Floral and trap volatiles

Volatile collections from flowers or traps of the three species were made both in the field study sites and in the laboratory under natural light and ambient temperature (18–25 °C) by using a dynamic headspace collection method followed by Tenax adsorbent extraction. Ten volatile samples from flowers, ten from traps, and ten control samples were collected from each of 10 individual plants of each species. For D. spatulata and D. arcturi, volatile collections were conducted in the field from December-January over two flowering seasons (2009/2010 and 2010/2011). For D. auriculata, volatile collections of ten samples were conducted in the field from October—November in two flowering seasons (2010 and 2011). All volatile collections were conducted on a sunny days from 10 AM to 4 PM. Additional samples were collected in the laboratory using potted mature plants collected as seedlings from the field. In the dynamic headspace collection (a), the intact flower head or trap was housed in a glass container (i.d. 4.0 cm, 6.0 cm high). The glass container consisted of two parts that tightly closed together using ground glass fittings. One part had a narrow slot (2 mm wide × 17 mm long) to allow insertion of the flower and trap into the glass container without damaging the stem. A charcoal–filtered air stream was pulled over the flower and the headspace was collected on an adsorbent containing 50 mg of Tenax-GR 35/60 (Alltech Associates Inc.) in a 15 mm long × 10 mm diameter glass tube. Tenax adsorbents were thermally conditioned at 200 °C under a stream of nitrogen before use. The airflow in the headspace collection system was 0.5 L/min and each collection session lasted for 6 h. The charcoal filter used to clean the incoming air was thermally activated before use in an oven at 200 °C. Control samples were collected with the above system but without flowers or traps to distinguish between floral and trap compounds and ambient contaminants. Immediately after volatile collection in the field, Tenax adsorbents were sealed in aluminium foils and transported to the laboratory in dry ice. Tenax adsorbents were extracted with 1 mL of hexane (5 × 200 μl aliquots, n-hexane analaR BDH, Laboratory Supplies, Poole, England). Eluted samples were sealed and stored at −80 °C until they were reduced to 10 μl at ambient temperature under a stream of argon immediately before GC/MS analysis.

The concentrated extracts of headspace from flowers and traps were analysed using a Saturn 2200 GC/MS (Varian Inc., Walnut Creek, CA, USA) equipped with a 30 m × 0.25 mm i.d. × 0.25 μm, VF5-MS capillary column (Varian Inc.). The spectra were recorded at an ionization voltage of 70 eV over a mass range m/z of 20 to 499. The transfer line and the ion trap were held at 250 °C and 180 °C respectively. The oven was programmed from 40 °C (held for 2 min), to 240 °C at 4 °C/min. Samples were injected in splitless mode with helium as carrier gas and the temperature of the injector was maintained at 220 °C. Compounds were identified by comparing their mass spectra with authentic standards (Sigma-Aldrich, MO, USA) and NIST 2005 MS library, as well as coincidence with Kovats retention indices published in the literature²².