Photosynthesis in perennial mixotrophic ... - Wiley Online Library

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 Île 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 Île 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.


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).


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


Solidago virgaurea

Convallaria majalis

E. helleborine albinos


Solidago virgaurea

E. helleborine albinos


–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);


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).


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


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 Île 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 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


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.


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 Île du Beurre Reserve team for help with experiments. They also thank the


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.

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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.
