A volatolomic approach for studying plant diversity : the case of Mediterranean Helichrysum (Asteraceae)

Among the aromatic plants of the Mediterranean area, Helichrysum species (Asteraceae, Gnaphalieae) are well-known for their secondary metabolite content (Morone-Fortunato et al. 2010; Leonardi et al. 2013). Helichrysum extracts are used in cosmetic industry and have potential pharmacological applications for their antioxidant, antimicrobial, and anti-inflammatory activities (Mancini et al. 2011). Investigations upon plant Volatile Organic Compounds (VOCs) represent an important field of research directed at special biological recognition and potential applications (Duradeva et al. 2013). From a systematic point of view, Helichrysum Miller is a large and taxonomically controversial genus, with a worldwide distribution. The circumscription of the species has undergone notable changes in time (Galbany-Casals et al. 2009, 2012) and is still debated. Three sections are currently recognized in the Mediterranean area: sect. Helichrysum, sect. Stoechadina (DC.) Gren. & Godr., and sect. Virginea (DC.) Gren. & Godr. With the aim of evaluating the potential contribution of the volatile fingerprint in solving taxonomic problems, the VOCs of annual leafy shoots of selected Mediterranean Helichrysum populations have been investigated. The research involved several populations of H. italicum (Roth) G. Don, H. litoreum Guss., and H. pseudolitoreum (Fiori) Brullo, all belonging to H. sect. Helichrysum. The study also included populations from Monte Capanne, Elba Island, which present morphological affinity with H. italicum (Roth) G. Don subsp. microphyllum (Willd.) Nyman, a subspecies currently reported for Sardinia, Sicily, Corse, and Baleares (Conti et al., 2005). H. stoechas (L.) Moench, a species belonging to a different section (H. sect. Stoechadina), was also taken into consideration to test the validity of the methodology. Helichrysum plants were collected from natural populations in April-May 2011, and cultivated under standard growing conditions in a cold greenhouse at the Botanical Garden of the University of Florence. The annual leafy shoots were collected in July 2012, in the post-blooming period. Three samplings were performed for each population. HS-SPME/GC-MS (HeadSpace-Solid Phase MicroExtraction/Gas Chromatography-Mass Spectrometry) analysis was applied to determine for the first time the VOC profiles of the Helichrysum populations. The VOC composition analysis revealed the emission of overall 386 different compounds, the most part belonging to terpenes, esters and alcohols. The terpenoidic fraction characterizing the vegetative bouquets revealed the predominance of sesquiterpene hydrocarbons in all the populations and the presence of monoterpene hydrocarbons, oxygenated monoterpenes and oxygenated sesquiterpenes invariably detected in descending order of amount. The results of the statistical analyses on both the presence/absence (PA) and relative quantitative data of VOCs allowed to group the populations on the basis of their volatile fingerprints, evidencing remarkable differences and affinities. The results of the ISA (Indicator Species Analysis) allowed to identify the volatile compounds that discriminate the single provenances. All of the Helichrysum populations, with one exception, are related to the presence of exclusive volatile compounds, with esters or ester derivatives being the most represented. H. stoechas is characterized by the occurrence of terpenes as indicator compounds. The results of CCA (Canonical Correspondence Analysis) on PA data and of RDA (Redundancy Analysis) on quantitative response data have revealed a degree of consistency within and significant differences across the populations, according to their genetic diversity, geographical origin and taxonomic treatment. Moreover, the results evidenced the potentiality of VOCs, in particular ester compounds, as valuable chemotaxonomic markers in discriminating among taxa. Although the limits of the “5 fold cross-validation” analysis - imposed by both the low number of replicates for each taxon and by the unbalance among such replicates (12 in H. litoreum, 6 in H. italicum subsp. italicum, and 3 in H. italicum subsp. microphyllum, H. pseudolitoreum and H. stoechas), the results indicated a high classification accuracy. In conclusion, the VOC profiles could represent a crucial feature for the systematics of Helichrysum populations in the Mediterranean area, also providing information for a wider exploitation of the secondary metabolite content of this genus.