Partial mycoheterotrophy in Pyroleae: nitrogen and carbon stable ...

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Partial mycoheterotrophy in Pyroleae: nitrogen and carbon stable isotope signatures during development from seedling to adult. Authors; Authors and affiliations.
Oecologia (2015) 177:203–211 DOI 10.1007/s00442-014-3137-x

PHYSIOLOGICAL ECOLOGY - ORIGINAL RESEARCH

Partial mycoheterotrophy in Pyroleae: nitrogen and carbon stable isotope signatures during development from seedling to adult Veronika A. Johansson · Anna Mikusinska · Alf Ekblad · Ove Eriksson 

Received: 11 April 2014 / Accepted: 26 October 2014 / Published online: 14 November 2014 © Springer-Verlag Berlin Heidelberg 2014

Abstract  Mycoheterotrophic plants (MHP) are divided into non-photosynthesizing full MHP and green-leaved partial or initial MHP. We investigated 13C and 15N isotope enrichment in five putatively partial MHP species in the tribe Pyroleae (Ericaceae): Chimaphila umbellata, Moneses uniflora, Orthilia secunda, Pyrola chlorantha and Pyrola minor, sampled from forest sites on Öland, Sweden. For M. uniflora and P. chlorantha, we investigated isotope signatures of subterranean seedlings (which are mycoheterotrophic), to examine how the use of seedlings instead of full MHP species (Hypopitys monotropa) as reference species affects the assessment of partial mycoheterotrophy. Our main findings were as follows: (1) All investigated Pyroleae species were enriched in 15N compared to autotrophic reference plants. (2) significant fungal-derived C among the Pyroleae species was found for O. secunda and P. chlorantha. For the remaining species of C. umbellata, M. uniflora and P. minor, isotope signatures suggested adult autotrophy. (3) C and N gains, calculated using seedlings as a full MHP reference, yielded qualitatively similar results as when using H. monotropa as a reference. However, the estimated differences in C and N gains became larger when Communicated by Rowan Sage. We dedicate this paper to the memory of our friend and colleague Ania Mikusinska, who tragically passed away during the preparation of the manuscript. V. A. Johansson (*) · O. Eriksson  Department of Ecology, Environment and Plant Sciences, Stockholm University, 106 91 Stockholm, Sweden e-mail: [email protected] A. Mikusinska · A. Ekblad  Department of Natural Sciences, Örebro University, 701 82 Örebro, Sweden

using seedlings as an MHP reference. (4) A previously unknown interspecific variation in isotope signature occurs during early ontogeny, from seed production to developing seedlings. Our findings suggest that there is a variation among Pyroleae species concerning partial mycoheterotrophy in adults. Adult autotrophy may be most common in Pyroleae species, and these species may not be as dependent on fungal-derived nutrients as some green orchids. Keywords  Dust seeds · Ericaceae · Mycoheterotrophy · Stable isotopes · Subterranean seedlings

Introduction Mycorrhizal interactions are one of the most important symbiotic relationships, and are based on exchanges of resources where the fungi supply plants with mineral nutrition in return for plant carbon assimilates (Smith and Read 2008). Mycoheterotrophic plants (MHP) lack chlorophyll and hence the capacity to photosynthesize (Leake 1994). Instead, they use various fungi as their source of organic carbon. The mycoheterotrophic plants associating with ectomycorrhizal fungi, and hence indirectly exploiting other plants associated with the same fungi, are considered cheaters in mycorrhizal symbioses (Björkman 1960; Cullings et al. 1996; Taylor and Bruns 1997; Bidartondo et al. 2002; Bidartondo 2005; Smith and Read 2008). Greenleaved relatives of fully mycoheterotrophic plants have revealed another form of mycoheterotrophy, where adult plants are able to receive carbon from both photosynthesis and from associated mycorrhizal fungi. These plants are termed partial mycoheterotrophs (Merckx 2013). MHP (hereafter referring to both partially and fully mycoheterotrophic plants) have exceptionally small dust seeds. These

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seeds lack or have very limited carbohydrate reserves and are therefore completely dependent on external sources of carbon during their germination and early development (Eriksson and Kainulainen 2011). Many species that are autotrophic as adults are thus initially mycoheterotrophic as seedlings (such as Orchidaceae; Leake 1994; Merckx 2013). Dust seeds have evolved independently in at least 12 plant families (Eriksson and Kainulainen 2011), of which Orchidaceae is the most well-studied family. A smaller, less-studied group of plants is the tribe Pyroleae (Ericaceae) (Kron et al. 2002). This group contains initially mycoheterotrophic species (Hynson et al. 2013b), partially mycoheterotrophic species (Tedersoo et al. 2007; Zimmer et al. 2007), and Pyrola aphylla, which remains fully mycoheterotrophic throughout its life cycle (Hynson et al. 2009a). By using stable isotopes, it is possible to trace the source of specific nutrients in food webs and to study fluxes in ecosystem processes (Dawson et al. 2002). This is because fractionation of heavy isotopes is common in biochemical processes and has been found to result in an accumulation of the heavier isotopes in higher trophic levels (Fry 2006). By applying an isotopic food chain model, studies have shown that full MHP have an isotopic signature more similar to mycorrhizal fungi, while green-leaved partial MHP are most often found in a range between full MHP and autotrophic plants (e.g., Gebauer and Meyer 2003; Trudell et al. 2003; Julou et al. 2005; Tedersoo et al. 2007; Zimmer et al. 2007; Hynson et al. 2012). For all Pyroleae species investigated to date, results suggest that the nitrogen demand of these plants is supplied by their fungal associates (e.g., Tedersoo et al. 2007; Zimmer et al. 2007). In contrast, investigations of fungal-derived carbon have yielded varying results, and the carbon sources used by Pyroleae species appear to be site-specific and possibly influenced by light availability (Zimmer et al. 2007; Hynson et al. 2012). Studies of C and N enrichment in partial MHP depend on the use of full MHP reference material, which is then, by necessity, another species. Another option, first used by Stöckel et al. (2014), and which might be associated with some advantage by reducing any unknown source of variation resulting from comparing the physiology of different species, is to use the subterranean seedlings developed by most species with dust seeds (Eriksson and Kainulainen 2011). These seedlings live underground for several years, during which time they are fully mycoheterotrophic. We investigated the C and N isotope signatures of five adult Pyroleae species (Chimaphila umbellata (L.) W. P. C. Barton, Moneses uniflora (L.) A. Gray, Orthilia secunda (L.) House, Pyrola chlorantha Sw. and Pyrola minor L.). For two of the species (M. uniflora and P. chlorantha), we investigated isotope signatures of seedlings. This is the first study of Pyroleae species we are aware of that uses seedling material to study 13C and 15N enrichment in these putatively

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partial MHP. This study is part of a larger project, including studies of recruitment limitation (Johansson and Eriksson 2013) and seed production and dispersal (Johansson et al. 2014) in Pyroleae species, and where molecular identifications are currently under way to determine the fungal hosts involved during germination and early development. Our main objectives were to answer the following questions: (1) How do adult Pyroleae species vary in C and N enrichment? (2) What are the isotopic signatures for seedlings and how do they relate to adult plants and other fully mycoheterotrophic species? (3) How does the use of seedlings instead of full MHP as a reference affect the assessment of partial mycoheterotrophy in Pyroleae? (4) Are there any difference among the Pyroleae species concerning C and N sources used for the production of seeds?

Methods Field site and sampling To examine the degree of mycoheterotrophy in Pyroleae, in the spring of 2012,we established plots around lake Hornsjön (N57°11.788′, E16°57.449′) on the island Öland in Sweden. The sites were open to semi-open old-growth nemoboreal–temperate coniferous forests, dominated by Pinus sylvestris L., with ground vegetation mainly consisting of Picea abies L., Quercus robur L., Corylus avellana L., Melampyrum pratense L. and various grasses and mosses, but also several species of orchids. We chose this area because it harbors five of seven species within the Pyroleae tribe existing in Sweden; C. umbellata, O. secunda, M. uniflora, P. chlorantha and P. minor, as well as the closely related fully mycoheterotrophic plant Hypopitys monotropa. We used 12 sampling plots in total, where samples were collected within areas of 3 × 3 m in order to get similar environmental conditions. The number of plots used per species was, however, dependent on the frequency of the study species. In the 12 plots used, C. umbellata, M. uniflora and P. chlorantha were found in ten plots, O. secunda in 11 plots and P. minor in four plots. For each study species, we sampled fresh top leaves of up to ten individuals and root material from three individuals at each plot. Where possible, we sampled five individuals of three autotrophic reference plant species within each plot (i.e., if three autotrophic species occurred in the plot). For detailed information on sampling and reference species, see Table 1. The autotrophic reference plants were chosen based on the following criteria, in accordance with Gebauer and Meyer (2003): (1) that they grew within the plot (to account for differences in the microhabitat possibly affecting isotopic signatures), (2) that they have a plot-abundance, enabling us to sample

Oecologia (2015) 177:203–211 Table 1  Sampling scheme of species present in respective plots from which material was collected and analyzed for δ13C and δ15N content

205 Species

Plot 1

2

3

4

5

6

7

10

10

8

9

10

11

12

 Study species 10

10

  Orthilia secunda

9

10

7

9

  Moneses uniflora

9

7

10

6

  Pyrola chlorantha

10

9

9

10

  Chimaphila umbellata

  Pyrola minor  Seedling materiala   Moneses uniflora

10

10

10

9

10

10

6

9

10

10

10

10

10

6

3

10

8

10

10

10

10

9 1

  Dactylorhiza maculata

4

  Epipactis helleborine

3

5 1

5

5

5

5

3

5

5

5

5

5

5

5

5

5

5 5

5 5

  Potentilla reptans

4

5

  Quercus robur

4

5

5

5

5

5

2 5

5

5

5

  Veronica officinalis  Fungal fruitbodies and hyphaea   Amanita citrina (Ecto)

5

5

5

5

5

5

5

1 1

1 1

  Russula queletii (Ecto) 1

  Russula sp. 1 (Ecto)

1

  Russula sp. 2 (Ecto)

1 1

  Russula xerampelina (Ecto) 1

  Thelephora terrestris (Ecto)

1

  Hygrophoropsis aurantiaca (Sap) 1

  Mycena rosella (Sap)

1

1

1

1

1

1

1

  Mycena sp. (Sap)   Stropharia aeruginosa (Sap)

1

  Fungal hyphae

1

five replicates, and (3) that they were part of the understory vegetation (that is, no larger trees were used). Furthermore, we collected leaves from orchids likely to be partially mycoheterotrophic (e.g., Selosse and Roy 2009), as well as material from the fully mycoheterotroph H. monotropa, when present in the plots.

5 5

  Picea abies

  Isotope analyses were performed on pooled samples

3

2

  Maianthemum bifolium

a

5 5

  Fragaria vesca

Mycorrhizal identities of collected mushroom species were classified into Ectomycorrhizal (Ecto) or saprophytic (Sap) according to Rinaldi et al. (2008)

10

5 5

5

  Corylus avellana

  Russula sanguinaria (Ecto)

10

1 5

  Goodyera repens

  Cortinarius sp. (Ecto)

9 10

1 1

  Hypopitys monotropa  Orchids: partial mycoheterotrophic references 5   Cephalanthera longifolia

  Betula pendula

9

10

  Pyrola chlorantha  Fully mycoheterotrophic reference

  Platanthera bifolia  Autotrophic references

10

1

1

1

1

1

1

1

All plant material was sampled between 8 and 12 August 2012. In addition, we also collected fruit bodies of various mushrooms present within the plots (see Table 1), as well as fungal hyphae extracted from mycelium in-growth bags (Wallander et al. 2001). These bags were cylindrical in shape with a diameter of 2 cm and a height of 10 cm,

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and were made of 50 µm mesh cloth filled with sterilized carbon free quartz sand (Mikusinska et al. 2013) that had been buried vertically in close proximity to the study plants during the spring of 2012. The different mushroom species that were collected were divided according to their function as either being ectomycorrhizal (Ecto) or saprophytic (Sap) based on Rinaldi et al. (2008). These collections were done in October 2012. In October 2012 and 2013, we also collected seedling material for two of the study species, M. uniflora and P. chlorantha, from an ongoing seed sowing experiment (work in progress). These were collected from four of the 12 study plots (see Table 1). All seedlings collected had ruptured testa, but the root-like seedling structure varyied in size. Seedlings larger than 2 mm could be found only for M. uniflora. Isotope signature analysis Plant leaves, roots and sporocarp samples were freezedried and ground to a fine powder, and 2 mg samples were weighed into tin capsules for analyses. Samples of seeds, seedlings and mycelia collected from the in-growth bags contained small amounts of materials, and 0.1 mg samples of seeds and seedlings and 0.2 mg samples of mycelium were weighed into tin capsules without grinding. The δ13C and δ15N values of plant and fungal materials were determined with an elemental analyser (model EuroEA3024; Eurovector, Milan, Italy) connected to an Isoprime isotoperatio mass spectrometer (GV Instruments, Manchester, UK). The results are presented as δ13C and δ15N, showing deviations in the ratio of the heavy to the light isotope relative to their respective international standards, Vienna Pee Dee Belemnite (V-PDB) and atmospheric N2; thus:

δ13 Corδ15 N =

     Rsample −Rstandard /Rstandard 1000 × 0/00 ,

where R is the molar ratio of 13C/12C or 15N/14N. For the 2 mg samples, the standard deviation of ten replicated samples was