Headspace Volatile Composition of the Flowers of Caralluma europaea N.E.Br. (Apocynaceae)

The volatile constituents of the flowers of Caralluma europaea (Guss.) N.E.Br (Apocynaceae) from Lampedusa Island were analyzed by a headspace GC method. The analyses allowed the identification and quantification of 41 compounds. The main components were, among the monoterpenoids, terpinolene (23.3%), α-terpinene (19.1%) and linalool (18.4%), whereas, among the carbonylic compounds the major constituents were heptanal (2.0%), octanoic acid (2.4%) and hexanoic acid (1.7%). The presence of a nitrogen containing compound, indole (0.8%) and of a sulphur containing compound, dimethylsulphide (t), noteworthy. The compounds found in the flowers of C. europaea have been compared with data available in the literature as regard to their odor, presence in other sapromyiophilous taxa, possible role as semiochemicals, and presence in decaying organic matter. 89.3% of total constituents have been described in other sapromyiophilous taxa. Some of the compounds are present in several types of decaying organic matter (excrements, decomposing bodies, and spoiled fish, etc). Several volatiles found in C. europaea flowers are used as semiochemicals by Hymenoptera, Coleoptera, Diptera, Lepidoptera and other insects. Sixteen volatiles, accounting for 32.4% of the total constituents, are described as attractants of some Diptera families, with a biology linked to decaying organic matter. Our data thus confirm that C. europaea floral bouquet falls within the sapromyiophilous pollination syndrome.


Introduction
Headspace GC analysis is widely used to study the volatiles composition of flowers and over 1,700 volatile compounds have been identified so far, as reported in a review by Knudsen et al. [1] describing volatiles present in the flowers of 991 taxa of the Angiosperms and a few Gymosperms. The volatile composition of flowers plays a major role, besides other tasks, in attracting insects involved in pollination [2]. Most floral scent are bouquets composed of at least a few, but usually many components. Although the blend is often dominated by one or a few main components, this does not necessarily mean that said component(s) provide the most important signal to the pollinators but instead it is the total scent [2]. Plant species with similar pollinators show similarities, not only in the visual attractants but also in the floral scent composition, regardless of the phylogenetic relatedness of the species.
Caralluma europaea (Guss.) N.E.Br [= Apteranthes europaea (Guss.) Plowes] belongs to the family Apocynaceae, subfamily Asclepiadoideae. In this subfamily the pollination system is phenotypically high specialized, with pollen aggragated in pollinia [3] like Orchidaceae, and almost all the taxa are insect pollinated. However, while the volatile composition of the flowers of Orchidaceae has been investigated by several authors, relatively few studies have been carried out on the Apocynaceae [1]. Recently Jürgens et al. [4,5] analysed the chemical volatile composition of some Asclepiadoideae and discussed their possible role in the biology of pollination. The chemical composition of flowers of the genus Caralluma is little studied, the only available data being those reported by Jürgens et al. [4] for Desmidorchis flava (N.E.Br) Meve & Liede (= Caralluma flava N.E.Br) and Apteranthes joannis (Maire) Plowes (= Caralluma joannis Maire).
In the present paper we present the volatile composition of flowers of C. europaea as determined by headspace analysis and discuss the possible role in its pollination biology. C. europaea, like most Asclepiadoideae, is pollinated by Diptera and falls within the sapromyiophilous syndrome where insects are attracted by the color and odor of the flowers simulating breeding sites or food sources [4].

Results and Discussion
In the flowers of C. europaea we detected 41 compounds (Table 1) belonging to nine different classes. The analyses allowed the identification of monoterpenoids, sesquiterpenoids, alcohols, aldehydes, ketones, acids and derivatives, nitrogen-and sulphur bearing compounds, and phenols. More than two thirds of the identified compounds were isoprenoids. In fact monoterpenoids were the main components, representing 77.0% of the compounds identified, accompanied by sesquiterpenoids (1.7%). Among these compounds, the monoterpene hydrocarbons were the most representative, amounting to 56.7%, with terpinolene (23,3%) and α-terpinene (19.1%) as the main components of this fraction. Linalool (18.4%) was the main oxygen containing monoterpene and represented almost the whole content of these compounds, being the remaining four compounds of this class present in lower amounts (0.2%-0.7%). Phenol, indole and dimethyl sulphide were the only compounds of the benzenoid, nitrogen and sulphur bearing compounds noted, the former two being present in the same amount (0.8%), while the latter was detected only in traces. Acids and derivatives and aldehydes were present in quite similar amounts but the aldehydes are present with a greater number of compounds.
The volatiles found give the floral bouquet of C. europaea sulfur, mushroom, woody, sweet, fish, smoky, rancid, woody, pungent, fecal, cheese and other odors (Table 1 and references therein). In the literature there are several reports about the pollinators of the genus Caralluma indicating that this genus is pollinated by flies [30,31,32,33] but there are few data on the volatiles of the flowers.
Flowers in taxa of the genus Caralluma show decaying organic matter odors, like C. arachnoidea with scents of rotting fruits and are pollinated by small Drosophilidae or Milichiidae, while the floral odor of Desmidorchis flava can be described as reminiscent of decaying urine or pungent and urinous and Coleoptera (Dermestidae) have been recorded as flower visitors, although it has not been determined whether they really act as pollinators [4]. Meve and Heneidak [3] state that C. europaea flowers have an odor of excrement without reporting any chemical analysis. Interstingly, 23 compounds found in C. europaea are also present in at least one fetid Asclepiadoideae [4], while 18 compounds are absent.
The analysis of the volatile composition of C. europaea ( Fig.1) combined with that of the taxa studied by Jürgens et al. [4] shows that twelve taxa share similar volatile composition and that C. europaea has similarity to the volatiles present in Hoodia gordonii, Desmidorchis flava and Orbea semota spp. orientalis.

Figure 1.
Cluster analysis based on a binary presence-absence matrix of the volatile profile of 15 taxa [4] and C. europaea using Euclidean distances between taxa. Unidenntified compounds were omitted from the analysis. By comparing our data with those of the 15 taxa studied by Jürgens [4] it is clear that C. europaea falls within the group of other three species: Hoodia gordonii, Desmidorchis flava and Orbea semota spp. orientalis.
The cluster analysis of latter group (Figure 2) confirms the similarity in volatiles of the four taxa.

Figure 2.
Cluster analysis based on a binary presence-absence matrix of the volatile profile of Hoodia gordonii, Desmidorchis flava and Orbea semota spp. orientalis [4] and C. europaea using Euclidean distances between taxa showing the close relationship of volatiles composition among the taxa.

Experimental
General Flowers of C. europaea were collected in Lampedusa Island (Italy, 35°29'28" and 35°21'39" N -12°30'54" and 12°37'55" E) in April 2009 from plants growing in the "Guitgia" area at an altitude of 20 m a.s.l. Clones of the plants are cultivated at the Botanical Garden of Palermo and a voucher specimen (N° PAL/MS/1119) was deposited in the Herbarium, Orto Botanico, Palermo, Italy. The flowers were removed from the plants with a single stroke of a razor blade, the cut surface was sealed with a drop of metacrylate (Attak ® ) to avoid the release of volatile compounds due to the cutting, placed in 20 mL autosampler vials with cripto caps and stored at -10 °C. The direct headspace analyses were performed after equilibrating the vials in a heated block. Headspace conditions were: equilibration time 35 min at 105 °C, pressurization time 1.0 min and loop fill time 1.0 min. The chemical composition was determined by using a HP 7694E headspace sampler coupled to a gas chromatograph interfaced with a HP 6890 GC SYSTEM flame ionization detector. Components were separated using two fused-silica capillary columns connected in series by press-fit: first column Carbowax EASYSEP connected to the detector, 30 m × 0.53 mm i.d., 1 µm film thickness and the second CP Sil 5CB connected to the injector; 25 m × 0.53 mm i.d., 5 µm film tickness. GLC conditions were: oven temperature 40°C with 8 min initial hold and then two ramps: the first from 40 °C to 150 °C at 2 °C/min and the second from 150 °C to 210 °C at 35 °C/min (6 min). The injector was maintained at 250 °C (splitless mode) and He was used as carrier gas (5 mL/min). Most constituents were identified by comparison of their retention indices (R i ) with either those of the literature [34,35] or with those of authentic compounds available in our laboratories. The retention indices were determined in relation to a homologous series of n-alkanes (C 8 -C 18 ) under the same operating conditions. Further identification was made by comparison of their mass spectra with either those stored in NIST 98 library or with mass spectra from the literature [34,36] and a home-made library. Pure commercial essential oil components used as standards for GC-FID analyses were obtained from Aldrich and Fluka. The comparison of the volatile composition of C. europaea with that of other taxa was performed by cluster analysis based on a binary presence-absence matrix of the volatile profile using Euclidean distances among taxa [37].

Conclusions
The volatile compounds found in the flowers of C. europaea seems to be a very attractive spectrum for the flies involved in pollination of this species. Flies are almost ubiquitous insects, occurring also in unfavourable habitats where other insects may be rare. Enabled by a highly sensitive olfaction system, flies are attracted to odors over long distances. C. europaea grows at the base of others plants or rocks or camouflaged in its environment, and flies may be the most abundant insects and therefore the sapromyiophilous syndrome reflects the adaptation of this species to this environment. Recently Pisciotta et al. [33] found that C. europaea in Lampedusa Island is pollinated by eight species of Diptera belonging to five families: Calliphoridae, Milichiidae, Muscidae (Figure 3), Sarcophagidae, and Trixoscelididae. It is interesting to note that all the Diptera families involved in pollination have a biology linked to decaying organic matters and therefore C. europaea falls within the sapromyiophilous pollination syndrome. According to Dobson [21] a single fly species may visit distinct flowers for different purposes (i.e. food versus oviposition) and therefore pollinate flowers. In particular flies prefer yellow in the presence of sweet scents, which signal food sources, and brownpurple in the presence of odor of excrements, which indicate egg-laying sites. Flowers of C. europaea are brown-purple with yellow stripes, they contain compounds with both sweet odors and compounds found in excrements, and in this way they may mimic both food resources and oviposition sites thus augmenting the spectrum of potential pollinators.