**3. Results**

*Orchis anthropophora* (Figure 1a) returned the results reported in Table 1 and Figure 2. The main volatile organic compounds detected were caryophyllene (11.32%), tetradecanal (57.17%) and hexadecanal (12.10%). Caryophyllene has a scent described as sweet, woody and terpenic, while the aroma of tetradecanal is described as fatty, waxy, amber, incense, citrus peel and musk.

The SPME analysis of *Orchis purpurea* (Figure 1b) showed that the main components of the aroma were aromatic compounds such as 3,5-dimethoxytoluene (35.29%) and elemicin (4.76%) (Table 1). The scent of elemicin is described as spicy and floral. When *Orchis italica* (Figure 1c) was examined, eucalyptol (3.93%), caryophyllene (47.29%) and 4-(3-hydroxy-2-methoxyphenyl)butan-2-one (4.96%) were the most abundant compounds found in the scent (Table 1). The scent of eucalyptol is recognized as eucalyptus, herbal, camphoreous and medicinal. *Orchis pauciflora* (Figure 1d) showed the presence of linalool (26.12%), 1,4-dimethoxybenzene (15.01%), germacrene D (9.34%) and 6,10,14-trimethyl-4,8,12-tetradecadrienal (3.60%). The scent of linalool is described as citrus, floral, sweet and woody, while that of 1,4-dimethoxybenzene is sweet, green and hay.

The SPME analysis of the scent of *Orchis mascula* (Figure 3a) showed the presence of eucalyptol (7.80%), linalool (21.28%), tetradecane (3.70%), pentadecane (4.41%) and 6,10,14-trimethyl-2-pentadecanone (Table 1 and Figure 4). *Orchis quadripunctata* (Figure 3b) had a scent composition where only hydrocarbons were found. Thus, tridecane (9.70%), tetradecane (9.38%), pentadecane (26.85%) and heptadecane were the main components of the scent (Table 1). In the case of *Orchis provincialis* (Figure 3c), the volatile organic compounds found in the SPME analysis were β-farnesene (44.16%), 3,7,11-trimethyl2,6,10-dodecatrienal (29.25%) and 6,10,14-trimethyl-2-pentadecanone (6.32%) (Table 1). β-Farnesene's scent is described as woody, citrus, herbal and sweet. Finally, the aroma components of *Orchis pallens* (Figure 3d) were α-zingiberene (14.67%), di-*epi*-<sup>α</sup>-cedrene (10.64%), β-curcumene (33.29%) and diethyltoluamide (13.95%) (Table 1). α-Zingiberene has a scent described as spicy, fresh and sharp, while that of di-*epi-*α-cedrene is described as woody, cedar, sweet and fresh.

**Figure 3.** (**a**) Orchis mascula; (**b**) Orchis quadripunctata; (**c**) Orchis provincialis; (**d**) Orchis pallens. Photos of V. A. Romano.

**Figure 4.** Chromatogram of volatile organic compounds from *Orchis mascula*; (a) β-terpinene; (b) 2,2,4,6,6-pentamethyl-3-heptene; (c) limonene; (d) eucalyptol; (e) linalool; (f) dodecane; (g) tridecane; (h) tetradecane; (k) germacrene D; (i) βsesquiphellandrene; (j) hexadecane; (l) heptadecane; (m) pristane; (n) octadecane; (o) 6,10,14-trimethyl-2-pentadecanone.


**Table 1.** SPME-GC-MS analysis of *Orchis* species.



## **4. Discussion**

It is interesting to note the large differences between our reported results and those reported in the Introduction section. For *O. anthropophora*, two different analyses are available [16,20]. While in the work of Cozzolino [16], only hydrocarbons with an extremely high molecular weight were found, the other article [20] reported that β-caryophyllene was the main component. In our study, β-caryophyllene was present, but the main component was tetradecanal. In the case of *O. purpurea*, no other results on the composition of the scent are available. For *O. italica*, only an article published by Cozzolino is available [16], and, also in this case, only high-molecular weight hydrocarbons were found. In our experiment, on the contrary, β-caryophyllene was the main component of the scent.

The scent of *O. pauciflora* has been determined through headspace analysis, showing the presence of 2-methyl-6-methylene-3,7-octadiene-2-ol as the main component [15]. However, in our analysis, linalool and 1,4-dimethoxybenzene were found as the main components. *O. mascula* was the object of an intense study where several different analytical methods were used. This way, headspace analysis returned *E*-ocimene as the main component of the scent [13,14]. This result was confirmed by SPME analysis [15,17]. In our analysis, as it is evident considering Figure 4, linalool was the main component of the aroma. For *O. quadripunctata*, the work of Schiestl and Cozzolino found only hydrocarbons [16]. Only hydrocarbons were found in this work, but with a significant difference in the molecular weight of the detected compounds. In the case of *O. provincialis*, the work of Schiestl and Cozzolino determined only the presence of hydrocarbons [17], while the presence of relevant amounts of β-farnesene was determined in this study. Finally, while an SPME analysis of the scent of *O. pallens* found phenethyl alcohol, farnesene and farnesol [21], our analysis of the same species found β-curcumene as the main component.

## **5. Conclusions**

This work shows the analysis of *Orchis* samples from Basilicata. The analyses were performed by using the same procedure and the same fiber in SPME-GC-MS, which allowed achieving a homogenous dataset. The analyses showed different scent compositions from those determined on samples deriving from different sites. These observed differences, when SPME of other headspace techniques is used, can depend both on the different absorption rates of the analytes on the fiber and on the variation in the scent due to natural adaptation of the plant to different environmental conditions, due, for example, to different pollination insects. A completely different consideration can be found in the work of Schiestl and Cozzolino, where a completely different analytical method was used (hexane extraction of labellum, and GC-MS analysis of the extracts). In their case, only hydrocarbons were determined. Probably, their analytical procedure was not the correct method for the determination of the orchid scent.

**Author Contributions:** Conceptualization, M.D. and V.A.R.; methodology, R.R.; investigation, R.R., L.V. and M.M.; data curation, R.L.; writing—original draft preparation, M.D. and V.A.R.; writing— review and editing, M.D., V.A.R. and R.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
