Determination of Volatile Organic Compounds in Some Epipactis, Neottia, and Limodorum Orchids Growing in Basilicata (Southern Italy)

Abstract

SPME analysis of the scent of Epipactis microphylla showed the presence of limonene as the main component of the scent. Other components were 2,4,4,6,6,8,8-heptamethyl-1-nonene, pentadecane, and heptadecane. The scent of Epipactis palustris was characterized by pentadecane, 2,4,4,6,6,8,8-heptamethyl-1-nonene, and heptadecane. The scent of Neottia nidus avis showed the presence of kaur-16-ene as the main component of the scent. Other components were heinecosane, tetradecane, pentadecane, hexadecane, heptadecane, and 5,9,13-trimethyl-4,8,12-tetradecanal. The scent of Neottia ovata is due to pentadecane, hexadecane, and heptadecane. The scent of Limodorum abortivum showed the presence of 2,4,4,6,6,8,8-heptamethyl-1-nonene, pentadecane, hexadecane, heptadecane, and 2-(dodecyloxy)-ethanol.

1. Introduction

The Orchidaceae family has a wide diversity of over 28,000 species, distributed in approximately 763 genera, spread from the Arctic tundra to Brazilian tropical rainforests [1,2].

It includes species that have developed very different life strategies, i.e., from epiphytic to terrestrial, from evergreen to completely, or partially, devoid of chlorophyll [3], adopting complex and different pollination strategies.

To attract pollinators, orchids have adapted the structure of their flowers and the spectrum of their fragrances. Many of them (30–40% of species) have developed deceptive tactics (mainly food or sexual deception) [4,5], while others reward pollinators (rewarding species) with nectar, fragrances, oils, resin, and wax [6].

Species that use deception, whether food or sexual, are linked to a single pollinator species or a single functional group, while species that offer rewards (generalist species) are pollinated by a wide range of animals from different groups, systematic and ecological [7,8,9].

In this work we analyzed the scents emitted by Epipactis microphylla (Ehrh.) Sw. 1800, Epipactis palustris (L.) Crantz 1769, Neottia nidus avis (L.) Rich. 1817, Neottia ovata (L.) Bluff and Fingerh. 1838, and Limodorum abortivum (L.) Sw. 1799, all species belonging to a relatively primitive group, the tribe Neottieae (subfamily Epidendroideae). They are all rewarding species that offer nectar as a reward and therefore often attract large numbers of different pollinator species.

The flower in the genus Epipactis is characterized by a bipartite labellum, with a more or less triangular part, which acts as a landing place for insects (the epichilus), and a basal part, cup-shaped, where the nectar is secreted (the hypochilus). Often, due to nectar fermentation, small quantities of alcohol are formed in the hypochilus of some species, which attracts many bees, making them slower and prolonging the time they spend on Epipactis plants, thus increasing the possibility of pollination of the flower [10].

E. microphylla (Ehrh.) Sw. 1800 is a very widespread species in Basilicata. It is considered autogamous but with residual allogamous characters [11]. Autogamy allows these species to reproduce even in beech forests, which are usually poor pollinators [12].

E. palustris (L.) Crantz 1769 manages to attract many pollinators (according to Nilsson (1978) [13] there are 103 species) including many Hymenoptera, mainly solitary wasps that feed on nectar (Hymenoptera: Eumenidae), bumblebees, honey bees, and Diptera (Empis sp., Episyrphus sp.) [14]. Among the Diptera, Sacrophaga carnaria (Diptera: Sacrophagidae) and Coelopa frigida (Diptera: Coelopidae) are also reported as pollinators [14]. Brantjes (1981) also reports ants as possible pollinators of E. palustris [15].

The composition of the scent released by E. palustris could be critical in initially attracting pollinators and other insect visitors. The discovered compounds, such as eugenol and vanillin, are strong aromatic attractants, as considerable numbers of dipterans visited the flowers of E. palustris [16]. It is a very rare species in Basilicata, where few stations with a good number of specimens are known [17].

Until a few years ago, the genus Neottia included only non-photosynthetic taxa. The long-known similarity between the species belonging to the genera Neottia and Listera, based on the structures of the reproductive systems [18], was confirmed by the phylogenetic classification based on DNA [1,19,20], leading to the recent expansion of the genus Neottia with the inclusion of photosynthetic Listera species.

N. nidus-avis Rich. 1817, is a perennial, non-photosynthetic, mycoheterotrophic herbaceous plant with short underground rhizomes. The tangle of adventitious roots, all of similar and limited length, form a tangled mass that resembles the nest of a bird that gives it its name, from neottiá (νεοττιά, which means nest in Greek) and nidus-avis (which means bird’s nest in Latin).

In the Euro-Mediterranean region it is found in shady beech forests and oak forests, mesophilous and sclerophilous, in pine forests with various native and planted subspecies of Pinus nigra [21,22]. The flowers of this species secrete nectar whose odor attracts insects. Its scent is reminiscent of honey or a musty, pleasant, and powerful odor [23,24,25].

The autogamy of this species [23] has been confirmed by countless observations [26] and already described by Darwin (1862) and Müller (1883) [27,28]. However, DNA analyses revealed that most of the populations studied are genetically diverse, which indicates that there is at least some allogamous pollination by insects that justifies the genetic diversity in the populations [23]. Several authors have observed flies, thrips, and ants as regular visitors [14,28,29,30,31].

N. nidus-avis is a very common species in all wooded areas of Basilicata.

N. ovata (L.) Bluff and Fingerh. 1838 is a long-lived forest herb, usually shade-tolerant but also present in full light, with an average half-life of 80 years [32]. It is a common species in Western Europe, and its geographical distribution reaches Eastern Siberia in Asia.

Although this common orchid can grow up to 60 cm, it is rather discreet; all parts of the flower are uniformly green, easily blending in with other plants. Easy to recognize by the two egg-shaped basal leaves, one in front of the other, with a long multiflora spike.

N. ovata is an allogamous plant that attracts numerous insects. Nilsson (1981) reports almost 300 different species, of which approximately 50 pollinia-bearing species, mainly Diptera, Hymenoptera, and some Coleoptera [33].

The yellow-green color of N. ovata flowers does not attract pollinators because it does not contrast with the surrounding vegetation. In these cases, other traits of the flower (smell and nectar) play a key role. Floral nectar (its concentration and composition) is rarely detectable, at a distance, by pollinators [34]. The fragrance of N. ovata attracts pollinating insects from afar [35], like the generic plants that accompany it and which often give off the same scents [33,36,37,38].

N. ovata is a species rarely present in Basilicata; only two stations with few specimens have been reported [17].

L. abortivum (L.) Sw. 1799 is a very common species in the woods of Basilicata; usually few specimens are found, but it is not difficult to find small populations with 10/20 plants.

L. abortivum is a common Mediterranean orchid, growing on decaying plant material. It forms small populations with irregular distribution in the woods and is characterized by very small leaves.

Although some chlorophyll pigmentation is observed near the leaves, reduced to scales, and in some places on the stems, it is assumed that its photosynthetic capacity is insufficient to support the entire energy needs of an adult plant [39].

The flowers open wide or remain closed, depending on the climatic conditions or the mode of pollination, allogamous or autogamous [12].

The open flowers are attractive, with large, divergent, and open lateral sepals, usually very colorful. The bipartite labellum is horizontal, which makes a good landing point for visiting insects.

The long, downward-curving, nectar-rich spur is much sought after by pollinating insects, mainly Anthophora males. Other reported pollinators are Bombus terrestris, Anthidium septemdentatum, and Lasioglossum sp. [28].

In cold, rainy, or very hot periods, the flowers can remain completely closed or open very little, in which case they can self-pollinate because the pollen masses in adverse conditions are rather friable and can lose coherence, emerge from the anther, and fall into the underlying stigmatic cavity, self-pollinating.

This work was a part of the study of the scent of the orchid species growing in Basilicata by using the solid-phase microextraction technique [40,41,42,43,44,45,46,47,48,49,50,51]. Our goal is to have an identification of the scent of spontaneous orchids obtained using the same methodology in order to have comparable data.

2. Materials and Methods

2.1. Plant Material

The sample of E. microphylla was collected at Abriola (Pz) 1425 m.a.s.l. (Serra Marlevante) 33TWE (64–82), on 16 June 2017. The sample of E. palustris was collected at Sasso di Castalda (Pz) 1040 m.a.s.l. (Fontana dei Meli) 33TWE (57–85), on 21 June 2017. The sample of N. nidus avi was collected at Brienza (Pz) 1000 m.a.s.l. (V. Cerasa) 33TWE (50–77), on 1 June 2018. The sample of N. ovata was collected at Marsicovetere (Pz) at 1281 m.a.s.l. (Fosso Salicone) 33TWE (70–72), on 5 June 2018. The sample of L. abortivum was collected at Laurenzana (Pz) at 1200 m.a.s.l. (Abetina di Laurenzana) 33TWE (79–73), on 1 June 2018. The plants were collected by Vito Antonio Romano (Dipartimento di Scienze, Università della Basilicata, 85100 Potenza, Italy).

The plants were harvested taking all the clod of earth, taking care not to damage the root system. All the plants had closed flowers to avoid using flowers that were already fertilized but not visible because they were at the beginning of fertilization. The plants were planted in special pots in the greenhouse of the University of Basilicata (Potenza 650 m.a.s.l.), in closed boxes with transparent cloth to avoid fertilization (even if occasional). The correct classification of the species was carried out on flowering plants. The plants were tested when the flowers were all open except the last two.

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.

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.

After the tests, the plants remained closed in the boxes to verify that at the end of flowering there were no fertile ovaries, and for this reason no herbarium samples were taken. The earthen bread with the bulbs was brought back to the site.

In view of the fact that the investigated taxa are rare wild plants, in order to preserve the species, we used a single plant for our analysis.

2.2. Analysis of Volatile Organic Compounds

The SPME analysis of all the samples has been performed. This way, the identified plants were collected and inserted in a glass jar for 24 h, where the fiber (DVB/CAR/PDMS) of the SPME syringe was also present. After this time, the fiber was desorbed in a gas chromatography 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 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. A single VOC peak was considered identified when its experimental spectrum showed greater than 90% similarity to the spectrum recorded in the library. All the analyses were performed in triplicate.

3. Results and Discussion

The analysis of volatile organic compounds emitted from the orchid species was performed by using a solid-phase microextraction (SPME) apparatus. The analyses were performed on Epipactis microphylla (Figure 1a), Epipactis palustris (Figure 1b), Neottia nidus avis (Figure 1c), Neottia ovata (Figure 1d), and Limodorum abortivum (Figure 1e).

The results of SPME analysis of the scent of the selected orchids are in

Table 1. Volatile organic compounds found in the scent of suitable orchid species.

Compound	r.t. a [min.]	KI a	Area% ± 0.03
			E. microphylla	E. palustris	N. nidus avis	N. ovata	L. abortivum
Mesityl oxide	4.97	804		4.95
2,2,4,6,6-Pentamethylhept-3-ene	9.31	1030	0.41	1.24		0.62	0.45
Limonene	10.60	1039	40.05	5.22	1.14	1.08	0.22
Linalool	10.65	1101				1.78
β-Terpineol	10.72	1130				0.62
Dodecane	13.09	1200		1.13
Decanal	13.23	1204	3.18			0.77
2-Dodecene	13.36	1222	0.80
Bornyl acetate	14.79	1285	1.70
Tridecane	14.97	1300				3.31	3.02
α-Terpinyl acetate	15.81	1355	2.69
2,4,4,6,6,8,8-Heptamethyl-2-nonene	16.12	1365			1.47		1.69
Farnesane	16.26	1378				1.54
2,4,4,6,6,8,8-Heptamethyl-1-nonene	16.45	1385	3.74	8.90		0.77	6.07
Tetradecane	16.55	1400	2.38	5.34	8.03	4.22	3.87
Methyleugenol	16.60	1408		1.31
Dodecanal	16.74	1415	1.36
1-Methoxydodecane	17.00	1424	1.02
Geranylacetone	17.46	1451	0.61
2,6-di-t-butylbenzoquinone	17.79	1472	1.17	2.78	1.74	2.06	2.32
Pentadecane	18.14	1500	5.51	12.03	7.79	15.22	6.86
β-Cadinene	18.74	1520					3.62
Elemicin	19.07	1554	1.24	7.94
Tetradecanal	19.86	1581	1.36	3.63
Hexadecane	20.36	1600			7.70	8.38	6.10
Heptadecane	21.06	1700	3.25	7.31	7.38	10.30	6.28
Pristane	21.14	1708	1.29	4.21	3.89		3.38
2-(Dodecyloxy)-ethanol	21.82	1731					6.32
2-Benzylideneoctanal	21.86	1746	2.94	1.59			2.90
Benzyl benzoate	22.15	1762	1.02
3,5-di-t-butyl-4-hydroxybenzaldehyde	22.22	1772		1.36			2.09
Octadecane	22.41	1800	1.58	3.89	4.84	5.62	4.55
Hexadecanal	22.67	1819		1.75
5,9,13-trimethyl-4,8,12-tetradecenal	23.01	1880	1.14	1.70	7.11	5.23	4.59
Nonadecane	23.70	1900	1.17	2.16	3.56	3.00	3.62
7,9-Di-t-butyl-1-oxaspiro [4,5]deca-6,9-diene-2,8-dione	24.19	1919					1.66
Palmitic acid	24.48	1950	1.09
Eicosane	24.93	2000	1.21	2.44	2.55	2.30	2.99
Kaur-16-ene	25.86	2031			19.67
Heinecosane	26.18	2100			10.60	4.99	5.26
Docosane	27.22	2200	0.65	1.20	1.61	2.23	2.45
9-Tricosene	28.12	2271				4.46
Tricosane	28.40	2300			1.21	4.85	1.48

^a r.t. = retention time, KI = Kovats index.

. E. microphylla showed the presence of limonene as the main component of the scent (40.05%). Other components were 2,4,4,6,6,8,8-heptamethyl-1-nonene (3.74%), pentadecane (5.51%), and heptadecane (3.25%). The analysis of the scent of E. palustris showed the presence of some hydrocarbons, i.e., pentadecane (12.03%), 2,4,4,6,6,8,8-heptamethyl-1-nonene (8.90%), and heptadecane (7.31%). In this case, the presence of elemicin (7.94%) was determined. SPME determination of volatile organic compounds in the scent of N. nidus avis showed the presence of kaur-16-ene as the main component of the scent (19.67%). Other components were heinecosane (10.60%), tetradecane (8.03%), pentadecane (7.79%), hexadecane (7.70%), heptadecane (7.38%), and 5,9,13-trimethyl-4,8,12-tetradecenal (7.11%). The scent of N. ovata is characterized by the presence of some hydrocarbons, i.e., pentadecane (15.22%), hexadecane (8.38%), and heptadecane (10.30%).

Finally, in the scent of L. abortivum, the main components were 2,4,4,6,6,8,8-heptamethyl-1-nonene (6.07%), pentadecane (6.86%), hexadecane (6.10%), heptadecane (6.28%), and 2-(dodecyloxy)-ethanol (6.32%).

While it is possible that hydrocarbons, such as the alkanes determined in this study, can act as insect attractors, in order to understand the nature of the scent of these orchid species, the role of minor components has to be considered. Limonene, a terpene with an intense smell, is the main component of the scent of E. microphylla. The following terpenes are present in the scent: α-terpinyl acetate (2.69%) and geranylacetone (0.61%) can have a role. Furthermore, several aldehydes are present in small quantities. Thus, the presence of decanal (3.18%), dodecanal (1.36%), tetradecanal (1.36%), 2-benzylideneoctanal (2.94%), and 5,9,13-trimethyl-4,8,12-tetradecenal (1.14%) has been determined. In the scent of E. palustris, the terpene elemicin is one of the main components. However, limonene (5.22%) and methyleugenol (1.31%) were determined in small quantities. Also, in this case, the following aldehydes have been determined in the aroma: tetradecanal (3.63%), 2-benzylideneoctanal (1.59%), 3,5-di-t-butyl-4-hydroxybenzaldehyde (1.36%), hexadecanal (1.75%), and 5,9,13-trimethyl-4,8,12-tetradecenal (1.70%) were observed as volatile components of the aroma. N. nidus avi showed the presence of the terpene kaur-16-ene as the main component of the scent. However, SPME determined the presence in the aroma of limonene (1.14%). Also, in this case, the following aldehyde was determined as a minor component: 5,9,13-trimethyl-4,8,12-tetradecanal was determined in 7.71%. N. ovata showed the presence of some alkanes as main components of the scent. However, some terpenes were determined as minor components, i.e., limonene (1.08%), linalool (1.78%), β-terpineol (0.62%), and farnesane (1.54%) were identified. Also, in this species, some aldehydes were present in the chromatogram, i.e., decanal (0.77%) and 5,9,13-trimethyl-4,8,12-tetradecenal (5.23%) were determined in small quantities. The scent of L. abortivum is characterized by the presence of some hydrocarbons. However, also in this case, some terpenes are present in the scent: in particular, limonene (0.22%) and β-cadinene (3.62%) were found to be minor components. Furthermore, some aldehydes could characterize the attractive properties of the scent, i.e., 2-benzylideneoctanal (2.90%), 3,5-di-t-butyl-4-hydroxybenzaldehyde (2.09%), and 5,9,13-trimethyl-4,8,12-tetradecenal (4.59%) have been determined.

It is possible to characterize the scent of every component found in SPME analysis. Obviously, the odor that is possible to find in the classifications usually present in the literature is the one perceived by man and has nothing to do with the odor and the attractive capacity that a compound can have towards an insect. In Table 1, it is possible to recognize the presence of several alkanes such as tridecane, tetradecane, pentadecane, hexadecane, heptadecane, octadecane, nonadecane, eicosane, heinecosane, docosane, and tricosane. These compounds have the typical odor of hydrocarbons, usually associated with that of gasoline. Mesityl oxide shows a musty, mildew, earthy, chemical, cardboard-like scent with nutty, chocolate, and woody nuances. 2,2,4,6,6-Pentamethylhept-3-ene has a strong fruity odor. Limonene shows a citrus, orange, fresh, sweet odor. Linalool has an odor characterized as citrus, orange, lemon, floral, waxy, aldehydic, and woody. β-Terpineol scent is defined as a pungent, earthy, woody odor. Decanal shows a sweet, aldehydic, orange, waxy, citrus odor. We did not find information on the odor 2-dodecene, while bornyl acetate has a woody, camphoreous, mentholic, cedar, woody, spicy odor. The scent of α-terpinyl acetate is characterized by a herbal, bergamot, lavender, lime, and citrus odor, while that of methyleugenol shows a spicy, earthy odor. Dodecanal has a soapy, aldehydic, citrus, green floral odor. We have no data on 1-methoxydodecane and 2,6-di-t-butylbenzoquinone, while the scent of geranylacetone has been characterized as fresh, green, fruity, waxy, rose, woody magnolia, and tropical. β-Cadinene has a green, woody odor, while that of elemicin has been described as spicy and floral. Tetradecanal shows a fatty, waxy, amber, incense dry, citrus peel, and musk odor. Pristane is an odorless compound such as palmitic acid. On the other hand, 2-(dodecyloxy)-ethanol has spice and floral scents such as jasmone and gardenia. Benzyl benzoate scent is due to sweet balsamic oily herbal odor. No data are available on 3,5-di-t-butyl-4-hydroxybenzaldehyde, 5,9,13-trimethyl-4,8,12-tetradecanal, and 7,9-di-t-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione. Hexadecanal scent is reported as cardboard odor. Tricosene is reported to have a fatty wax odor.

Considering the possible role that the determined compounds could have in the pollination processes, E. microphylla showed the presence of limonene, while other components were 2,4,4,6,6,8,8-heptamethyl-1-nonene, pentadecane, and heptadecane. Limonene is rarely involved in pollination [52]. As described above, E. microphylla is an autogamous species and does not need to attract pollinator insects. However, alkanes and alkenes could be involved in residual sexual deception [52]. On the contrary, as reported above, E. palustris is an allogamous species, and the scent showed the presence of pentadecane, 2,4,4,6,6,8,8-heptamethyl-1-nonene, and heptadecane, compounds probably involved, as in E. microphylla, in a sexual deception strategy, where the compounds mimic the sexual pheromones of the insect [52]. The scent N. nidus avis showed kaur-16-ene, heinecosane, tetradecane, pentadecane, hexadecane, heptadecane, and 5,9,13-trimethyl-4,8,12-tetradecenal. As reported above, it is an autogamous species. The role of kaur-16-ene is not evident because it was not involved in the scent of other orchids, while the hydrocarbon mixture can be involved in a sexual deception strategy [52]. The scent of N. ovata is due to pentadecane, hexadecane, and heptadecane. As described in the introduction section, N. ovata is an allogamous species that must attract pollinator insects. The determined compounds are in agreement for a sexual deceptive attraction of the pollinators [52]. L. abortivum showed in the scent 2,4,4,6,6,8,8-heptamethyl-1-nonene, pentadecane, hexadecane, heptadecane, and 2-(dodecyloxy)-ethanol. As reported in the introduction, the species can be allogamous or autogamous depending on the external conditions. The presence of alkanes and alkenes in the scent is in agreement with the sexual deceptive behavior of the species.

4. Conclusions

The above reported data represent the first determination of the scent of Epipactis microphylla, Epipactis palustris, Neottia nidus avis, Neottia ovata, and Limodorum abortivum. The aim of this work is to give significant help in the determination of the components relevant for their attractive properties in order to facilitate the pollination of the species. As reported above, most of the species studied are allogamous or self-pollinating. In the first case, they have to attract the pollinator insects by simulating sexual signals emitted by the female or miming the food aroma. Alternatively, the orchid can mimic the shape and the odor of a rewarding plant. To understand the actual attractive processes, it is important to know the composition of the scent. The scent composition of the species studied was not known. This is the first contribution in this field in order to eliminate this information deficit.