**1. Introduction**

The determination of the volatile organic compounds emitted from a natural source is smell and taste are the oldest of our senses. They probably developed in very primitive organisms as means of obtaining information about chemical changes in the organism's environment. Animals use smell and taste to find food and to assess its quality. The smell of food has a powerful effect on animals.

Living organisms use the chemical sense as a means of communications. If the communication is between different parts of the same organism, the messenger is referred to as a hormone. Chemicals used to carry signals from one organism to another are known as semiochemicals. In the case of flowers, the aroma components are mainly devoted to attracting pollinator insects.

In recent years, *Himantoglossum* s.l. has included other taxa of considerable interest and conservation [1,2]. Currently, the expanded genus *Himantoglossum* is composed of the subgenus *Himantoglossum* including all the species of the former genus *Himantoglossum*, the subgenus *Barlia*, consisting of the two species of the genus *Barlia*, and the subgenus *Comperia* consisting only of the former species *Comperia comperiana* (Table 1).

The attraction of the pollinator in orchids generally occurs first through the air diffusion of scents, then through sight, as the pollinator approaches its target inflorescence, and finally through tactile signals and hardly volatile, extractable compounds when it lands on the chosen flower.

*H. hircinum* and *H. adriaticum* have large, showy flowers with a long, captivating lip, often adorned with showy tufts of colored papillae that provide footholds acting as a guide for pollinators. Vöth (1990) speculates that these papillae could also be the seat of osmophores responsible for most of the volatile organic compounds emitted by the plant [3]. The scent emitted by these two species can be strong, unpleasant, or sweet. They

**Citation:** Mecca, M.; Racioppi, R.; Romano, V.A.; Viggiani, L.; Lorenz, R.; D'Auria, M. The Scent of *Himantoglossum* Species Found in Basilicata (Southern Italy). *Compounds* **2021**, *1*, 164–173. https://doi.org/ 10.3390/compounds1030015

Academic Editor: Juan Mejuto

Received: 9 November 2021 Accepted: 7 December 2021 Published: 20 December 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

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are allogamous species that do not offer food reward; in fact, their short and sack-like spur does not contain nectar [4].

Teschner (1980) has shown that the spur of *H. hircinum* and *H. adriaticum* may contain small amounts of glucose in some populations [5]. Kropf and Renner (2008) chemically demonstrated the presence of nectar in *H. hircinum* [6]. Little is known about the pollination of the various species of the *H. hircinum* group; pollinators are thought to differ locally, with Teschner (1980) suggesting that solitary bees are the true pollinators [5].

In this work, we have dealt with the only two species present in Basilicata belonging to the old genus *Himantoglossum* Spreng. 1826, *Himantoglossum hircinum* (L.) Sprengel 1826 and *Himantoglossum adriaticum* H. Bauman 1978, while, in previous works we analyzed the perfumes of *Barlia robertiana* [7], a species until a few years ago considered to belong to the monospecific genus *Barlia* and now merged into the new clade *Himantoglossum*.

*H. hircinum* (Figure 1) has a Mediterranean-Atlantic distribution, from southern Great Britain to northern Africa. Present in Italy, the species is reported to be widespread in Sicily between 150 and 1750 m [8], sporadically in the southern regions, also reported in Tuscany, Liguria, and southern Piedmont [9].

**Table 1.** Taxonomy of the genus *Himantoglossum* s.l. generated by integrating the results of the present study with those of Sramkó [10].


**Figure 1.** *Himantoglossum hircinum.* Photo of V. A. Romano.

From the observations of one of the authors of the following work (VAR), we report a wide diffusion for Basilicata of this species, so much so that it is very common in the hilly and mountainous area of the Province of Potenza from 400 to 1500 m where it forms large populations with dozens of plants. It blooms from early May to early June.

*H. adriaticum* (Figure 2) has a Euro-Mediterranean distribution, present in southern Italy up to the Alpine regions, Slovenia, and Croatia; its northeastern limit also touches Austria, Hungary, and Slovakia [9].

**Figure 2.** *Himantoglossum adriaticum.* Photo of V. A. Romano.

In Basilicata it was reported by Gölz and Reinhard (1982), Conti et al. (2005), Fascetti et al. (2008), Romano et al. (2013) [11–14]. It blooms from mid-June to mid-July from 1300 to 1600 m. Many plants have never been observed on the same site, maximum 10 plants on an area of 0.5 ha. Generally, there are 1–3 isolated plants distant from other single specimens even a few km. The richest area is that of the Pollino National Park between Basilicata and Calabria [15].

For the perfume tests, *H. hircinum* plants from Basilicata and *H. adriaticum* plants from Basilicata and Abruzzo were used. All the plants were collected before anthesis and planted in the gardens of the University of Basilicata and tested, many days later, when they were in full bloom.

The scent of a flower can be an important factor determining the pollination of a plant. The study of the scent of the orchids was the object of several works in the past [16]. Unfortunately, several different approaches have been used in order to determine the composition of the aroma of an orchid. Extraction, headspace analysis, and SPME have used. Often, different chemical procedures allowed to obtain different results. On the basis of these considerations, a research project started devoted to the determination of the scent of the orchids found in Basilicata using the same procedure, solid phase microextraction coupled with gas chromatography and mass spectrometry. This way, the composition of

the scent of *Platanthera bifolia* subsp. *osca* [17,18], *Platanthera chlorantha* [18], *Cephalanthera* orchids [19], *Serapias* orchids [20], *Gymnadenia* orchids [21], *Barlia robertiana* [7], *Neotinea* orchids [22], and *Orchis* species [23] has been investigated.

Some studies report some data on the scent of *H. hircinum*. (*E*)-Ocimene, elemicin, (*E*)-3-methyl-4-decenoic acid, (*Z*)-4-decenoic acid, and lauric acid were considered as the main components of the scent after absorption on charcoal [24]. (*E*,*<sup>Z</sup>*)-2,6-dimethyl-3,5,7- octatrien-2-ol and the (*E*,*E*) isomer were claimed as major constituents of the aroma [25,26]. Finally, hexane extraction of the labella showed the presence of high molecular weight alkanes such as pentacosane, heptacosane, and nonacosane [27].

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

## *2.1. Plant Material*

The samples of *H. adriaticum* were collected at Comune di Cocullo, Prov dell'Aquila (Abruzzo), 1070 m. a.s.l., on 20 May 2017 (Sample 1), at Fontana delle Brecce, Marsico Nuovo (Pz), 1439 m. a.s.l., on 10 June 2017 (Sample 2), at Piano Visitone, Viggianello (Pz), 1500 m. a.s.l., on 12 June 2018 (samples 3 and 4). The sample of *H. hircinum* was collected at Contrada Manta, Potenza, 1000 m. a.s.l., on 1 May 2017. The plants were collected by Vito Antonio Romano.

The plants were harvested about two weeks before flowering by taking all the clod of earth, taking care not to damage the root system, planted in special pots in the gardens of the University of Basilicata (Potenza 650 m. a.s.l.), in waiting for their full bloom. Two days before the tests the plants were transferred to an air-conditioned room at 22 ◦C. The plants were tested, whole without being damaged, under a cylindrical glass bell (12 cm × 45 cm) in which only the inflorescence and the SPME probe are inserted (Figure 3).

**Figure 3.** The apparatus used to collect the scent of the plants used in this study.

To avoid contamination, the interior of the bell was isolated from the external environment with appropriate closing and sealing systems during the 24 h of the test (from eight in the morning to 8 the following day).

In order to be sure that the internal environment of the bell was isolated from the external environment, various blank tests were carried out.

The plants were successively used for further studies on pollination, fertility, and germination of the plants. After these studies, the plants 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 Figures 2 and 3. In view of the fact that the investigated taxa are rare wild plants, in order to preserve the species, we have chosen to use a single plant for our analysis.

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

The SPME [4] analysis of five different samples of *Himantoglossum* has been performed. This way, the identified plants were collected and inserted in glass jar for 24 h where was present also the fiber (DVB/CAR/PDMS) of and SPME syringe. 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 1 cm fiber (57328-U, Supelco, Milan, Italy) was employed to determine VOCs. 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 in the range 1 to 800 m/z (Agilent) was utilized with helium at 0.8 mL/min as the carrier gas. The EI source was used at 70 eV. 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. Tentatively identification of aroma components was based on mass spectra and Wiley 11 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.
