Supplementary

The Composition of the Aroma of Serapias orchids in Basilicata (Southern Italy)

Maurizio D'Auria,a Richard Lorenz,b Marisabel Mecca,a Rocco Racioppi,a Vito Antonio Romano,a a) Dipartimento di Scienze, Università della Basilicata, Viale dell'Ateneo Lucano 10, 85100 Potenza, Italy b) Arbeitskreis Heimische Orchideen Baden-Württemberg, Leibnizstr. 1, 69469 Weinheim, Germany

The SPME-GC-MS analysis of the aroma components of , Serapias cordigera subsp. lucana, , , and were reported. The relevant components of the scent of Serapias cordigera were -amorphene, ethyl oleate, heneicosane, heptadecane, 2-heptadecanone , ethyl elaidate, pentadecane, octadecane, farnesylacetaldehyde, nonadecane, and ethyl stearate. Serapias cordigera subsp. lucana gave -amorphene, pentadecane, propyl undecanoate, heptadecane, ethyl elaidate and oleate, ethyl stearate, and 8-heptadecene. Serapias vomeracea showed pentadecane, 5- nonadecene, 1-nonadecene, 3-heptadecene, benzyl benzoate, 2-undecanone, and octadecane. Serapias lingua showed the presence of heptadecane, 3-heptadecene, isopropyl myristate, pentadecane, nonadecane, isopropyl palmitate, octadecane, and benzyl benzoate. Serapias parviflora gave pentadecane, pentadecene, hexadecane, heptadecane, nonadecane, and ethyl elaidate.

Keywords: Orchids; aroma; Serapias; SPME; gaschromatography-mass spectrometry.

3. Experimental

3.1. material

The sample of Serapias cordigera was collected at Tuoppo dell’Acera at Savoia di Lucania (1060 m amsl) (PZ) at June 6th 2017. The sample of Serapias cordigera subsp. lucana was collected at Moliterno (912 m amsl) (PZ) at May 29th 2017. The sample of Serapias vomeraces was collected at the University of Basilicata (712 m amsl) (PZ) at June 1st 2017. The sample of Serapias lingua was collected at Borgo Ralle at Satriano di Lucania (992 m amsl) (PZ) at June 12th 2017. The sample of Serapias parviflora was collected at the University of Basilicata (718 m amsl) (PZ) at May 25th 2018. The were collected by Vito Antonio Romano.

The plants were successively used for further sudies on impollination, fertility, and germination of the plants. After these studies the pant were not in condition to be collected in an herbarium. However, these species can be recognized without ambiguities on the basis of their properties, well documented by the Fig. S1-S5.

To prevent plant damage the whole plant from a population in Basilicata, a large portion of soil all around the plant was removed by its habitat and placed in greenhouse for few days of acclimatization.

Following this period, for three days, the plant was placed under a bell jar, taking care to observe both during a day if perceptible aromas, were developed (fig. S6).

In view of the fact that it is a rare wild plant, in order to preserve the species we have chosen to use a single plant for our analysis.

3.2. GC-MS analysis

A 50/30-μm DVB/CAR/PDMS module (57328-U, Supelco, Milan, Italy) was employed to determine VOCs. SPME fiber was maintained in the bell jar for 24 h. Analyses were accomplished with an HP 6890 Plus gas chromatograph equipped with a Phenomenex Zebron ZB-5 MS capillary column (30-m x 0.25-mm i.d. x 0.25 μm FT) (Agilent, Milan, Italy). An HP 5973 mass selective detector (Agilent) was utilized with helium at 0.8 mL/min as the carrier gas. A splitless injector was maintained at 250 °C and the detector at 230 °C. The oven was held at 40 °C for 2 min, then gradually warmed, 8 °C/min, up to 250 °C and held for 10 min. Tentatively identification of aroma components was based on mass spectra and NIST 14 library comparison. Single VOC peak was considered as identified when its experimental spectrum matched with a score over 90% that present in the library. All the analyses were performed in triplicate.

Figure S1. Serapias cordigera L. 1753.

Figure S2. Serapias cordigera subsp. lucana R. Lorenz & V.A. Romano 2014.

Figure S3. Serapias vomeracea subsp. longipetala (Ten.) H. Baumann & Künkele 1989.

Figure S3. Serapias lingua L. 1753.

Figure S3. Serapias parviflora Parl. 1837.

Figure S6. The experimental apparatus. Table S1. SPME-GC/MS analysis of Serapias species.

Compound KI KI Area [%] ± 0.02 lit.a S. cord. S. S. vom. S. lingua S. parv. lucana

Eucalyptol 1030 1033 0.74 0.47 0.96 Limonene 1031 1036 0.81 Dodecane 1200 1200 0.32 Decanal 1209 1211 0.51 Pelargonic acid 1280 1275 1.35 2-Undecanone 1291 1285 1.61 3.31 Tridecane 1300 1300 1.01 1.91 Undecanal 1310 1309 0.35 4-Octen-3-one 1320 1320 0.84 1,2,4-Trimethoxy- 1331 1331 1.23 benzene Methyl 4- 1337 1335 0.95 methoxybenzoate 2,4,4,6,6,8,8- 1343 1343 1.26 Heptamethyl-2- nonene Ethyl decanoate 1378 1377 0.66 Tetradecane 1400 1400 1.32 2.13 Dodecanal 1409 1401 1.29 0.64 0.60 0.68 Geranylacetone 1436 1434 0.29 -Farnesene 1451 1458 0.45 allo-Aromadendrene 1465 1461 1.67 2.39 Pentadecene 1493 1491 7.05 Pentadecane 1500 1500 2.46 11.13 25.43 7.41 11.03 -Amorphene 1506 1522 28.85 38.06 Lauric acid 1571 1568 1.47 3-Hexadecene 1586 1589 0.69 Tetradecanal 1590 1591 0.45 Ethyl laurate 1597 1595 1.65 1.49 Hexadecane 1600 1600 1.71 3.96 Benzophenone 1604 1604 1.71 1.41 3-Heptadecene 1685 4.33 8.73 Pentadecanal 1687 1690 1.22 0.77 1.32 8-Heptadecene 1689 1680 3.02 2.84 1-Heptadecene 1692 1690 2.16 Heptadecane 1700 1700 3.96 4.55 13.89 11.70 6.38 Pristane 1710 1707 0.78 2.03 2-Ethylhexyl 1735 1735 0.81 1.55 benzoate Farnesal 1738 1737 0.91 1.01 Myristic acid 1754 1758 1.50 Benzyl benzoate 1762 1765 4.18 3.80 Guaiazulene 1772 1778 2.10 3,5-di-t-Butyl-4- 1772 1774 1.04 hydroxy- benzaldehyde Octadecane 1800 1800 1.92 1.40 3.24 4.49 2.97 Phytane 1804 1809 0.72 Hexadecanal 1819 1822 1.17 2.28 1.92 1.86 Isopropyl myristate 1836 1832 0.81 7.80 Farnesyl- 1860 1.80 2.31 0.99 1.79 3.15 acetaldehyde 5-Nonadecene 1880 1918 14.48 1-Nonadecene 1890 1893 5.16 0.87 Nonadecane 1900 1900 1.76 0.92 2.57 6.38 4.28 2-Heptadecanone 1915 1910 3.42 0.54 0.75 Methyl palmitate 1926 1928 1.67 Palmitic acid 1950 1963 1.64 Eicosane 2000 2000 1.17 0.66 2.70 1.91 Isopropyl palmitate 2011 2012 4.86 3,13-Octadien-1-ol 2022 0.35 10-Heinecosene 2082 0.45 Propyl undecanoate 2093 5.15 Heneicosane 2100 2100 4.14 4.31 Ethyl elaidate 2174 2174 3.39 3.47 Ethyl oleate 2179 2180 15.33 2.66 1.64 Ethyl stearate 2190 2193 2.42 3.33 Docosane 2200 2200 2.58 1.38 Tridecyl benzoate 2253 2253 1.68 1.89 9-Tricosene 2271 2273 2.63 Tricosane 2300 2300 0.75 1.26 1.04 Behenic alcohol 2443 2470 1.61 a a) http://webbook.nist.gov; b) http://www.pherobase.com; c) https://pubchem.ncbi.nlm.nih.gov

Table S2. Volatile organic compounds determined in Serapias species divided into the class of the compounds.

Compound r.t [min.] Area [%] ± 0.02 S. cord. S. lucana S. vom. S. lingua S. parv. Fatty acids Pelargonic acid 14,08 1,35 Lauric acid 19,1 1,47 Myristic acid 21,92 1,5 Palmitic acid 24,48 1,64 Aldehydes Decanal 13,35 0,51 Undecanal 15,04 0,35 Dodecanal 16,75 1,29 0,64 0,6 0,68 Tetradecanal 18,35 0,45 Pentadecanal 20,8 1,22 0,77 1,32 Farnesal 21,76 0,91 1,01 3,5-di-t-Butyl-4- hydroxy-benzaldehyde 22,34 1,04 Hexadecanal 22,68 1,17 2,28 1,92 1,86 Farnesyl-acetaldehyde 23,01 1,8 2,31 0,99 1,79 3,15 Alkanes Dodecane 13,2 0,32 Tridecane 14,87 1,01 1,91 Tetradecane 16,55 1,32 2,13 Pentadecane 18,14 2,46 11,13 25,43 7,41 11,03 Hexadecane 19,64 1,71 3,96 Heptadecane 21,07 3,96 4,55 13,89 11,7 6,38 Pristane 21,15 0,78 2,03 Octadecane 22,41 1,92 1,4 3,24 4,49 2,97 Phytane 22,55 0,72 Nonadecane 23,7 1,76 0,92 2,57 6,38 4,28 Eicosane 24,94 1,17 0,66 2,7 1,91 Heneicosane 26,1 4,14 4,31 Docosane 27,23 2,58 1,38 Tricosane 28,32 0,75 1,26 1,04 Aromatic compounds Limonene 9,93 0,81 1,2,4-Trimethoxy- benzene 16,17 1,23 Methyl 4- methoxybenzoate 16,23 0,95 Benzophenone 20,27 1,71 1,41 2-Ethylhexyl benzoate 21,32 0,81 1,55 Benzyl benzoate 4,18 3,8 Guaiazulene 22,05 2,1 Tridecyl benzoate 27,34 1,68 1,89 Fatty acids esters Ethyl decanoate 16,49 0,66 Ethyl laurate 19,57 1,65 1,49 Isopropyl myristate 22,77 0,81 7,8 Methyl palmitate 24,04 1,67 Isopropyl palmitate 25,24 4,86 Propyl undecanoate 26,09 5,15 Ethyl elaidate 26,89 3,39 3,47

Ethyl oleate 26,99 15,33 2,66 1,64 Ethyl stearate 27,19 2,42 3,33 Alkenes 2,4,4,6,6,8,8- Heptamethyl-2-nonene 16,3 1,26 b-Farnesene 17,52 0,45 Pentadecene 17,97 7,05 allo-Aromadendrene 17,76 1,67 2,39 a-Amorphene 18,27 28,85 38,06 3-Hexadecene 19,35 0,69 3-Heptadecene 20,77 4,33 8,73 8-Heptadecene 20,84 3,02 2,84 1-Heptadecene 20,89 2,16 5-Nonadecene 23,43 14,48 1-Nonadecene 23,5 5,16 0,87 10-Heneicosene 25,91 0,45 9-Tricosene 28,13 2,63 Alcohols Eucalyptol 9,87 0,74 0,47 0,96 3,13-Octadien-1-ol 25,66 0,35 Behenic alcohol 29,18 1,61 Ketones 2-Undecanone 14,81 1,61 3,31 4-Octen-3-one 15,87 0,84 Geranylacetone 17,46 0,29 2-Heptadecanone 23,77 3,42 0,54 0,75 fatty acids 0 0 0 5,96 0 aldehydes 4,19 5,88 4,11 6,64 7,24 alkanes 16,16 19,71 45,79 40,35 43,65 aromatic compounds 1,71 5,96 4,18 7,91 2,36 fatty acid esters 21,14 17,73 0 15,97 1,49 alkenes 30,52 43,47 27,26 10,29 13,55 alcohols 0 0,74 0,35 0,47 2,57 ketones 4,26 1,61 4,14 0,75 0 77,98 95,1 85,83 88,34 70,86

Further (undeterminated) compounds 22,02 4,9 14,17 11,66 29,14