Appendix 1S Detailed Methods to Describe the Structure and Secretion of the Extrafloral

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Appendix 1S – Detailed methods to describe the structure and secretion of the extrafloral nectaries

To characterize the general organization of leaf glands, mature leaves were fixed in FAA (Formalin-Acetic-Alcohol, Johansen 1940), embedded and sectioned into slices of 6 µm using standard historesin microtechnique, and stained with toluidine blue (O’Brien et al. 1964). For SEM (scanning electron microscopy) studies, samples were fixed in FAA (Johansen 1940), post-fixed in osmium tetroxide 1% and dehydrated in an ethyl alcohol series. Specimens were critical point dried and gold coated. To characterize the main classes of substances encountered in the EFNs of A. album and A. scabriusculum, histochemical analyses were performed. Fresh hand-cut sections were subjected to eight different histochemical tests: (a) Fehling’s solution to detect reducing sugars (Purvis et al. 1964); (b) Sudan IV to detect total lipids (Johansen, 1940); (c) α-naphthol dimethyl-paraphenylenediamine (NADI) to detect terpenes (David and Carde 1964); (d) 0.02% ruthenium red aqueous solution to detect mucilage/pectin (Johansen 1940); (e) 10% ferric trichloride aqueous solution to label phenolic compounds (Johansen 1940); (f) Dragendorff reagent to detect alkaloids (Svendsen and Verpoorte 1983), and (g) mercuric bromophenol blue to detect total proteins (Mazia et al. 1953). Standard control procedures were carried out simultaneously, following the recommended protocols.

Fehling’s qualitative test was further complemented with additional tests to determine the presence of glucose. Complementary analyses were conducted by collecting a sample of the secretion produced by the EFNs using 1µl and 5 µl microcaps obtained from Drummond (USA) and Merck (Germany), respectively. We used urine test strips (Combut-Test, England) to detect the presence of sugar. In addition, the sugar concentration (and volume) of the secretions were quantified with a portable Eclipse refractometer (Standley, England).

Specific References:

David R, Carde JP (1964) Coloration différentielle des inclusions lipidique et terpeniques des pseudophylles du Pin maritime au moyen du reactif Nadi. Comptés Rendus de l’Académie des Sciences Paris 257: 1338-1340.

Johansen DA (1940) Plant microtechnique. McGraw-Hill Book Co. Inc. New York.

Mazia D, Brewer PA, Alfert M (1953) The cytochemical staining and measurement of protein with mercuric bromophenol blue. The Biological Bulletin 104: 57-67.

O’Brien TP, Feder N, McCully ME (1964) Polychromatic staining of plant cell walls by toluidine blue. Protoplasma 59: 368-373.

Purvis M, Collier D, Walls D (1964) Laboratory techniques in botany. London, Butterworths.

Svendensen AB, Verpoorte R (1983) Chromatography of alkaloids. Elsevier Scientific Publishing Company.

Appendix 2S - Morphological description and development of foliar EFNs

The EFNs encountered in both species of Anemopaegma (Bignonieae, Bignoniaceae) are circular, disc-shaped, and present a concave surface, with three distinct cell layers: (i) Secretory layer: Formed by 32-40 columnar cells in a palisade-like arrangement that is densely cytoplasmic and thin-walled. Cells are covered by a thin and smooth cuticle and lack visible pores; (ii) Intermediate cell layer: Consists of one large, ellipsoid and vacuolated cell, with anticlinal walls that are thick and highly suberized; (iii) Foot or basal layer: Pluricellular, consisting of smaller and more quadrangular in shape than the adjacent epidermal cells. These cells present thickened anticlinal walls that are covered by cuticle. The nectaries lack vascularization.

Although these glands are already visible in very young leaflets (17-25 mm), they just start secreting in the intermediate stage of leaflet expansion (>50 mm); the secretory phase is, however, active until the senescence of the leaflet. Active nectaries are yellowish-green and turgid, while inactive nectaries are brownish and dry. The secretion is hyaline and somewhat viscous. After released, the secretion accumulates on the concave EFNs surface, from where it can be gathered by insects, mostly Camponotus ants.

Appendix 3S – Abundance of EFNs on 17 species of Anemopaegma from different habitats (rainforest N=10; and savannas N=7).

In the vegetative plant parts of Anemopaegma species, EFNs are distributed on leaflets (six leaflets considered on each count) and prophylls (four prophylls considered on each count), but absent on stems and petioles. In general, the abundance of EFNs was greater in forest species than in savanna species. Asterisks indicate species with many non-glandular trichomes (denser on abaxial side of leaflets). Bold indicates species studied in savannas.

Figure 1S – Most common herbivores found in A. album and A. scabriusculum.