Ecology, 94(9), 2013, pp. 2019–2029 Ó 2013 by the Ecological Society of America Do fungivores trigger the transfer of protective metabolites from host plants to arbuscular mycorrhizal hyphae? MARIE DUHAMEL, 1,2,5 ROEL PEL, 2 ASTRA OOMS, 2 HEIKE BU ¨ CKING, 3 JAN JANSA, 4 JACINTHA ELLERS, 2 NICO M. VAN STRAALEN, 2 TJALF WOUDA, 2 PHILIPPE VANDENKOORNHUYSE, 1 AND E. TOBY KIERS 2 1 Universite´ de Rennes I, CNRS UMR6553 EcoBio, Campus Beaulieu, F-35042 Rennes, France 2 Institute of Ecological Science, Vrije Universiteit, 1081 HV Amsterdam, The Netherlands 3 Department of Biology and Microbiology, South Dakota State University, Brookings, South Dakota 57007 USA 4 Institute of Microbiology, Academy of Sciences of the Czech Republic, Vı´de ˇ nska ´ 1083, 14220 Praha 4, Czech Republic Abstract. A key objective in ecology is to understand how cooperative strategies evolve and are maintained in species networks. Here, we focus on the tri-trophic relationship between arbuscular mycorrhizal (AM) fungi, host plants, and fungivores to ask if host plants are able to protect their mutualistic mycorrhizal partners from being grazed. Specifically, we test whether secondary metabolites are transferred from hosts to fungal partners to increase their defense against fungivores. We grew Plantago lanceolata hosts with and without mycorrhizal inoculum, and in the presence or absence of fungivorous springtails. We then measured fungivore effects on host biomass and mycorrhizal abundance (using quantitative PCR) in roots and soil. We used high-performance liquid chromatography to measure host metabolites in roots, shoots, and hyphae, focusing on catalpol, aucubin, and verbascoside. Our most striking result was that the metabolite catalpol was consistently found in AM fungal hyphae in host plants exposed to fungivores. When fungivores were absent, catalpol was undetectable in hyphae. Our results highlight the potential for plant-mediated protection of the mycorrhizal hyphal network. Key words: cooperation; defense; Folsomia candida; Glomus sp.; mutualism; networks; Plantago lanceolata; species interactions; symbiosis. INTRODUCTION All mutualistic interactions are embedded in larger ecological webs (Bascompte 2009). This means that external species, including predators, parasites, herbi- vores, and even other mutualists (e.g., Palmer et al. 2010) can influence the benefit : cost ratios of mutual- isms, and alter their ecological and evolutionary outcomes (Afkhami and Rudgers 2009). Anthropogenic disturbances are increasingly linked to the disruption of species networks (Kiers et al. 2010), and this has prompted a call to focus on understanding how cooperative strategies evolve and are maintained in species networks (Bascompte 2009). The 450-million-year-old arbuscular mycorrhizal (AM) symbiosis is likely the world’s most prevalent mutualism (van der Heijden et al. 2008). It primarily involves the exchange of carbohydrates from plants for mineral nutrients from the fungal partner (Parniske 2008). Estimates suggest that up to 20% of total host carbon can be transferred to AM fungi (for review see Bago et al. 2000). In return, AM fungi improve the host plant’s supply of phosphorus (Parniske 2008) and nitrogen (Fellbaum et al. 2012), and provide a diversity of other benefits to the host plant (van der Heiden et al. 2008). The symbiosis contributes to massive global nutrient transfer, global carbon sequestration, and soil stabilization (Rillig and Mummey 2006). These features make it paramount to health and ecosystem function. Like all mutualisms, the mycorrhizal symbiosis exists in a rich web of interactions. A given host is colonized by multiple AM fungal species (e.g., Vandenkoornhuyse et al. 2002), and a single fungus can simultaneously colonize several plant individuals belonging to different plant species (e.g., Vandenkoornhuyse et al. 2007, Mikkelsen et al. 2008). This common mycelial network represents a dynamic underground environment: AM fungal hyphae can account for up to 30% of the total soil microbial biomass (for review see Leake et al. 2004). The plant–AM fungal network coexists with popula- tions of soil microarthropods (Hishi et al. 2008) that feed on rhizosphere fungi, including AM fungal hyphae (Jonas et al. 2007). Collembola, known collectively as springtails, are among the most abundant soil arthro- pods (Petersen and Luxton 1982), and most Collembola species feed on fungal hyphae (Fountain and Hopkin 2005). Depending on their densities, fungivores may either enhance or degrade the symbiosis (Gange 2000). At low densities, the presence of fungivores has been shown to increase AM fungal colonization and hyphal development by acting as a transporting agent for Manuscript received 7 November 2012; revised 20 February 2013; accepted 27 February 2013. Corresponding Editor: J. D. Hoeksema. 5 E-mail: duhamel.marie14@gmail.com 2019