Oecologia (2014) 175:875–885 DOI 10.1007/s00442-014-2940-8 PLANT-MICROBE-ANIMAL INTERACTIONS - ORIGINAL RESEARCH Nutritional regulation in mixotrophic plants: new insights from Limodorum abortivum Alessandro Bellino · Anna Alfani · Marc-André Selosse · Rossella Guerrieri · Marco Borghetti · Daniela Baldantoni Received: 15 January 2014 / Accepted: 28 March 2014 / Published online: 11 May 2014 © Springer-Verlag Berlin Heidelberg 2014 Abstract Partially mycoheterotrophic (mixotrophic) about this nutrition, and especially about the physiologi- plants gain carbon from both photosynthesis and their cal balance between photosynthesis and fungal C gain. To mycorrhizal fungi. This is considered an ancestral state in investigate possible compensation between photosynthesis the evolution of full mycoheterotrophy, but little is known and mycoheterotrophy in the Mediterranean mixotrophic orchid Limodorum abortivum, fungal colonization was experimentally reduced in situ by fungicide treatment. We Electronic supplementary material The online version of this article (doi:10.1007/s00442-014-2940-8) contains supplementary measured photosynthetic pigments of leaves, stems, and material, which is available to authorized users. ovaries, as well as the stable C isotope compositions (a proxy for photosynthetic C gain) of seeds and the sizes of Communicated by Caroline Müller. ovaries and seeds. We demonstrate that (1) in natural con- Alessandro Bellino began this research, then Daniela Baldantoni ditions, photosynthetic pigments are most concentrated worked alongside him in every phase of this project, from in ovaries; (2) pigments and photosynthetic C increase devising the experimental plan to the samplings, analyses, and in ovaries when fungal C supply is impaired, buffering C interpretation of data. Marc-André Selosse proposed additional limitations and allowing the same development of ova- experiments and measurements, and carried out some of them in his lab. Rossella Guerrieri and Marco Borghetti performed ries and seeds as in natural conditions; and (3) responses 13 the δ13C measurements. Anna Alfani coordinated the work. All to light of pigment and C contents in ovaries shift from contributed to writing the paper. null responses in natural conditions to responses typical of autotrophic plants in treated L. abortivum, demonstrating A. Bellino (*) · A. Alfani · D. Baldantoni (*) Dipartimento di Chimica e Biologia, Università degli Studi di photoadaptation and enhanced use of light in the latter. L. Salerno, Via Giovanni Paolo II 132, 84084 Fisciano, Salerno, abortivum thus preferentially feeds on fungi in natural con- Italy ditions, but employs compensatory photosynthesis to buffer e-mail: [email protected] fungal C limitations and allow seed development. D. Baldantoni e-mail: [email protected] Keywords Mycoheterotrophy · Evolution · Photosynthetic pigments · δ13C · Orchids M.-A. Selosse Département Systématique et Evolution (UMR 7205 OSEB), Muséum national d’Histoire naturelle, CP 50, 45 rue Buffon, 75005 Paris, France Introduction R. Guerrieri · M. Borghetti Dipartimento di Scienze dei Sistemi Colturali, Forestali e Mycoheterotrophic (MH) plants obtain organic carbon dell’Ambiente, Università della Basilicata, Viale dell’Ateneo from their mycorrhizal symbionts, which allow C transfer Lucano 10, 85100 Potenza, Italy from surrounding autotrophic plants via common mycor- rhizal networks (Leake 1994, 2004; Selosse and Cameron R. Guerrieri Earth Systems Research Center, University of New Hampshire, 8 2010) or, for some tropical orchids associated with saprobic College Road, Durham, NH 03824, USA fungi, from soil organic matter (Martos et al. 2009; Hynson 1 3 876 Oecologia (2014) 175:875–885 et al. 2013). Mycoheterotrophy evolved independently in the possibility that photosynthesis compensates for fun- many plant lineages, including liverworts, lycopods, ferns, gal C is indirectly suggested by the increase in photosyn- and angiosperms, and represents a common strategy for thetic efficiency and photosynthetic C in the shoots of C. overcoming the competition for light (Bidartondo 2005; damasonium during ovary ripening, when fungi are rare in Merckx and Freudenstein 2010). Several relevant features roots (Roy et al. 2013). However, such compensation has of many MH plants have already been clarified, such as never been experimentally investigated. A compensatory the identity of fungal species involved in mycorrhiza for- photosynthesis in PMH plants is particularly interesting mation, their organic C sources, and some aspects of their since it would indicate a preference for fungal C nutrition physiology and development (Hynson et al. 2013). It was and an underexpression of photosynthetic capabilities in discovered that MH species often evolved within clades of natural conditions, which would be used as a bet-hedging green, partially mycoheterotrophic (PMH) plants, i.e., mix- resource. The establishment of photosynthesis as a second- otrophic plants that cumulate photosynthetic C gain with ary C source in PMH species relying mostly on fungal C MH nutrition (Gebauer and Meyer 2003; Bidartondo et al. would have straightforward evolutionary implications. In 2004; Julou et al. 2005; Selosse and Roy 2009; Selosse and environmental conditions favoring mycorrhizal associa- Cameron 2010), indicating that PMH nutrition is an inter- tion and fungal C supply (such as shadiness and wetness), mediate phase in the evolution of MH nutrition (Tedersoo the balance between the two C gains could constantly tip et al. 2007; Selosse and Roy 2009; Hynson et al. 2013). toward mycoheterotrophy, making photosynthesis less and The shifts to full MH nutrition, however, seem to be rare, less required. Such conditions could favor the evolution of and PMH nutrition was proposed to be an evolutionary MH plants by limiting the impacts on fitness of mutations metastable trait (Selosse and Roy 2009; Roy et al. 2013). affecting photosynthetic performance, and allowing suc- The processes and evolutionary paths that led from PMH cessful transitions to MH nutrition. Conversely, PMH nutri- to MH plants are currently unknown. Studies on the PMH tion could be preserved in more variable environments, orchid Cephalanthera damasonium highlighted that this where compensatory photosynthesis could be required to transition is unlikely to have resulted from a simple loss of buffer fungal C limitations. photosynthesis, as indicated by the lower fitness of achlo- In order to investigate the potential for compensa- rophyllous variants, but requires the joint and progressive tory photosynthesis in PMH plants, we studied in situ, for evolution of many physiological and morphological traits 2 years, the effects of limited access to fungal C on the (Julou et al. 2005; Roy et al. 2013). Clarification of the evo- photochemical apparatus and the photosynthetic C gain of lutionary path to MH nutrition and of the metastability of the Mediterranean PMH orchid Limodorum abortivum (L.) PMH nutrition could arise from deeper investigation of the Swartz. We expected that these traits would be enhanced eco-physiology of PMH species, and especially of the rela- in response to reduced access to fungal C, highlighting a tionship between photosynthesis and mycoheterotrophy. contrario their downregulation in natural conditions. Due The results of a few studies investigating plants in dif- to its nutritional physiology and phylogenetic position, this ferent light conditions (Preiss et al. 2010; Matsuda et al. species is a particularly useful model for our experiments. 2012) have suggested that PMH plants in the light incorpo- First, the photosynthetic rate of L. abortivum, measured on rate more photosynthetic C in their biomass than those in stem and scaly leaves, is below the compensation point in the shade do, indicating a possible compensation between full light (Girlanda et al. 2006), making it easier to detect the two C gains. This was assessed by measuring the nat- even small changes in photosynthetic pigments and C gain ural stable C isotope composition (13C/12C), since C aris- using 13C content as a proxy (Gebauer and Meyer 2003; ing from photosynthesis is depleted in 13C as compared to Bidartondo et al. 2004; Julou et al. 2005; Selosse and Roy fungal C (Selosse and Roy 2009; Hynson et al. 2013). In 2009). Second, it is phylogenetically close to the well-stud- PMH species, it is possible that either mycoheterotrophy ied Cephalanthera and Epipactis genera (within the Neot- compensates for photosynthesis, which is usually limited tieae tribe). Specifically, in the first year, we focused on the by the light conditions in the shade environments colo- changes in photosynthetic pigments of leaves, stems, and nized, or that photosynthesis compensates for mycohetero- ovaries; then, in the second year, on the changes in seed 13C trophy. The two processes, moreover, could be expressed at content, following fungicide-induced reductions of myc- different stages of the continuum of adaptations from near orrhizal colonization. It was hypothesized (Montfort and full autotrophy to near full mycoheterotrophy exhibited Küsters 1940) that photosynthesis in Corallorhiza trifida, by PMH species. In orchids at least, the survival of non- a PMH orchid relying mostly on fungal C, like L. aborti- chlorophyllous individuals, although with a lower fitness, vum (Zimmer et al. 2008; Cameron et al. 2009), could be shows that fungal C has a limited ability to compensate preferentially involved in reproduction. To investigate this