Carbotrap

Floral scent samples of C. maranhense were collected using dynamic headspace methods for two purposes: (1) to chemically characterise its scent bouquet and (2) to compare scent traits (i.e., chemical composition and total amount) in female and male flowers. Single male (n = 8) and female (n = 5) flowers, each from a different individual were individually enclosed with polyester bags (Toppits^®, Minden, Germany), and the scented air was drawn for 3 min through an adsorbent filter (ChromatoProbe quartz micro vials; 15 mm × 2 mm i.d.; containing 1.5 mg Tenax-TA (mesh 60–80, Supelco, Bellefonte, PA, USA) and 1.5 mg Carbotrap (mesh 20–40, Supelco, Bellefonte, PA, USA); fixed using glass wool). The adsorbent filter was connected to a membrane pump (G12/01 EB, Rietschle Thomas, Puchheim, Germany) using silicone tubing. The pump worked at a constant flow rate of 200 mL/min. All samples were collected around 09:00 a.m., which was the time of highest scent emission as perceived by the human nose. The number of samples per sex depended on the availability of flowering individuals. To detect environmental contaminants, negative controls (empty bags) were collected simultaneously at a distance of ca. 2 m from the target inflorescence using the same methods described above. The samples were kept in the freezer at −20 °C until the chemical analyses.

Volatiles emitted by flowers were sampled from six out of the ten studied populations during the flowering season in 2012, from a total of 16 individuals (see Table 1 for sampling details). In the field, we selected intact plants and flowers and sampled their volatiles using a dynamic headspace method. A stem bearing three freshly opened flowers was enclosed in a polyacetate bag (19×19×24 cm) soon after dusk (18.00–19.00 h), after which the air was pumped out from the bag for 60 min at 200 mL/min through a quartz tube (15 mm long; 2 mm diameter) containing a 1∶1 mixture of 3 mg Tenax-TA (mesh 60–80, Supelco) and Carbotrap (mesh 20–40, Supelco) using a portable membrane pump (Spectrex PAS-500). A negative control was obtained repeating the same procedure with a stem bearing no flower. Scent samples were subsequently analysed by direct mass spectrometry (MS) coupled to gas chromatography (GC) analyses as described in [50] (link). The GC-MS data were processed using MS Worksation 7 Software. Compounds were identified thanks to the library NIST 02 mass spectral through a comparison of the retention times with published data [51] . Differences in relative emission rate of the 10 major compounds among habitat types (with population as a nested factor) were investigated using a permutational MANOVA (based on non-normal distributions).

Volatile collection from the whole lettuce plants was done on the 60th day after sowing. The plant was wrapped with a Nalophane NA foil tube (20 µm; Kalle Hungaria Kft., Budapest, Hungary) a day before measurements. Continuous charcoal-filtered airflow (1 L min⁻¹) was pulled through the system using a vacuum pump (Thomas G 2/02 EB, Garder Denver Thomas GmbH, Fürstenfeldbruck, Germany). Volatile collection traps filled with 50 mg of Porapak Q (80–100 mesh), 50 mg of HayeSep Q (60–80 mesh), and 50 mg of Carbotrap (20–40 mesh) adsorbents (Supelco, Sigma-Aldrich, 595 North Harrison Road, Bellefonte, PA, USA) were used to collect the headspace volatiles for 1, 2, 4, or 6 h. The sampling temperature of all volatile collections was maintained at 25 ± 1 °C. Before each volatile collection, the adsorbent filters were cleaned, as described by Molnár [15 (link)]. The collected volatiles were immediately extracted with 300 µL of n-hexane into a 1.5 mL vial and kept at −18 °C until gas chromatography-mass spectrometry analysis.