This is a repository copy of From mycoheterotrophy to mutualism: Mycorrhizal specificity and functioning in Ophioglossum vulgatum sporophytes. White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/86085/ Version: Accepted Version Article: Field, K.J., Leake, J.R., Tille, S. et al. (5 more authors) (2015) From mycoheterotrophy to mutualism: Mycorrhizal specificity and functioning in Ophioglossum vulgatum sporophytes. New Phytologist, 205 (4). 1492 - 1502. ISSN 0028-646X https://doi.org/10.1111/nph.13263 This is the peer reviewed version of the following article: Field, K.J., Leake, J.R., Tille, S., Allinson, K.E., Rimington, W.R., Bidartondo, M.I., Beerling, DJ. and Cameron, D.D. (2015) From mycoheterotrophy to mutualism: Mycorrhizal specificity and functioning in Ophioglossum vulgatum sporophytes. New Phytologist, 205 (4). 1492 - 1502, which has been published in final form at http://dx.doi.org/10.1111/nph.13263. 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[email protected] https://eprints.whiterose.ac.uk/ 1 From mycoheterotrophy to mutualism: mycorrhizal specificity and functioning in 2 Ophioglossum vulgatum sporophytes 1* 1 1 1 3 Katie J. Field , Jonathan R. Leake , Stefanie Tille , Kate E. Allinson , William R. 2,3,4 2,3 1 1 4 Rimington , Martin I. Bidartondo , David J. Beerling and Duncan D. Cameron 5 1 6 Department of Animal and Plant Sciences, University of Sheffield, Western Bank, Sheffield, 7 UK 2 8 Department of Life Sciences, Imperial College London, London SW7 2AZ, UK 3 9 Jodrell Laboratory, Royal Botanic Gardens, Kew, TW9 3DS, UK 4 10 Department of Life Sciences, Natural History Museum, Cromwell Road, London SW7 5BD, 11 UK 12 13 14 15 16 * Corresponding author: 17 Katie J. Field ([email protected]) 18 Tel: +44-(0)114 2220093 19 Fax: +44-(0)114 2220002 20 21 Summary 22 • Mycorrhizal functioning in the fern Ophioglossum is complex and poorly understood. 23 It is unknown whether mature O. vulgatum sporophytes form mutualistic associations 24 with fungi of the Glomeromycota and with what specificity. Are green sporophytes 25 able to ‘repay’ fungal carbon invested in them by mycorrhizal partners during the 26 initially heterotrophic gametophyte and early sporophyte stages of the lifecycle? 27 • We identified fungal partners of O. vulgatum sporophytes using molecular techniques 33 14 28 and supplied them with P-orthophosphate and O. vulgatum sporophytes with CO2. 29 We traced the movement of fungal-acquired nutrients and plant-fixed carbon between 13 15 30 symbionts and analysed natural abundance C and N isotope signatures to assess 31 nutritional interactions. 32 • We found fungal specificity of O. vulgatum sporophytes towards a mycorrhizal 33 fungus closely related to Glomus macrocarpum. Our radioisotope tracers revealed 34 reciprocal carbon-for-phosphorus exchange between fern sporophytes and fungal 35 partners, despite competition from surrounding vegetation. Monocultures of O. 13 15 36 vulgatum were enriched in C and N, providing inconclusive evidence of 37 mycoheterotrophy when experiencing competition from the surrounding plant 38 community. 39 • We show mutualistic and specific symbiosis between a eusporangiate fern and fungi 40 of the Glomeromycota. Our findings suggest a ‘take now, pay later’ strategy of 41 mycorrhizal functioning through the lifecycle O. vulgatum, from mycoheterotrophic 42 gametophyte to mutualistic above-ground sporophyte. 43 44 Key words: Competition, mycoheterotrophy, mycorrhiza, Ophioglossaceae, Ophioglossum 45 vulgatum, pteridophyte, specificity, symbiosis 46 47 Introduction 48 The symbiosis between plants and arbuscular mycorrhizal fungi dates back more than 450 49 million years to the colonisation of the land by plants (Read et al., 2000; Bonfante & Selosse, 50 2010). To better understand the role of mycorrhizal fungi in land plant evolution, there is 51 increasing interest in resolving relationships between plants and their fungal partners to 52 determine how these associations may have changed both across the land plant phylogeny 53 and functionally through coevolution (Bidartondo et al., 2004; Merckx & Bidartondo 2008; 54 Arnold et al., 2010; Merckx et al., 2012; Merckx et al., 2013). Advances in molecular and 55 physiological ecology have provided new insights into the evolutionary history of the 56 symbiosis in the major clades of plants and fungi (Wang et al., 2010; Bidartondo et al., 2011, 57 Field et al., 2012, 2014). However, important knowledge-gaps regarding the fungal partners 58 of plants in key nodes of the land plant phylogeny remain (Fig. 1). In particular, neither the 59 identity of mycorrhizal fungi nor their functional roles have been determined in the widely 60 distributed (Singh et al., 2009) basal euphyllophyte (“true-leaved plant”) genus 61 Ophioglossum, thought to have evolved prior to the break-up of Gondwana (Parris, 2001). 62 In common with >1,000 species of lycophytes and ferns, the subterranean gametophyte 63 generations of Ophioglossum are achlorophyllous, nourished with organic carbon and 64 nutrients via mycorrhizal fungi (Boullard, 1979; Leake, 1994; Winther & Friedman, 2007). 65 This form of nutrition, termed mycoheterotrophy, has evolved many times in land plants 66 (Leake, 1994; Bidartondo, 2005; Merckx & Freudenstein, 2010) with examples ranging from 67 a liverwort, to lycopods, ferns and angiosperms (Fig. 1). The initial developmental stages of 68 Ophioglossum sporophytes are also achlorophyllous (Bruchmann, 1904) and 69 mycoheterotrophic. However, mature sporophytes consist of a characteristic blade-like green 70 photosynthetic shoot (the trophophore) (Fig. S1a), often accompanied by an epiphyllous 71 fertile sporophore, in which the functional role of mycorrhiza has not been investigated. 72 Because of the life-stage changes from heterotrophy to autotrophy mycorrhizal functioning in 73 Ophioglossum is both complex and poorly understood. 74 Ophioglossum vulgatum L. (Fig. S1a), is one of the most widespread and abundant grassland 75 species in the Ophioglossaceace (GRIN taxonomic database). Sporophytes of O. vulgatum 76 are initially subterranean, achlorophyllous and colonised by aseptate fungi that form irregular 77 hyphal swellings in the plant tissues (Bruchmann, 1904; Boullard, 1979). These fungi must 78 provide the main carbon and nutrient supplies required to enable development of the 79 underground root axis from which shoots develop (Bruchmann, 1904). 80 Recent studies of achlorophyllous gametophyte and photosynthetic sporophyte generations of 81 lycopods (Lycopodium and Huperzia) and ophioglossoid ferns (Botrychium) have revealed 82 specificity and intergenerational fidelity in their arbuscular mycorrhizal fungal associates 83 (AMF) (Winther & Friedman, 2007, 2008, 2009). This suggests carbon invested by AMF 84 partners in supporting a mycoheterotrophic gametophyte and early subterranean sporophyte 85 may be repaid by established green sporophytes. Such ‘take now, pay later’ mycorrhizal 86 functioning has been suggested as the basis of fungal specificity and overall mutualism 87 through the mycoheterotrophic-to-autotrophic life stages of many green-leaved orchids 88 (Cameron et al., 2008). However, experimental evidence for photosynthate ‘pay back’ to 89 fungal symbionts of any of the lower tracheophytes is currently lacking. Studies of 90 mycorrhiza specificity and functioning in these plants are of particular interest for species 91 like O. vulgatum that often inhabit plant species-rich permanent grasslands that host a highly 92 diverse community of AMF ranging from 24 to more than 70 phylotypes (Vandenkoornhuyse 93 et al., 2002; Johnson et al., 2004; Dumbrell et al., 2011). 94 Intergenerational fungal specificity opens the possibility of intergenerational carbon subsidy 95 from green-leaved sporophytes to achlorophyllous gametophytes via a shared fungal partner, 96 a form of ‘parental nurture’ suggested by Leake et al. (2008). This contrasts with the 97 suggestion that stabilization of mutualistic interactions in AM symbioses with autotrophs 98 involves the plants providing organic carbon strictly in proportion to the nutrients delivered 99 by the fungus (Fitter, 2006; Kiers et al., 2011). Under the latter model of mutualism in the 100 chlorophyllous sporophyte, any fungal ‘reward’ for carbon investment in the gametophyte 101 and early stages of sporophyte establishment would be conditional upon ongoing nutrient 102 demand by the plant and its supply from the fungus. However, tight regulation of carbon-for- 103 nutrient exchange is not universal but represents only one position along the mutualism- 104