Volatile collection and analyses of floral scents

In the sympatric zone in Botton, we sampled the volatile compounds emitted by flowers (the entire inflorescence) (Table 1). Floral scents emitted by the sympatric Platanthera species and the intermediate morphotypes were sampled for chemical analyses in the same phenological flower state, using a dynamic headspace adsorption technique during peak flowering time (June–July) and between 21:00 and 01:00 h local time, thereby matching the peak feeding times of most nocturnal moths (Nilsson, 1978). The same individuals were used to sample plant material for genetic analyses. The intact inflorescences were carefully enclosed in modified polyacetate bags (Pingvin frying bags, Art.nr 352: Kalle Nalo GmbH, Wiesbaden, Germany). The air, together with volatiles, was drawn through the bag by a battery-operated membrane pump, with a flow of 100 ml/min, into Teflon-PTFE cartridges containing 85 mg of the adsorbent Tenax-GR, mesh 60/80 (Andersson et al., 2002) for 60 min. Trapped scent compounds were eluted with 100 µL of cyclohexan and all samples were stored at −20 °C. Then, extracts were analysed by Gas Chromatography/Mass Spectrometry (GC-MS) on a Finnigan Trace Ultra GC coupled to a Finnigan POLARIS Q ion trap mass and equipped with a Restek RXI-5 MS column (30 m length × 0.25 mm diameter × 0.25 µm film thickness).

Aliquots of 1 µL of the extracts were injected in splitless mode first at 35 °C (4 min, followed by a programmed increase of oven temperature to 200 °C at a rate of 5 °C/min⁻¹) then at 200 °C for 1 min with an oven temperature to 270 °C at a rate of 10 °C/min. Helium was used as carrier. The proportional abundance of floral scent compounds (relative amounts with respect to aggregate peak areas, excluding contaminants) was calculated on the absolute amounts of compounds. Component peaks in the GC-MS chromatograms were quantified by integration of selected ion currents relative to one internal standard (IS) (2-phenylethanol, C₈H₁₀O). The Xcalibur™ Software was used and 2 µL of the internal standard was added for quantification of five samples of each group randomly chosen. Components were identified by their mass spectral patterns and chromatographic retention data (retention time and relative retention time). Furthermore, components were identified by comparing recorded mass spectra with the NIST08 and Wiley275 spectral databases with a probability of match >90%.