**1. Introduction**

Within the families of flowering plants, the Orchidaceae family is one of largest, with more than 28,000 species. The scent of some orchids has a relevant importance in perfume industries. However, the emission of volatile organic compounds from an orchid can have a relevant role in the life of the plant considering the possible effect of these compounds in attracting pollinators, or in defense against pathogens. One third of all the orchid species are food-deceptive species because the flowers do not contain nectar and the volatile organic compounds emitted mimic the floral signal of rewarding plants to attract pollinators. Furthermore, many compounds show antimicrobial and antifungal activities [1]. Volatile organic compounds are mainly terpenes, phenylpropanoid derivatives and fatty acid derivatives.

Some years ago, we started a project devoted to determining the floral scent of all the orchid species found in Basilicata (Southern Italy). The main feature of this study is the use of the same chemical method in order to determine the scent. Several methods can be used to determine VOCs. For example, FT-IR has been used to determine the composition of volatile mixtures [2,3], and, probably, GC-MS and FT-IR can be considered complementary methods in the analysis of complex mixtures [4]. We decided to use the solid-phase microextraction (SPME) procedure [5]. SPME analysis needs the exposure of a fiber, contained in the needle of a syringe, to a scent. The adsorbed components are then thermally desorbed into the injection sector of the gas chromatographic apparatus. The use of a single procedure allows obtaining a homogeneous dataset, also considering that SPME can suffer from the different absorption rates of the single components in the fiber [5]. This way, significant results were obtained in the characterization of the scent of *Platanthera bifolia* subsp. *osca* [6–8], *Platanthera chlorantha* [7,8], *Cephalanthera* orchids [9], *Serapias* orchids [8,10], *Gymnadenia* orchids [11], *Barlia robertiana* [8] and *Neotinea* orchids [12].

In this article, we want to continue with the realization of our project, showing the chemical composition of *Orchis* species found in Basilicata. This orchid genus is a very common one, where it is diffused in all of Europe, and in Basilicata, there are nine species, *Orchis anthropophora*, *O. italica*, *O. mascula*, *O. pallens*, *O. pauciflora*, *O. provincialis*, *O. purpurea*, *O. quadripunctata* and *O. simia*. In this study, we will report the results obtained in the determination of the scent in *O. anthropophora*, *O. purpurea*, *O. italica*, *O. pauciflora*, *O. mascula*, *O. quadripunctata*, *O. provincialis* and *O. pallens*.

In some of these species, some previous results have been reported in the literature. Nilsson reported a data headspace analysis of *O. mascula*, showing the presence of tricyclene (23.6%), α-pinene (15.6%) and *E*-ocimene (30.5%) as the main components, and linalool in a low quantity (2.4%) [13,14]. The same species, in a headspace analysis of the scent, showed the presence of limonene (8.37%), 1,8-cineole (11.74%), *E*-ocimene (23.25%) and linalool (11.89%) [15]. In a work where hexane extracts were considered, pentacosane (12.07%), heptacosane (46.00%) and nonacosane (27.97%) were determined by the same research group [16]. The same species has been analyzed using SPME (with PDMS-DVB fiber), showing the presence of limonene (11.67%), *E*-ocimene (26.68%) and linalool (13.15%) [17,18]. In an analysis of *O. mascula* through SPME (with PDMS-DVB fiber) where white and purple flowers were analyzed, the authors found in purple flowers (*Z*)-3-hexenyl acetate (6.46%), limonene (10.67%), *E*-ocimene (22.68%) and linalool (13.15%), while in the white flowers, the same compounds were found in a different ratio (12.20%, 12.88%, 16.30%, 3.46%) [19]. In *O. italica*, only one article reported the composition of the scent, obtained through hexane extraction, where tricosane (37.05%), pentacosane (16.88%), heptacosane (20.65%), nonacosane (8.14%) and 5-pentacosene (5.44%) were observed [16]. The same article also examined the scent of *O. provincialis*, showing the presence of pentacosane (12.07%), heptacosane (46.00%) and nonacosane (27.97%) [16]. Schiestl and Cozzolino also examined the scent of *O. quadripunctata* and found tricosane (14.86%), pentacosane (29.62%), heptacosane (32.51%) and nonacosane (9.21%) [16]. Headspace analysis of *O. pauciflora* showed in its scent nonanal (4.88%), 2-methyl-6-methylene-3,7-octadien-2-ol (30.49%), myrcene (25.87%) and *E*-ocimene (8.26%) [15]. Two articles were related to the analysis of the aroma components of *O. simia*: In the first one, where dynamic headspace analysis was performed, ethyl acetophenone (6.79%), α-pinene (32.68%), β-pinene (6.10%), sabinene (5.23%), myrcene (5.45%), eucalyptol (7.89%) and linalool (7.41%) were found [20]. In the second article, where SPME was used, nonanal (5.47%), (*Z*)-3-hexenyl acetate (3.21%), decanal (2.15%), α-pinene (11.49%), myrcene (7.12%), limonene (4.26%) and β-phellandrene (9.03%) were found [19]. The scent of *O. pallens* has been determined in a work where SPME (with Carbowax-PDMS fiber) was used; in this case, phenethyl alcohol, β-farnesene, α-farnesene and farnesol were determined [21]. Finally, in a headspace analysis of *O. anthropophora*, nonanal (9.46%), undecane (8.72%), benzeneacetaldehyde (4.87%), α-pinene (5.77%), limonene (5.22%), 1,8-cineole (7.49%), β-caryophyllene (22.33%) and caryophyllodienol (9.91%) were found as components of its aroma [22,23], while in a study where hexane extracts were examined, tricosane (10.82%), pentacosane (24.47%), heptacosane (26.92%), nonacosane (8.00%), 9-pentacosene (8.50%) and 9-heptacosene (7.68%) were found [16].

The above-reported data show that very different results can be obtained by using different GC-MS analytical methods able to characterize the aroma components, showing that the use of a homogenous method can provide valuable information on the scent of these species. In this work, the same HS-SPME-GC-MS method was used in order to characterize the scent of eight species of the *Orchis* genus.

#### **2. Experimental Section**

#### *2.1. Plant Material*

The sample of *Orchis anthropophora* was collected at Tolve (PZ) (359 m a.s.l.) on 18 April 2018. The sample of *Orchis italica* was collected at Tolve (PZ) (343 m a.s.l.) on 23 April 2018. The sample of *Orchis mascula* was collected at Sasso di Castalda (PZ) (1090 m a.s.l.) on 22 May 2018. The sample of *Orchis pallens* was collected at Serra di Crispo at Terranova del Pollino (PZ) (1882 m a.s.l.) on 18 June 2018. The sample of *Orchis pauciflora* was collected at Madonna di Sasso at Sasso di Castalda (PZ) (1882 m a.s.l.) (1330 m a.s.l.) on 9 May 2018. The sample of *Orchis provincialis* was collected at Sasso di Castalda (PZ) (1069 m a.s.l.) on 30 April 2018. The sample of *Orchis purpurea* was collected at the campus of the University of Basilicata at Macchia Roma in Potenza (714 m a.s.l.) on 5 May 2018. The sample of *Orchis quadripunctata* was collected at Bosco Ralle at Satriano di Lucania (PZ) (1034 m a.s.l.) on 11 June 2018. The plants were collected by Vito Antonio Romano.

The plants were successively used for further studies on the impollination, fertility and germination of the plants. After these studies, the plants were not in condition to be collected in a herbarium. However, these species can be recognized without ambiguities on the basis of their properties, well documented in Figures 1 and 2.

**Figure 1.** (**a**) Orchis anthropophora; (**b**) Orchis purpurea; (**c**) Orchis italica; (**d**) Orchis pauciflora. Photos of V. A. Romano.

**Figure 2.** Chromatogram of volatile organic compounds from *Orchis anthropophora*: (a) tridecane; (b) tetradecane; (c) caryophyllene; (d) humulene; (e) pentadecane; (f) tridecanal; (g) 1-(1-methylethyl)-5-methyl-1,2,3,4- tetrahydronaphthalene; (h) 2-allyl-4-methylphenol; (k) ethyl dodecanoate; (i) hexadecane; (j) tetradecanal; (l) megastigmatrienone; (m) 4-(1-methylethyl)-1,6-dimethyl-1,2,3,4-tetrahydronaphthalene; (n) heptadecane; (o) pristane; (p) pentadecanal; (q) ethyl tetradecanoate; (r) octadecane; (s) hexadecanal; (t) nonadecane; (u) isopropyl palmitate; (v) heneicosane.

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

Following this period, for three days, the plant was placed under a bell jar. In view of the fact that the investigated taxa are rare wild plants, in order to preserve the species, we chose to use a single plant for our analysis.

#### *2.2. Analysis of Volatile Organic Compounds*

SPME [4] analysis of eight different samples of Orchis was performed. This way, the identified plants were collected and inserted in a glass jar for 24 h where a fiber (DVB/CAR/PDMS) and SPME syringe were also present. After this time, the fiber was desorbed in a gas chromatographic apparatus equipped with a quadrupole mass spectrometer detector. A 50/30 μm DVB/CAR/PDMS module with a 1 cm fiber (57328-U, Supelco, Milan, Italy) was employed to determine VOCs. The SPME fiber was maintained in the bell jar for 24 h. The analytes were desorbed in the splitless injector at 250 ◦C for 2 min. Analyses were accomplished with an HP 6890 Plus gas chromatograph equipped with a Phenomenex Zebron ZB-5 MS capillary column (30 m × 0.25 mm i.d. × 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. The analyses were performed by using a splitless injector. The 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. Tentative identification of aroma components was based on mass spectra and Wiley 11 and NIST 14 library comparison. A single VOC peak was considered as identified when its experimental spectrum matched with a score over 90% present in the library. All the analyses were performed in triplicate.

To avoid contamination on the sample due, for example, to volatile organic compounds emitted from the soil, analysis of *Orchis anthropophora* was conducted on a single flower without the presence of soil, showing that this type of contamination does not exist. Otherwise, all the analyses were carried out by inserting the flowering plant in a glass bell jar and isolating the plant from the soil.
