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Citation: Mecca, M.; Racioppi, R.;
Romano, V.A.; Viggiani, L.; Lorenz,
R.; D’Auria, M. Volatile Organic
Compounds in Dactylorhiza Species.
Compounds 2022,2, 121–130. https://
doi.org/10.3390/compounds2020009
Academic Editor: Juan Mejuto
Received: 21 February 2022
Accepted: 29 March 2022
Published: 12 April 2022
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Communication
Volatile Organic Compounds in Dactylorhiza Species
Marisabel Mecca 1, Rocco Racioppi 1, Vito Antonio Romano 1, Licia Viggiani 1, Richard Lorenz 2
and Maurizio D’Auria 1, *
1Dipartimento di Scienze, Universitàdella Basilicata, V.le dell’Ateneo Lucano 10, 85100 Potenza, Italy;
marisabelmecca@libero.it (M.M.); rocco.racioppi@unibas.it (R.R.); vitoantonio_romano@libero.it (V.A.R.);
licia.viggiani@unibas.it (L.V.)
2AHO Baden-Württemberg, 64686 Lautertal, Germany; lorenz@orchids.de
*Correspondence: maurizio.dauria@unibas.it; Tel.: +39-0971-205-480
Abstract: HS-SPME-GC–MS analysis of the scent of Dactylorhiza viridis revealed the presence of ver-
benone (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%). Anal-
ysis 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-epi-aristolochene
(10.18%).
Keywords:
Dactylorhiza; volatile organic compounds; solid phase microextraction;
gas chromatography
;
mass spectrometry
1. Introduction
The taxonomy of the genus Dactylorhiza Necker ex Nevski is one of the most studied
in the Orchidaceae family [
1
–
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
–
16
], while others [
17
–
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
–
25
] and molecular genetics
studies [10,26–30]
.
The scents emitted by five species of Dactylorhiza present in Southern Italy, four present in
Basilicata, Dactylorhiza viridis (L.) R. M. Bateman, Pridgeon, and M. W. Chase (
Figure 1a
),
Dactylorhiza romana (Sebastiani) Soó(Figure 1b), Dactylorhiza sambucina (L.) Soó(Figure 2a),
and one present in Campania, Dactylorhiza saccifera (Brongn.) Soó(Figure 2b), and Dacty-
lorhiza incarnata (L.) Soó(Figure 2c) were analyzed.
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,
Compounds 2022,2, 121–130. https://doi.org/10.3390/compounds2020009 https://www.mdpi.com/journal/compounds
Compounds 2022,2122
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
–
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.
Figure 1.
(
a
): Dactylorhiza viridis, Moliterno (Pz), 23 May 2018; (
b
): Dactylorhiza romana, Rifreddo (Pz),
10 April 2017. Photos of V. A. Romano.
The color, smell, size, and shape of flowers are key signals that attract pollinators in
search of rewards [
34
–
36
]. Floral color is often included in studies on the evolutionary
models of pollination systems while floral scents, whose characterization requires sophis-
ticated and complex tools, have been little studied, even if they are often considered the
main attraction for pollinators [
37
–
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 [41–47].
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 grat-
ifying 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
–
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
–
60
], just emerged from the soil, without experience
which, after the first attempts at pollination, abandon the plant, moving away in search
Compounds 2022,2123
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.
Figure 2.
(
a
): Dactylorhiza sambucina, Moliterno (Pz), 21 May 2018; (
b
): Dactylorhiza saccifera Abriola
(Pz), 30 June 2017; (
c
): Dactylorhiza incarnata, Mandranello, Padula (Sa), 6 June 2018. Photos of
V. A. Romano.
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-
Compounds 2022,2124
trometry. Until now, the volatile organic compounds constituents of the scent of the orchid
species have been determined on several orchid species [62–69].
2. Materials and Methods
2.1. Plant Material
The sample of D. viridis was collected at Moliterno (Pz), 1100 m a.s.l., on 23 May 2018.
The sample of D. romana was collected at Rifreddo (Pz), 1120 m a.s.l., on 10 April 2017. The
sample of D. sambucina (yellow color) was collected at Moliterno (Pz), 1200 m a.s.l., on 21
May 2018. The sample of D. saccifera was collected at Abriola (Pz), 1200 m a.s.l., on 30 June
2017. The sample of D. incarnata was collected at Mandranello, Padula (Sa), 1200 m a.s.l.,
on 3 June 2018. 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 were inserted.
To avoid contamination, the interior of the bell was isolated from the external environ-
ment 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 1and 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.
2.2. 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-
µ
m 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
µ
m 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.
3. Results and Discussion
The scent of Dactylorhiza orchids has been the object of some studies in the past. Analy-
sis of D. sambucina showed the presence of limonene,
β
-myrcene,
α
-pinene,
α
-bergamotene,
β
-bisabolene, caryophyllene, and
β
-selinene [
70
]. Dichloromethane extracts of flowers of
D. incarnata showed the presence of 4-hydroxybenzaldehyde (16.88%), 4-hydrozybenzyl
Compounds 2022,2125
alcohol (30.60%), and methyl 4-hydroxyphenylacetate (34.37%) [
71
]. Pentane, diethyl ether
extraction of flowers of D. incarnata showed the presence of nonanal, 9-(Z)-heptacosene,
9-(Z)-nonacosene, tricosane, pentacosane, and heptacosane [
72
] The analysis of the scent of
D. romana (red flower), obtained by head space analysis of the scent absorbed on Porapak Q,
showed the presence of several components, including nonanal (9.19%), sabinene (10.89%),
(E)-ocimene (15.63%), and linalool (7.99%) [
31
]. Such different results, obtained by using
different analytical techniques, allowed us to analyze the scent of these species by using
SPME (solid phase microextraction) technique. The results are reported in Table 1.
The never studied before D. viridis contains, as its main components, verbenone
(28.86%) and caryophyllene (25.67%), while other compounds present in relevant amounts
are β-terpineol (9.48%) and δ-cadinene (6.94%) (Table 1and Figure 3).
Figure 3. Main components of the scent of D. viridis.
In the scent of D. romana several compounds were found:
β
-ocimene (18.69%, found
also in [
31
]), pentadecane (18.40%),
α
-farnesene (14.65%), and isopropyl 14-methylpentadec-
anoate (14.32%) (Table 1). Previous works published on the aroma of D. incarnata found
phenolic compounds, in one case, while long chain alkanes and alkenes, were found in
another case [
71
,
72
]. SPME analysis of the scent detected the presence of a mixture of
hydrocarbons, but with lower molecular weight than those found in [
72
]. Tetradecane
(11.07%), pentadecane (28.40%), hexadecane (19.53%), and heptadecane (17.33%) were the
main components, together with α-cubenene (11.48%) (Table 1).
D. saccifera contained, as a main component, caryophyllene (17.38%), while other
significant compounds were hydrocarbons, pentadecane (6.43%), hexadecane (6.13%), and
heptadecane (5.08%) (Table 1). Finally, the aroma components found in D. sambucina
were caryophyllene (12.90%, found also in [
70
]),
β
-sesquiphellandrene (32.16%, Figure 4),
4,5-di-epi-aristolochene (10.18%, Figure 4) (Table 1).
Figure 4. Main components of the scent of D. sambucina.
Compounds 2022,2126
Table 1. SPME-GC–MS analysis of Dactylorhiza species.
Compound r.t. (a) (min.) KI (b) D. viridis D. romana D. incarnata D. saccifera D. sambucina
Area % ±0.03
Mesityl oxide 4.94 782 2.03
α-Pinene 7.78 933 0.20 0.30
Sabinene 8.77 972 2.27 2.44
β-Pinene 8.86 979 0.79
β-Myrcene 9.12 989 2.42
2,2,4,6,6-pentamethyl-3-heptene 9.29 1018 0.60
Limonene 9.79 1028 1.88
Eucalyptol 10.00 1032 2.65
β-Ocimene 10.33 1044 18.69
β-Terpineol 10.74 1085 9.48
Linalool 11.22 1100 0.85
Lilac aldehyde A 12.09 1145 0.20
Lilac aldehyde B 12.25 1154 0.90
Dodecane 13.08 1200 0.45
α-Terpineol 13.16 1209 2.07
Lilac alcohol A 13.52 1221 2.08
Verbenone 13.55 1223 28.86
Lilac alcohol B 13.84 1235 4.00
Citral 14.55 1265 4.21
Tridecane 14.88 1300 2.88 0.85
2,4,4,6,6,8,8-Heptamethyl-2-nonene 16.02 1343 1.48
α-Cubebene 16.42 1360 11.48 0.34
Tetradecane 16.64 1400 11.07 3.90 2.13
β-Elemene 16.69 1403
Caryophyllene 17.22 1420 25.67 17.38 12.90
Methyl 2-phenylcyclopropanecarboxylate 17.31 1435 0.90
cis-α-Bergamotene 17.38 1440 7.61
β-Farnesene 17.48 1458 0.68
Geranylacetone 17.61 1468 6.62
Humulene 17.76 1473 0.42
4,5-Di-epi-aristolochene 18.23 1485 10.18
Compounds 2022,2127
Table 1. Cont.
Compound r.t. (a) (min.) KI (b) D. viridis D. romana D. incarnata D. saccifera D. sambucina
Area % ±0.03
Pentadecane 18.25 1500 2.09 18.40 28.40 6.43
β-Selinene 18.40 1511 1.28
α-Farnesene 18.45 1518 14.65 3.30
β-Bisabolene 18.50 1523 2.64
δ-Cadinene 18.78 1530 6.94
7-Hexadecenal 18.64 1555 1.00
β-Sesquiphellandrene 18.76 1557 32.16
Elemicin 19.07 1566 3.30
Hexadecane 19.71 1600 19.53 6.13 1.06
Methyl dihydrojasmonate 20.32 1650 2.55 1.23
Cyclotetradecane 20.47 1673 1.58
Heptadecane 21.14 1700 17.33 5.08 0.64
3,5-Di-t-butyl-4-hydroxybenzaldehyde 22.13 1772 4.38
Octadecane 22.48 1800 8.24 3.40 0.25
Phytone 23.14 1848 0.58
Nonadecane 23.70 1900 2.93
Isopropyl 14-methylpentadecanoate 25.35 1915 14.32 1.94
7,9-Di-t-butyl-1-oxaspiro[4.5]deca-6,9-
diene-2,8-dione 24.11 1929 1.88
Eicosane 24.93 2000 1.83
13-epi-Manoyl oxide 25.49 2015 3.42
Docosane 27.22 2200 1.93
Tricosane 28.31 2300 2.28
Bis(1-phenylethyl)phenol 30.98 2426 2.08
(a) R.T. retention time; (b) KI Kovats index.
Compounds 2022,2128
4. 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
–
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.
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, V.A.R. and M.D.; 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.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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