Optimal Defense Theory in an ant‐plant mutualism: extrafloral nectar as an induced defense is maximized in the most valuable plant structures

We selected 45 plants with similar phenotypic characteristics (1.5-2 m in height, producing leaves with EFNs but not yet producing flowers) and at least 10 m apart. We randomly allocated the plants to one of three treatments (N = 15 plants per treatment; treatments are summarized in Table 1). In the first treatment (Foliar control), no manipulation was conducted. In the other two treatments, herbivory was simulated by cutting the apical part of leaves with scissors. In one treatment (Foliar 10%), 10 % of the leaf area was removed from all leaves (including young and mature leaves) of each plant, and in the other treatment (Foliar 40%), we removed 40 % of the leaf area from all leaves in each plant. In addition, we selected a different set of 30 similar plant individuals (1.5-2 m in height, developed leaves, 10-15 inflorescences), but now during flowering and without EFNs active on leaves and with EFNs active on inflorescences. We randomly allocated the plants to one of two treatments (N = 15 plants per treatment). In the first treatment (Floral control), no experimental manipulation was conducted; in the second treatment (Floral 10%), we cut 10 % of the apical part of all buds and flowers of each plant with the aid of a pair of scissors. We did not do a 40 % cutting treatment on buds and flowers as we had on the leaves, due to the small size of floral buds and the associated difficulty in their handling. Leaves, buds and flowers were cut in the evening at 2100 h, during the period of highest productivity of extrafloral nectar in Q. multiflora (Lange, Calixto, & Del-Claro, 2017). Simulated herbivory has been used in many studies to test induced plant responses, including the response of extrafloral nectaries (Heil, Fiala, Baumann, & Linsenmair, 2000; Jones & Koptur, 2015; Wäckers & Wunderlin, 1999). In the case of Q. multiflora, natural foliar herbivory rates vary from 2.64 ± 1.9 % (mean ± SD) in ant-attended plants to 8.16 ± 4.08 % in plants without ants (Calixto et al., unpublished). Thus, our treatments mimicked natural herbivory rates. On each individual, we selected one EFN. If studying leaf EFNs, we selected an EFN on the adaxial surface of a young leaf near the apical meristem (Fig. 1b), and if studying inflorescence EFNs, we chose the most basal EFN of an inflorescence (Fig. 1c). The marked EFNs were isolated with a mesh bag and Tanglefoot resin strip (Tanglefoot Company®, Rapids, Michigan), decreasing dilution by rain and dew and reducing access to and removal of nectar by ants and other arthropods. Both factors (dilution by rain and dew, and removal by ants and other arthropods) might influence the amount of nectar present at the time of assessment. Foliar experiments and data collection occurred during October, while Floral experiments occurred in January. Nectar produced in all selected EFNs on plants in all five treatments was collected 1, 6, 24, 48, 72, and 96 hours after cutting (method adapted from Heil, Fiala, Baumann, & Linsenmair, 2000). At each census, we measured the volume of nectar produced and the quantity of sugar (Brix % - mg sugar per ml solution) with the aid of 5μL graduated microcapillary tubes and manual refractometer (Eclipse® model, Bellingham & Stanley, Tunbridge Wells, UK). All evaluated EFNs were washed with distilled water and dried with filter paper immediately after simulated herbivory and after each evaluation. During evaluations, we recorded ant abundance and richness on plants at the time of nectar collection. An individual of each ant species was collected, fixed in 70% alcohol, and identified with confirmation by specialists from the Universidade Federal do Paraná, in Curitiba, Brazil. Data on ant identity are presented elsewhere (Supporting Information Table S1).