the growth season at KH and IB, and even increased at M€a
(where drought stress may even have occurred late in the
growth season). In M€a, leaf biomass shifted over the growth
season from 100% heterotrophic at emergence to <20% heterotrophic at fruiting (a value likely overestimated because
H. monotropa is a conservative baseline, and because structural C with low turnover likely buffers the contribution of
autotrophic C late in the season; Hynson et al. 2012, 2013).
This fits the shift from 80% to 20% heterotrophy in the MX
C. damasonium over the growth season (Roy et al. 2013).
The intensity of this shift may, however, be modulated
depending on the light environment (Preiss, Adam & Gebauer
2010), since at low light level, lower photosynthesis gives
more weight to the fungal C contribution. Accordingly, fruiting IB E. fibri significantly differed from autotrophic plants at
the shaded site, but not at the sunny one.
The decreasing fungal colonization over the growth season
at Ch may limit the uptake of fungal C towards the end of
the growth season, although the correlation between colonization and nutrient flow is debatable (Matsuda et al. 2012).
Such a decrease fits observations on C. damasonium (Roy
et al. 2013) and on autotrophic orchids (Rasmussen & Whigham 2002; Kohout et al. 2013). In orchids, we do not know
whether C flows from living pelotons or from old pelotons at
the stage of their final breakdown (Trudell, Rygiewicz &
Edmonds 2003; Bougoure et al. 2014): although this issue is
still debated (see discussion in Selosse 2014), lower colonization reduces both the living symbiotic interface and the quantity of pelotons undergoing breakdown. Beyond quantitative
aspects, fungal integrity and activity vary over the year, often
with a minimum in dry summer conditions (Koide et al.
2007; Querejeta, Egerton-Warburton & Allen 2009), especially for ectomycorrhizal fungi whose host trees invest C in
fruits rather than in mycorrhizas at that time of the year
(Barbaroux, Breda & Dufr^ene 2003).
Thus, photosynthesis, perhaps together with decreasing fungal availability, favours a shift to autotrophy at the fruiting
stage. Since fruits and flowers did not differ from leaves in
d13C, d15N and N content at M€a and KH, seed production
clearly relies on photosynthates. Such a role for photosynthesis in seed production is congruent with the reduction of seed
number and quality in C. damasonium albinos (Roy et al.
2013).
BELOW-GROUND ORGANS AND EMERGING SHOOTS
MOSTLY RELY ON FUNGAL C (PREDICTIONS #4 AND #5)
The high d13C values of emerging shoots as compared with
fully expanded leaves (>3.4& at M€a, >2.9& at KH and
>2.7& at Ch) can be explained by the mobilization either of
plant reserves, which are usually enriched in 13C (Cernusak
et al. 2009), or of 13C-enriched fungal C (Hynson et al.
2013). The first source is unlikely to act alone, since young
leaves of autotrophic species at M€a and IB, which also have
rhizomatous reserves, are more 13C-depleted at the same sampling date. Most importantly, E. helleborine mycorrhizas,
which contain fungal tissues, are enriched in 13C as compared
with rhizomes (+0.21&, non-significant, at Ch; Fig. S5) and
© 2014 The Authors. Journal of Ecology © 2014 British Ecological Society, Journal of Ecology, 102, 1183–1194
Resource allocation in mixotrophic orchids 1191
with non-mycorrhizal roots (+1.22&, P < 0.001, at KH; Fig.
S4), which both store starch. This supports the hypothesis that
13
C enrichment in E. helleborine emerging shoots is higher
than in autotrophs because of mobilization of fungal C, rather
than simply due to a particularly high 13C enrichment of plant
reserves. In the future, measurements of starch d13C in
Epipactis spp. could corroborate this hypothesis. Moreover,
the mobilization of starch cannot account for the high d15N
and N content of emerging shoots.
Two other observations directly support the use of
13
C-enriched fungal C in emerging shoots, with little or no
contribution of photosynthetic reserves. First, at M€a, green
and albino emerging shoots (and mycorrhizas at the first two
sampling dates) did not significantly differ in d13C, d15N or
N content, as previously reported for C. damasonium by Roy
et al. (2013): since albinos only access fungal C, photosynthetic C is thus undetectable in emerging green shoots (prediction #5). Secondly, shoots of individuals dormant or not in
the previous year(s) at IB did not significantly differ in d13C
and N content, so that photosynthetic C from the previous
year(s) has no detectable impact (prediction #4).
Photosynthetic C was also undetectable in MX belowground organs, whose d13C remained stable over the growth
season at M€a. Since Epipactis roots develop synchronously
with above-ground organs (Tatarenko & Kondo 2003), our
measurements encompassed both established and newly
grown tissues. To further corroborate this, we re-analysed
C. damasonium rhizomes sampled by Julou et al. (2005) at
flowering time in 2003 (rhizomes are devoid of fungi). Rhizomes of two albinos, two green individuals and two dormant
individuals did not significantly differ in d13C, d15N and N
content (Table S1 in Supporting Information), so that they did
not contain detectable photosynthetic C. That experimental
defoliation of the MX Cephalanthera longifolia (L.) Fritsch
did not affect survival (Shefferson, Kull & Tali 2005) also
supports the idea that photosynthetic C plays little part in MX
below-ground organs and reserves, in sharp contrast with
autotrophic plants, which depend on photosynthetic C in this
regard (Chapin, Schulze & Mooney 1990).
However, at the two IB sites, emerging shoots of individuals that formed shoots in the previous year had lower d13C
than the individuals that were dormant, although not significantly (Fig. 4). Thus, we cannot exclude a contribution of
photosynthates to below-ground organs, albeit limited and
without significant d13C shift. This would explain the more
frequent dormancy observed in albinos (Roy et al. 2013) and
defoliated individuals of the MX C. longifolia (Shefferson,
Kull & Tali 2005), which lack photosynthates. It would also
mean an adaptive role for the non-flowering shoots observed
in MX species.
IMPAIRED PHOTOSYNTHESIS REDUCES AUTOTROPHY
(PREDICTION #2), BUT NOT REPRODUCTION
(REJECTION OF PREDICTION #3)
PAR reduction leads to 13C depletion in autotrophic plants
(Zimmerman & Ehleringer 1990; Preiss, Adam & Gebauer
2010; see Introduction for mechanism), as observed here for
autotrophs shaded at KH and IB. By contrast, d13C values of
MX plants did not vary at KH after experimental shading,
and even increased at the shaded IB site, in line with our prediction #2. A higher proportion of 13C-enriched fungal C thus
buffered the expected 13C depletion in shaded MX biomass,
congruently with previous reports on MX plants (Preiss,
Adam & Gebauer 2010; Matsuda et al. 2012).
Defoliation was expected to abruptly reduce photosynthesis,
thus shifting biomass towards higher d13C, and to impair seed
production (prediction #3), since fruits rely on photosynthetic
C as demonstrated above. Unexpectedly, the lack of difference between defoliated and control plants after 54 days
invalidated prediction #3, and defoliation even increased fruit
dry weight. Similarly, defoliation of the MX C. longifolia did
not impair flowering (Shefferson, Kull & Tali 2006). Identical
d13C suggests similar photosynthetic C contributions in defoliated and control plants, so that some photosynthetic compensation occurred after defoliation. Indeed, stems, flowers
and fruits of defoliated plants are green and may explain why
defoliated plants did not undergo the low fruiting success typical of albinos (Salmia 1989; Roy et al. 2013). Stems significantly contribute to the photosynthetic budget of plants
(Nilsen 1995; Hoyaux et al. 2008), even in MX orchids
(Zimmer, Meyer & Gebauer 2008). Although the efficiency
of their photosynthesis is lower than that of leaves (by at least
one half, on a biomass basis), green flowers and fruits contribute up to 60% of the C requirements of the reproductive
structures of autotrophic plants (see Aschan & Pfanz 2003;
for review). Although they are not optimized for capturing
direct sunlight, stems and fruits might efficiently capture the
diffuse understorey light, especially after defoliation. Nonfoliar photosynthesis often compensates for the C loss after
experimental or phytophagous defoliation (Thomson et al.
2003; Li et al. 2012), even in terms of seed production
(Lennartsson, Nilsson & Tuomi 1998; Thomson et al. 2003),
for which over-compensation is sometimes observed (Obeso
2002). Lastly, mobilization of reserves for seed production, as
shown by Primack and Stacy (1998) in the lady’s slipper
orchid (Cypripedium acaule Aiton), could buffer the impact
of defoliation: the consequences would then only be detectable in the following year(s). Even if fungal C and thus
resources for below-ground survival are not massively mobilized, the impact of the observed compensation on dormancy
and long-term survival and fecundity remains to be assessed.
Notably, d13C values for fruits show that fungal C does
not detectably contribute to this compensation, either because
it is unavailable (perhaps due to decreased colonization, see
above), or because there is no pathway for massive reallocation of fungal C to fruits. However, we cannot exclude a
marginal compensatory contribution, at the isotopic detection
limit, since fruits and stems became non-significantly
enriched in 13C after defoliation. Such a marginal contribution of fungal C would explain a contrario the persistence of
a seed production in albinos (Salmia 1989; Julou et al. 2005;
Roy et al. 2013) and should be further investigated in green
individuals.
© 2014 The Authors. Journal of Ecology © 2014 British Ecological Society, Journal of Ecology, 102, 1183–1194
1192 C. Gonneau et al.
Stem
Green
leaf
Fruit
Stem
Rhizome
Roots
FUNGUS
Among mechanisms accounting for above-ground photosynthetic compensation, recent work on the related MX
Limodorum abortivum (Bellino et al. 2014) demonstrates the
plasticity of fruit photosynthesis: after fungicide treatment,
photosynthetic pigments become more concentrated in ovaries, enhancing fruit photosynthesis and maintaining C nutrition and seed production. Thus, the fruits of MX orchids may
have a potential for higher photosynthetic capacity, selected
as a bet-hedging strategy against fluctuations of fungal C flow
or of light levels in their forest habitats. Plasticity of fruit
photosynthesis deserves further investigation in MX plants.
Conclusion – a MX model for C sources
and allocation
Data on MX Epipactis spp. and C. damasonium (Roy et al.
2013) congruently suggest temporal change of C flows
(Fig. 5) that differ from the usual paradigm for autotrophs.
Fungal C (brown lines in Fig. 5) is used for shoot formation
and emergence, as well as in below-ground organs over the
growth season. MH nutrition may also support dormancy and
winter survival, although these periods should be studied
more directly. Photosynthetic C from leaves, stems and fruits
is mainly used above-ground (green lines in Fig. 5), for
example, for seed production. As stated above, small amounts
of photosynthetic C may flow to below-ground organs (especially in non-flowering stems), while small amounts of fungal
C may support above-ground organs late in the season (especially in albinos). Direct evidence for these fluxes (dotted
lines in Fig. 5) and their role in some special conditions is so
far lacking. MX orchids thus mainly allocate photosynthetic
Fig. 5. A schematic model of C flows in MX
orchids for fungal C (light brown in winter;
darker brown at shoot emergence and deep
dark brown at fruiting) and photosynthetic C
(green). Dotted thin lines indicate possible,
smaller C flows.
C to seeds (fitness by reproduction) and fungal C to belowground persistence (fitness by survival).
Our model, based on isotopic investigations, and thus indirect evidence, should be challenged by finer assessments of C
flows at various developmental stages by labelling photosynthates or fungal C (which could be performed by labelling the
photosynthates of the donor tree using in- or ex situ treefungus-MX plant tripartite designs; Yagame et al. 2012; Bougoure et al. 2014). The dotted fluxes in Fig. 5 may then be
confirmed. However, such labelling provides an instantaneous
record, and successive experiments will be required over the
growth season. Conversely, isotopic composition offers an
integrated view of the biomass origin. In the future, labelling
experiments will estimate the reliability of conclusions based
on d13C and N content approaches and further assess the plasticity of MX physiology after shading, defoliation or in albinos.
Our model should also be phylogenetically challenged in
other MX orchid and non-orchid lineages (e.g. MX Ericaceae). If general, this pattern may explain why achlorophyllous individuals do not often invade MX populations:
photosynthesis occurs at a perfect place and time to meet fruiting costs, but any shift to pure MH nutrition would reduce
seed production. This mechanism is of major importance in
understanding what limits the evolution of MH species and
pure C sinks in mycorrhizal symbioses.
Acknowledgements
The authors thank Tamara Tesitelová, Marie-Pierre Dubois, Benjamin Coll and
the ^Ile du Beurre Reserve team for help with experiments. They also thank the
© 2014 The Authors. Journal of Ecology © 2014 British Ecological Society, Journal of Ecology, 102, 1183–1194
Resource allocation in mixotrophic orchids 1193
Societe Francßaise d’Orchidophilie members, namely Jean Koenig, Claire Damesin, Jean-Louis Gatien, Jean-Jacques Guillaumin, Chantal Riboulet and Gil
Scappaticci, for their support. The authors dedicate this paper to Jean Koenig,
who passed away last year after many decades of support of orchid research.
Data used in this paper were partly produced through use of the molecular
genetic analysis technical facilities of the Centre Mediterraneen de l’Environne No.
ment et de la Biodiversite. J. Jersakova was supported by Project GACR
14-21432S, and I. Till-Bottraud by a grant from the Conseil General de l’Isere.
We thank three anonymous referees and Richard Shefferson for useful and
detailed comments on this manuscript.
References
Abadie, J.C., Puttsepp, U., Gebauer, G., Faccio, A., Bonfante, P. & Selosse,
M.-A. (2006) Cephalanthera longifolia (Neottieae, Orchidaceae) is mixotrophic: a comparative study between green and nonphotosynthetic individuals. Canadian Journal of Botany, 84, 1462–1477.
Aschan, G. & Pfanz, H. (2003) Non-foliar photosynthesis – a strategy of additional carbon acquisition. Flora, 198, 81–97.
Barbaroux, C., Breda, N. & Dufr^ene, E. (2003) Distribution of above-ground
and below-ground carbohydrate reserves in adult trees of two contrasting
broad-leaved species (Quercus petraea and Fagus sylvatica). New Phytologist, 157, 605–615.
Bellino, A., Alfani, A., Selosse, M.-A., Guerrieri, R., Borghetti, M. & Baldantoni, D. (2014) Nutritional regulation in mixotrophic plants: new insights
from Limodorum abortivum. Oecologia, 175, 875–885.
Bougoure, J., Ludwig, M., Brundrett, M., Cliff, J., Clode, P., Kilburn, M. &
Grierson, P. (2014) High-resolution secondary ion mass spectrometry analysis of carbon dynamics in mycorrhizas formed by an obligately myco-heterotrophic orchid. Plant, Cell & Environment, 37, 1223–1230.
Cameron, D.D., Preiss, K., Gebauer, G. & Read, D.J. (2009) The chlorophyllcontaining orchid Corallorhiza trifida derives little carbon through photosynthesis. New Phytologist, 183, 358–364.
Cernusak, L.A., Tcherkez, G., Keitel, C., Cornwell, W.K., Santiago, L.S.,
Knohl, A. et al. (2009) Why are non-photosynthetic tissues generally 13C
enriched compared with leaves in C3 plants? Review and synthesis of current
hypotheses. Functional Plant Biology, 36, 199–213.
Chapin, F.S., Schulze, E.-D. & Mooney, M.A. (1990) The ecology and economics of storage in plants. Annual Review of Ecology and Systematics, 21,
423–447.
Farquhar, G., Ehleringer, J. & Hubick, T. (1989) Carbon isotope discrimination
and photosynthesis. Annual Review of Plant Physiology and Plant Molecular
Biology, 40, 503–537.
Gebauer, G. & Meyer, M. (2003) N-15 and C-13 natural abundance of autotrophic and mycoheterotrophic orchids provides insight into nitrogen and carbon gain from fungal association. New Phytologist, 160, 209–223.
Girlanda, M., Selosse, M.-A., Cafasso, D., Brilli, F., Delfine, S., Fabbian, R.,
Ghignone, S., Pinelli, P., Segreto, R., Loreto, F., Cozzolino, S. & Perotto, S.
(2006) Inefficient photosynthesis in the Mediterranean orchid Limodorum
abortivum is mirrored by specific association to ectomycorrhizal Russulaceae.
Molecular Ecology, 15, 491–504.
Girlanda, M., Segreto, R., Cafasso, D., Liebel, H.T., Rodda, M., Ercole, E.,
Cozzolino, S., Gebauer, G. & Perotto, S. (2011) Photosynthetic Mediterranean meadow orchids feature partial mycoheterotrophy and specific mycorrhizal associations. American Journal of Botany, 98, 1148–1163.
Hashimoto, Y., Fukukawa, S., Kunishi, A., Suga, H., Richard, F., Sauve, M. &
Selosse, M.-A. (2012) Mycoheterotrophic germination of Pyrola asarifolia
dust seeds reveals convergences with germination in orchids. New Phytologist, 195, 620–630.
Hoyaux, J., Moureaux, C., Tourneur, D., Bodson, B. & Aubinet, M. (2008)
Extrapolating gross primary productivity from leaf to canopy scale in a winter wheat crop. Agricultural and Forest Meteorology, 148, 668–679.
Hynson, N.A., Mambelli, S., Amend, A.S. & Dawson, T.E. (2012) Measuring
carbon gains from fungal networks in understory plants from the tribe Pyroleae (Ericaceae), a field manipulation and stable isotope approach. Oecologia,
169, 307–317.
Hynson, N.A., Madsen, T.P., Selosse, M.-A., Adam, I.K.U., Ogura-Tsujita, Y.,
Roy, M. & Gebauer, G. (2013) The physiological ecology of mycoheterotrophy. Mycoheterotrophy, the Biology of Plants Living on Fungi (ed. V. Merckx), pp. 297–342. Springer, Berlin.
Julou, T., Burghardt, B., Gebauer, G., Berveiller, D., Damesin, C. & Selosse,
M.-A. (2005) Mixotrophy in orchids, insights from a comparative study of
green individuals and nonphotosynthetic individuals of Cephalanthera
damasonium. New Phytologist, 166, 639–653.
Kohout, P., Tesitelova, T., Roy, M., Vohnık, M. & Jersakova, J. (2013) A
diverse fungal community associated with Pseudorchis albida (Orchidaceae)
roots. Fungal Ecology, 6, 50–64.
Koide, R., Shumway, D.L., Xu, B. & Sharda, J.N. (2007) On temporal partitioning of a community of ectomycorrhizal fungi. New Phytologist, 174,
420–429.
Leake, J.R. (2004) Myco-heterotroph/epiparasitic plant interactions with ectomycorrhizal and arbuscular mycorrhizal fungi. Current Opinion in Plant
Biology, 7, 422–428.
Lennartsson, T., Nilsson, P. & Tuomi, J. (1998) Induction of overcompensation
in the field gentian, Gentianella campestris. Ecology, 79, 1061–1072.
Li, W., Luo, J., Tian, X., Peng, C. & Zhou, X. (2012) Patterns of defoliation
and their effect on the plant growth and photosynthetic characteristics of
Ipomoea cairica. Weed Biology and Management, 12, 40–46.
Light, M.H.S. & MacConaill, M. (2006) Appearance and disappearance of a
weedy orchid, Epipactis helleborine. Folia Geobotanica, 41, 77–93.
Matsuda, Y., Shimizu, S., Mori, M., Ito, S.I. & Selosse, M.-A. (2012) Seasonal
and environmental changes of mycorrhizal associations and heterotrophy levels in mixotrophic Pyrola japonica (Ericaceae) growing under different light
environments. American Journal of Botany, 99, 1177–1188.
Mayor, J.R., Schuur, E.A.G. & Henkel, T.W. (2009) Elucidating the nutritional
dynamics of fungi using stable isotopes. Ecology Letters, 12, 171–183.
Nilsen, E.T. (1995) Stem photosynthesis, extent, patterns, and role in plant carbon economy. Stems and Trunks in Plant form and Function (ed. B. Gartner), pp. 223–240. Academic Press, San Diego.
Obeso, J.R. (2002) The costs of reproduction in plants. New Phytologist, 155,
321–334.
Preiss, K., Adam, I.K.U. & Gebauer, G. (2010) Irradiance governs exploitation
of fungi, fine-tuning of carbon gain by two partially mycoheterotrophic orchids. Proceedings of the Royal Society B: Biological Sciences, 277, 1333–
1336.
Primack, R. & Stacy, E. (1998) Cost of reproduction in the pink lady’s slipper
orchid (Cypripedium acaule, Orchidaceae): an eleven-year experimental study
of three populations. American Journal of Botany, 85, 1672–1679.
Querejeta, J.I., Egerton-Warburton, L.M. & Allen, M.F. (2009) Topographic
position modulates the mycorrhizal response of oak trees to interannual rainfall variability. Ecology, 90, 649–662.
Rasmussen, H.N. & Whigham, D.F. (2002) Phenology of roots and mycorrhiza
in five orchid species differing in phototrophic strategy. New Phytologist,
154, 797–807.
Roy, M., Gonneau, C., Rocheteau, A., Berveiller, D., Thomas, J.C., Damesin,
C. & Selosse, M.-A. (2013) Why do mixotrophic plants stay green? A comparison between green orchid individuals in situ. Ecological Monographs,
83, 95–117.
Salmia, A. (1989) General morphology and anatomy of chlorophyll-free and
green forms of Epipactis helleborine (Orchidaceae). Annales Botanici Fennici, 26, 95–105.
Scappaticci, G. & Till-Bottraud, I. (2010) Epipactis fibri Scappaticci & Robatsch, une espece atypique et mal connue. Cahier de la Societe Francßaise d’Orchidophilie, 7, 59–65.
Selosse, M.-A. (2014) The latest news from biological interactions in orchids:
in love, head to toe. New Phytologist, 202, 337–340.
Selosse, M.-A. & Roy, M. (2009) Green plants eating fungi, facts and questions about mixotrophy. Trends in Plant Science, 14, 64–70.
Selosse, M.-A., Weiss, M., Jany, J.L. & Tillier, A. (2002) Communities and
populations of sebacinoid basidiomycetes associated with the achlorophyllous
orchid Neottia nidus-avis (L.) L.C.M. Rich. and neighbouring tree ectomycorrhizae. Molecular Ecology, 11, 1831–1844.
Selosse, M.-A., Faccio, A., Scappaticci, G. & Bonfante, P. (2004) Chlorophyllous and achlorophyllous specimens of Epipactis microphylla (Neottieae, Orchidaceae) are associated with ectomycorrhizal septomycetes, including
truffles. Microbial Ecology, 47, 416–426.
Shefferson, R.P. (2009) The evolutionary ecology of vegetative dormancy in
mature herbaceous perennial plants. Journal of Ecology, 97, 1000–1009.
Shefferson, R.P., Kull, T. & Tali, K. (2005) Adult whole-plant dormancy
induced by stress in long-lived orchids. Ecology, 86, 3099–3104.
Shefferson, R.P., Kull, T. & Tali, K. (2006) Demographic response to shading
and defoliation in two woodland orchids. Folia Geobotanica, 41, 95–106.
St€ockel, M., Meyer, C. & Gebauer, G. (2011) The degree of mycoheterotrophic
carbon gain in green, variegated and vegetative albino individuals of Cephalanthera damasonium is related to leaf chlorophyll concentrations. New Phytologist, 189, 790–796.
Tatarenko, I.V. & Kondo, K. (2003) Seasonal development of annual shoots in
some terrestrial orchids from Russia and Japan. Plant Species Biology, 18,
43–55.
© 2014 The Authors. Journal of Ecology © 2014 British Ecological Society, Journal of Ecology, 102, 1183–1194
1194 C. Gonneau et al.
Tedersoo, L., Pellet, P., K~oljalg, U. & Selosse, M.-A. (2007) Parallel evolutionary paths to mycohetereotrophy in understorey Ericaceae and Orchidaceae,
ecological evidence for mixotrophy in Pyroleae. Oecologia, 151, 206–217.
ıhova, G. & Selosse, M.-A. (2012)
Tesitelova, T., Tesitel, J., Jersakova, J., R
Symbiotic germination capability of four Epipactis species (Orchidaceae) is
broader than expected from adult ecology. American Journal of Botany, 99,
1020–1032.
Thomson, V.P., Cunningham, S.A., Ball, M.C. & Nicotra, A.B. (2003) Compensation for herbivory by Cucumis sativus through increased photosynthetic
capacity and efficiency. Oecologia, 134, 167–175.
Trudell, S.A., Rygiewicz, P.T. & Edmonds, R.L. (2003) Nitrogen and carbon
stable isotope abundances support the myco-heterotrophic nature and
host-specificity of certain achlorophyllous plants. New Phytologist, 160,
391–401.
Yagame, T., Orihara, T., Selosse, M.-A., Yamato, M. & Iwase, K. (2012)
Mixotrophy of Platanthera minor, an orchid associated with ectomycorrhizaforming Ceratobasidiaceae fungi. New Phytologist, 193, 178–187.
Zimmer, K., Meyer, C. & Gebauer, G. (2008) The ectomycorrhizal specialist
orchid Corallorhiza trifida is a partial myco-heterotroph. New Phytologist,
178, 395–400.
Zimmer, K., Hynson, N.A., Gebauer, G., Allen, E.B., Allen, M.F. & Read, D.J.
(2007) Wide geographical and ecological distribution of nitrogen and carbon
gains from fungi in pyroloids and monotropoids (Ericaceae) and in orchids.
New Phytologist, 175, 166–175.
Zimmerman, J.K. & Ehleringer, J.R. (1990) Carbon isotope ratios are correlated
with irradiance levels in the Panamanian orchid Catasetum viridiflavum. Oecologia, 83, 247–249.
Received 27 November 2013; accepted 21 May 2014
Handling Editor: Richard Shefferson
Supporting Information
Additional Supporting Information may be found in the online version of this article:
Figure S1. d15N of leaves from green and albino Epipactis helleborine at M€a.
Figure S2. d13C and d15N of various organs from green and albino
Epipactis helleborine at M€a.
Figure S3. Response of net CO2 assimilation to irradiance in unshaded
versus shaded Epipactis helleborine at KH.
Figure S4. d13C of different organs in unshaded versus shaded
Epipactis helleborine at KH.
Figure S5. d13C of different Epipactis helleborine organs at Ch.
Figure S6. d15N of Epipactis fibri individuals with different life histories at IB.
Table S1. d13C, d15N and N content in rhizomes from albino, green
and dormant Cephalanthera damasonium individuals.
© 2014 The Authors. Journal of Ecology © 2014 British Ecological Society, Journal of Ecology, 102, 1183–1194