Volatile Organic Compounds in Dactylorhiza Species

: HS-SPME-GC–MS analysis of the scent of Dactylorhiza viridis revealed the presence of verbenone (28.86%), caryophyllene (25.67%), β -terpineol (9.48%), and δ -cadinene (6.94%). In the scent of Dactylorhiza romana β -ocimene (18.69%), pentadecane (18.40%), α -farnesene (14.65%), and isopropyl 14-methylpentadecanoate (14.32%) were found. Dactylorhiza incarnata contained tetradecane (11.07%), pentadecane (28.40%), hexadecane (19.53%), heptadecane (17.33%), and α -cubenene (11.48%). Analysis of Dactylorhiza saccifera showed the presence of caryophyllene (17.38%), pentadecane (6.43%), hexadecane (6.13%), and heptadecane (5.08%). Finally, the aroma components found in Dactylorhiza sambucina were caryophyllene (12.90%), β -sesquiphellandrene (32.16%), 4,5-di- ep i-aristolochene (10.18%).


Introduction
The taxonomy of the genus Dactylorhiza Necker ex Nevski is one of the most studied in the Orchidaceae family [1][2][3][4][5][6][7][8], widespread in Eurasia, North Africa, Alaska [9,10], with a great variety of confused and difficult to classify forms in precise taxa. It includes a number of species that varies strongly between authors, from twelve [1] to seventy-five species [6], or to thirty-six species and forty-six subspecies [11]. Traditional classifications recognize four sections of the genus Dactylorhiza: Aristatae, Sambucinae, Iberanthus, and Dactylorhiza s.s. [6]. In the early 2000s, new genetic work led to the inclusion of Coeloglossum viride, the only species of the genus Coeloglossum Hartm. 1820, in the genus Dactylorhiza as Dactylorhiza viridis (L.) [12,13]. Several authors have used this new classification in recently published monographs [14][15][16], while others [17][18][19] have been reluctant to include Coeloglossum viride within Dactylorhiza, arguing that current evidence is insufficient.
In this work it was decided to consider Coeloglossum in Dactylorhiza recognizing D. viride, as reported in recent molecular biology [20][21][22][23][24][25] and molecular genetics studies [10,[26][27][28][29][30] D. viridis is an uncommon species in Basilicata; it always grows in small populations (from 1 to 10 plants) in open woods and pastures from 600 m a.s.l. at 2100 m a.s.l. It has a very variable morphology, due to its stature (plants more than 30 cm high in the open woods and meadows of the mid-hill, or a few centimeters low in altitude meadows), due to its color (from light green to reddish yellow), and the number of flowers. D. viridis is the only species of the group that has a rewarding strategy by rewarding pollinators with nectar, even if in minimal quantities [27], while, on the contrary, the other species of Dactylorhiza do not produce nectar by pursuing a strategy of food deception. Food-deceptive orchids are more common than sexually deceptive ones; their flowers provide signals of food gratification but do not provide re-compensation [31][32][33]. In Basilicata the populations of D. romana and D. sambucina are characterized by the simultaneous presence of flowers colored from yellow to red with more or less frequent various intermediate forms, while the populations of D. saccifera, and D. incarnata, have flowers with pinkish, white, or fleshy pinks often faded and attenuated in their features. The color, smell, size, and shape of flowers are key signals that attract pollinators in search of rewards [34][35][36]. Floral color is often included in studies on the evolutionary models of pollination systems while floral scents, whose characterization requires sophisticated and complex tools, have been little studied, even if they are often considered the main attraction for pollinators [37][38][39][40]. Different types of floral scent variations can be distinguished. The floral odor can vary in composition (when the flowers emit different compounds or distinct ratios of the same compounds) and/or emission speed (that is, when the flowers emit the same bouquet but in different quantities). In many cases, it has been reported that the chemical profiles of floral scents vary both between individuals of the same population and between different populations, through an almost infinite number of combinations of volatile compounds [41][42][43][44][45][46][47].
Comparative analysis of the emission of scents revealed a greater variation of the compounds among the same individuals in the deceptive orchids compared to the gratifying orchids [48,49]. The flower bouquet varies significantly between food deceptive orchids, even within the same genus, and can affect pollinator species and their behaviors in multiple ways [31,41]. The analysis of floral odors in orchids has been mainly carried out to characterize olfactory signals presumably attractive to pollinators, but only in a few cases have these studies been coupled, with the analysis of the pollinator response, to the volatile compounds emitted using electrophysiological techniques [48,50,51] and behavioral tests [52][53][54][55]. Therefore, there is an increasing need for a better understanding of the evolution of floral scents in order to obtain a more complete view of the mechanisms and patterns of evolution of pollination systems.
Food-deceptive orchids are pollinated by generalist pollinators [56], mainly bumblebee queens and various other bees [57][58][59][60], just emerged from the soil, without experience which, after the first attempts at pollination, abandon the plant, moving away in search of other inflorescences [61]. It is thought that the wide variability of the morphology of flowers and scents in deceptive species for food favors cross-pollination by reducing the learning in the recognition of flowers by newly emerged, inexperienced pollinators, thus limiting self-pollination and geitonogamy and favoring outbreeding. While D. romana, sambucina, and saccifera are widespread in the territory of Basilicata, also forming large populations, D. incarnata has been identified in a single station on the Maddalena Mountains (Padula. Salerno) on the border between Basilicata and Campania where it forms a large population mixed with D. saccifera and where it is possible to find many hybrid forms between the two species and also many plants of D. incarnata var. ochroleuca. This population of D. incarnata is the southernmost station reported in Italy.
The purpose of this work is to complete the collection of data on the various perfumes emitted by the species of orchids in Basilicata by using the same technique, solid phase microextraction performed on intact plats followed by gas chromatography-mass spec-trometry. Until now, the volatile organic compounds constituents of the scent of the orchid species have been determined on several orchid species [62][63][64][65][66][67][68][69]. 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 were inserted.

Materials and Methods
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 8 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 1 and 2. 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.

Analysis of Volatile Organic Compounds
The SPME analysis of five different samples of Dactylorhiza has been performed. This way, the identified plants were collected and inserted in glass jars for 24 h where the fiber (DVB/CAR/PDMS) and SPME syringe were also placed. 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 (Agilent, Milan, Italy) in the range 0-800 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. Tentative 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.

Conclusions
The results described above can give us some useful information. First, the results of our analysis, performed by using SPME technique, are not in agreement with previous described scent composition [31,[70][71][72]. This difference may depend on several factors: first of all, the different location of the plants under study. The different environmental conditions could induce plants to adopt different strategies for pollination. Second, the different analysis methodology could play a significant role. Furthermore, we can observe that every species adopts a different strategy. D. viridis has a scent where terpenes are the main components. This statement is applicable also to D. romana; however, it is noteworthy that the terpenes involved in the scent are different from those observed in the other species. The scent of D. incarnata only included hydrocarbons as components; hydrocarbons are present in the scent of D. saccifera but, in this case, caryophyllene is also present in relevant amount. Finally, terpenes were detected in the scent of D. sambucina, as in D. viridis and in D. romana, but the compounds involved in the scent, with the exception of caryophyllene, are different from those observed in the other species.