RESEARCH PAPER Similar local, but different systemic, metabolomic responses of closely related pine subspecies to folivory by caterpillars of the processionary moth A. Rivas-Ubach 1,2,3 , J. Sardans 2,3 , J. A. H  odar 4 , J. Garcia-Porta 5 , A. Guenther 6,7 , M. Oravec 7 , O. Urban 7 & J. Pe ~ nuelas 2,3 1 Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, USA 2 CSIC, Global Ecology Unit CREAF-CEAB-CSIC-UAB, Cerdanyola del Vall es, Catalonia, Spain 3 Cerdanyola del Vall es, CREAF, Catalonia, Spain 4 Grupo de Ecolog  ıa Terrestre, Departamento de Biolog  ıa Animal y Ecolog  ıa, Facultad de Ciencias, Universidad de Granada, Granada, Spain 5 Institute of Evolutionary Biology, CSIC-Universitat Pompeu Fabra, Barcelona, Spain 6 Department of Earth System Science, University of California, Irvine, CA, USA 7 Global Change Research Centre, Academy of Sciences of the Czech Republic, Brno, Czech Republic Keywords Folivory; metabolomics; phenolics; plant–insect; stoichiometry; systemic responses. Correspondence A. Rivas-Ubach, Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 3335 Innovation Boulevard, Richland, 99354, WA, USA. E-mail: albert.rivas.ubach@gmail.com Editor K. Leiss Received: 27 October 2015; Accepted: 25 November 2015 doi:10.1111/plb.12422 ABSTRACT Plants respond locally and systemically to herbivore attack. Most of the research con- ducted on plant–herbivore relationships at element and molecular levels have focused on the elemental composition or/and certain molecular compounds or specific fami- lies of defence metabolites showing that herbivores tend to select plant individuals or species with higher nutrient concentrations and avoid those with higher levels of defence compounds. We performed stoichiometric and metabolomics, both local and systemic, analyses in two subspecies of Pinus sylvestris under attack from caterpillars of the pine processionary moth, an important pest in the Mediterranean Basin. Both pine subspecies responded locally to folivory mainly by increasing relative concentra- tions of terpenes and some phenolics. Systemic responses differed between pine sub- species, and most of the metabolites presented intermediate concentrations between those of the affected parts and unattacked trees. Our results support the hypothesis that foliar nutrient concentrations are not a key factor for plant selection by adult female processionary moths for oviposition, since folivory was not associated with any of the elements analysed. Phenolic compounds generally did not increase in the attacked trees, questioning the suggestion of induction of phenolics following folivory attack and the anti-feeding properties of phenolics. Herbivory attack produced a gen- eral systemic shift in pines, in both primary and secondary metabolism, which was less intense and chemically different from the local responses. Local pine responses were similar between pine subspecies, while systemic responses were more distant. INTRODUCTION Carbon (C) to nitrogen (N) and C to phosphorus (P) biomass ratios are lower in herbivores than in plants (Fagan et al. 2002). Foliar nutrient concentration has been widely reported in recent decades as an important factor in the selection of foliage by insect herbivores, who usually choose plants with the high- est nutrient concentrations for maintaining their internal C:N: P stoichiometric homeostasis (Elser et al. 2000; Sterner & Elser 2002; Ngai & Jefferies 2004; Cosme et al. 2011; Sardans et al. 2012) and for ensuring larval survival (H odar et al. 2002). The location of oviposition is crucial for most herbivorous insects to ensure larval survival. However, the role of element compo- sition on host selection by herbivores remains unclear (Rivas-Ubach et al. 2014; Jactel et al. 2015); other chemical and physical barriers could play even more significant roles (Trem- mel & M€ uller 2013; Onodera et al. 2014). Onodera et al. (2014) reported that insects selected organs of a plant containing less defence compounds than plants with higher nutrient concen- trations, thus demonstrating the importance of defence metabolites in host selection by herbivores. Plants have devel- oped a wide array of resistance mechanisms against herbivores (Hanley et al. 2007; Heil 2009). Secondary plant metabolic compounds are examples of defence compounds (Herms & Mattson 1992; Kessler & Baldwin 2001) and can be constitutive or induced by a specific stressor. Herbivore attack induces the synthesis of defence compounds at both local and systemic levels (Karban & Baldwin 1997; Sticher et al. 1997; Heil & Bueno 2007; Heil 2009) through induced internal plant sig- nalling (Howe & Jander 2008; Wu & Baldwin 2009) and pro- duction of reactive oxygen species (ROS) (Orozco-Cardenas & Ryan 1999; Wu & Baldwin 2010). Studies of plant-induced chemical responses to herbivore attack have generally focused on a single compound or families of metabolites (Sardans et al. 2011). The role of volatile organic compounds, such as terpenes, in plant defence has been Plant Biology © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands 1 Plant Biology ISSN 1435-8603