Two new species of Inocybe from Mediterranean Cistaceae heathlands.

This study explored a heathland region in Portugal, and through morphology, biogeography, and multilocus phylogeny, two new species of Inocybaceae are described. The first species, Inocybe iberilepora, belongs to "I. flocculosa group," whereas the second species, Inocybe phaeosquamosa, belongs to a relatively isolated and understudied clade, distantly related to I. furfurea and allies. Both species are tied to a west Mediterranean distribution and ecology, associating with the local Cistaceae ecosystems. By characterizing these new species, our research contributes to the understanding of European Funga and enriches the knowledge of the genus Inocybe on a global scale.

The Wound-Healing Potential of the Endemic Plant Helianthemum canum (L.) Baumg: Preclinical Studies Supported with Phytochemical Profiling.

The study's objective is to clarify the probable mechanisms underlying the wound-healing properties of Helianthemum canum L. (Cistaceae), a traditional anti-inflammatory and wound-healing medicine. LC/MS-MS was used to perform phytochemical analyses on a 70 % methanol extract of the plant's aerial parts. In vivo, linear incision and circular excision models were used to evaluate the wound healing activity. For anti-inflammatory effect, in vivo acetic acid capillary permeability assay and in vitro Interleukin 1, Interleukin 6, and Interferon É£ levels in LPS-induced FR skin fibroblast cell line were also evaluated. The extract significantly improved wound healing in experimental models, with tensile strength values of 27.8 % and a contraction value of 35.09 %. Histopathological examinations, hydroxyproline estimation, hyaluronidase, collagenase, and elastase enzyme inhibitory assays confirmed wound healing potential. Inflammatory cytokines were significantly inhibited in the LPS-induced FR cell line, with the highest effect seen on IL-6 (34.5±2.12 pg/mL). This study offered the first concrete proof that H. canum can be used to treat wounds by suggesting that the myricetin and quinic acid content identified by LCMS-MS analysis may be accountable for the effect of H. canum on wound contraction and hydroxyproline production.

Strong conservatism of floral morphology during the rapid diversification of the genus Helianthemum.

PREMISE: Divergence of floral morphology and breeding systems are often expected to be linked to angiosperm diversification and environmental niche divergence. However, available evidence for such relationships is not generalizable due to different taxonomic, geographical and time scales. The Palearctic genus Helianthemum shows the highest diversity of the family Cistaceae in terms of breeding systems, floral traits, and environmental conditions as a result of three recent evolutionary radiations since the Late Miocene. Here, we investigated the tempo and mode of evolution of floral morphology in the genus and its link with species diversification and environmental niche divergence. METHODS: We quantified 18 floral traits from 83 taxa and applied phylogenetic comparative methods using a robust phylogenetic framework based on genotyping-by-sequencing data. RESULTS: We found three different floral morphologies, putatively related to three different breeding systems: type I, characterized by small flowers without herkogamy and low pollen to ovule ratio; type II, represented by large flowers with approach herkogamy and intermediate pollen to ovule ratio; and type III, featured by small flowers with reverse herkogamy and the highest pollen to ovule ratio. Each morphology has been highly conserved across each radiation and has evolved independently of species diversification and ecological niche divergence. CONCLUSIONS: The combined results of trait, niche, and species diversification ultimately recovered a pattern of potentially non-adaptive radiations in Helianthemum and highlight the idea that evolutionary radiations can be decoupled from floral morphology evolution even in lineages that diversified in heterogeneous environments as the Mediterranean Basin.

Phytochemistry and Antihyperglycemic Potential of Cistus salviifolius L., Cistaceae.

Cistus salviifolius has been previously reported as a traditional remedy for hyperglycemia. However, the plant has been scarcely investigated from scientific point of view. Thus, the aim was to examine the chemical composition and to evaluate its antioxidant and antihyperglycemic potential in vitro. Aqueous and ethanolic extracts were evaluated for total phenolic, tannin, and flavonoid content using spectrophotometric methods. Detailed chemical characterization was performed by high-performance liquid chromatography (HPLC-DAD). The volatile organic compounds (VOCs) profile was assessed by gas chromatography technique. The potential in diabetes treatment was evaluated through tests of free radicals neutralization, inhibition of lipid peroxidation process, and test of ferric ion reduction; activity in tests of inhibition of α-amylase, α-glucosidase and dipeptidyl peptidase-4 was also evaluated. High content of phenolics (majority being tannins) was detected; detailed HPLC analysis revealed high content of gallic acid, followed by rutin, chlorogenic and caffeic acids. The VOCs analysis determined sesquiterpene hydrocarbons and oxygenated sesquiterpenes as the main groups of compounds. The assays classified extracts as potent neutralizers of 2,2-diphenyl-1-picrylhydrazil and nitroso radicals formation and potent inhibitors of α-amylase and α-glucosidase. In conclusion, Cistus salviifolius represents a rich source of phenolics and essential oil with sesquiterpenes. The established results suggested its promising antioxidant and antihyperglycemic activities.

First report on phytochemical investigation, antioxidant and antidiabetic activities of Helianthemum getulum.

A new flavonoid, 5,7,2',4',5'-pentahydroxyflavone 3-O-ß-D-galactopyranoside (12) and twelve known derivatives: an aryltetralin-lignan (3), seven flavonoids (4-5, 7-10, 13) and four phenolic acids (1-2, 6, 11) have been isolated from the aerial parts of Helianthemum getulum Pomel. (Cistaceae family) an endemic species to the septentrional Sahara that is being studied for the first time. Structure elucidation of the isolated compounds was established by means of spectroscopic methods especially NMR and Mass Spectrometry. In vitro antioxidant (DPPH, ABTS, GOR and CUPRAC assays) and antidiabetic (micro-dilution method) activities of the crude extract, fractions and isolated compounds were performed. The new flavonol (12) and Compounds (2, 3, 7, 9) were found to be the most active, some of them exhibiting better activity than the antioxidant standards. Compounds 7, 9 and 3 showed higher α-glucosidase inhibitory activity compared to standard acarbose (IC50= 2.70 ± 0.03 µM, 3.09 ± 0.03 µM, 37.28 ± 1.20 µM and 275.43 ± 1.59 µM, respectively).

Phyto-Extract-Mediated Synthesis of Silver Nanoparticles Using Aqueous Extract of Sanvitalia procumbens, and Characterization, Optimization and Photocatalytic Degradation of Azo Dyes Orange G and Direct Blue-15.

Green synthesis of silver nanoparticles (AgNPs) employing an aqueous plant extract has emerged as a viable eco-friendly method. The aim of the study was to synthesize AgNPs by using plant extract of Sanvitalia procumbens (creeping zinnia) in which the phytochemicals present in plant extract act as a stabilizing and reducing agent. For the stability of the synthesized AgNPs, different parameters like AgNO3 concentration, volume ratios of AgNO3, temperature, pH, and contact time were studied. Further, AgNPs were characterized by UV-visible spectroscopy, FT-IR (Fourier Transform Infrared Spectroscopy), XRD (X-ray Diffraction), SEM (Scanning Electron Microscopy), and EDX (Energy Dispersive X-ray Spectrometer) analysis. FT-IR analysis showed that the plant extract contained essential functional groups like O-H stretching of carboxylic acid, N-H stretching of secondary amides, and C-N stretching of aromatic amines, and C-O indicates the vibration of alcohol, ester, and carboxylic acid that facilitated in the green synthesis of AgNPs. The crystalline nature of synthesized AgNPs was confirmed by XRD, while the elemental composition of AgNPs was detected by energy dispersive X-ray analysis (EDX). SEM studies showed the mean particle diameter of silver nanoparticles. The synthesized AgNPs were used for photocatalytic degradation of Orange G and Direct blue-15 (OG and DB-15), which were analyzed by UV-visible spectroscopy. Maximum degradation percentage of OG and DB-15 azo dyes was observed, without any significant silver leaching, thereby signifying notable photocatalytic properties of AgNPs.

Biogeographic history and environmental niche evolution in the palearctic genus Helianthemum (Cistaceae).

The biogeographic history and the degree of environmental niche conservatism provide essential clues to decipher the underlying macroevolutionary processes of species diversification and to understand contemporary patterns of biodiversity. The genus Helianthemum constitutes an excellent case study to investigate the impact of the geo-climatic changes and the environmental niche shifts on the origins of plant species diversity in the Mediterranean hotspot. It is a palearctic species-rich lineage with c. 140 species and subspecies mostly belonging to three distinct evolutionary radiations, almost confined to the Mediterranean region and occurring across varied environmental conditions. In this work, we studied the ample and rapid diversification of the genus Helianthemum across its whole distribution range by performing phylogenetic reconstructions of ancestral ranges and environmental niche evolution. We observed a striking synchrony of biogeographic movements with niche shifts between the three major clades of the genus Helianthemum, likely related to the geo-climatic events occurred in the Mediterranean Basin since the Upper Miocene. In particular, Late Miocene and Early Pliocene were dominated by episodes of range expansions, the Late Pliocene by range contraction and vicariance events, and Pleistocene by most intense environmental niche shifts and in-situ diversification. Our study also provides evidence for four main environmental niches in Helianthemum (i.e., Mediterranean, subdesert, humid-montane and subtropical-insular) and a tendency toward environmental niche conservatism within different subclades, with few niche shifts mostly occurring from Mediterranean ancestors. The relative longer time spent in Mediterranean areas by the ancestors of Helianthemum suggests that the larger species diversity observed in the Mediterranean (i.e. Northern Africa and Southern Europe) may have been generated by a time-for-speciation effect reinforced by environmental niche conservatism. Overall, our work highlights the role of the Mediterranean Basin as a 'cradle of diversity' and an 'evolutionary hub', facilitating the environmental transitions and determining the building up of a global plant biodiversity hotspot.

Screening of Plant Pollen Sources, Polyphenolic Compounds, Fatty Acids and Antioxidant/Antimicrobial Activity from Bee Pollen.

In this study, the botanical origin, total flavonoid and phenolic content, antioxidant activity, phenolic profile and fatty acid composition of mixed bee pollen loads collected in Bayburt, Turkey, were determined. In addition to these assays, antibacterial activity of bee-collected pollen extract (BCPE) against a variety of food-borne pathogenic bacteria was determined in vitro. Pollen loads were classified into five botanical families based on their color: Asteraceae, Fabaceae, Campanulaceae, Cistaceae and Rosaceae. Total flavonoid, total phenolic, CUPRAC and CERAC concentrations were 173.52 mg GAE/g, 79.21 mg QE/g, 85.59 mg Trolox/g and 118.13 mg Trolox/g, respectively. Twenty-three phenolic compounds were scanned in bee pollen extract by LC-MS/MS, with rutin being the most abundant. Cis-4,7,10,13,16,19 docosahexaenoic acid was the predominant fatty acid, followed by cis-11-eicosenoic acid, palmitic acid, and alfa linolenic acid. In addition, the agar well diffusion (AWD) and micro-broth dilution methods were used to determine of the antibacterial activity of the BCPE sample. MIC values were observed to vary between 2.5-5 mg/mL for Gram-positive bacteria and 5-10 mg/mL for Gram-negative bacteria. These findings indicate that bee pollen could be a potential source of antioxidants and antimicrobials.

Desert truffle genomes reveal their reproductive modes and new insights into plant-fungal interaction and ectendomycorrhizal lifestyle.

Desert truffles are edible hypogeous fungi forming ectendomycorrhizal symbiosis with plants of Cistaceae family. Knowledge about the reproductive modes of these fungi and the molecular mechanisms driving the ectendomycorrhizal interaction is lacking. Genomes of the highly appreciated edible desert truffles Terfezia claveryi Chatin and Tirmania nivea Trappe have been sequenced and compared with other Pezizomycetes. Transcriptomes of T. claveryi × Helianthemum almeriense mycorrhiza from well-watered and drought-stressed plants, when intracellular colonizations is promoted, were investigated. We have identified the fungal genes related to sexual reproduction in desert truffles and desert-truffles-specific genomic and secretomic features with respect to other Pezizomycetes, such as the expansion of a large set of gene families with unknown Pfam domains and a number of species or desert-truffle-specific small secreted proteins differentially regulated in symbiosis. A core set of plant genes, including carbohydrate, lipid-metabolism, and defence-related genes, differentially expressed in mycorrhiza under both conditions was found. Our results highlight the singularities of desert truffles with respect to other mycorrhizal fungi while providing a first glimpse on plant and fungal determinants involved in ecto to endo symbiotic switch that occurs in desert truffle under dry conditions.

Results of a randomized controlled phase III trial: efficacy of polyphenol-containing cystus® tea mouthwash solution for the reduction of mucositis in head and neck cancer patients undergoing external beam radiotherapy.

PURPOSE: To determine the effect of Cystus® tea (Naturprodukte Dr. Pandalis GmbH & Co. KG) as mouthwash compared to sage tea on oral mucositis in patients undergoing radio(chemo)therapy for head and neck cancer. METHODS: In this randomized, prospective phase III study, 60 head and neck cancer patients with primary or postoperative radio(chemo)therapy were included between 04/2012 and 06/2014. They received either sage or Cystus® tea for daily mouthwash under therapy. Mucositis was scored twice a week following the Radiation Therapy Oncology Group and the European Organization for Research and Treatment Cancer (RTOG/EORTC) scoring system. Dental parameters were also recorded. Statistical evaluation of the primary endpoint was performed using t­test and log rank test. RESULTS: Data from 57 patients could be evaluated. Patient characteristics showed no significant difference between the two groups (n = 27 sage; n = 30 Cystus®). A total of 55 patients received the prescribed dose (60-66 Gy postoperative; 70-76.8 Gy primary). Mucositis grade 3 was observed in 23 patients (n = 11 sage; n = 12 Cystus®) and occurred between day 16 and 50 after start of therapy. There was no significant difference between the two groups in latency (p = 0.75) and frequency (p = 0.85) of the occurrence of mucositis grade 3. The self-assessment of the oral mucosa and the tolerability of the tea also showed no significant differences. Occurrence of dental pathologies appeared to increase over time after radiotherapy. CONCLUSION: Cystus® and sage tea have a similar effect on the occurrence of radiation-induced mucositis regarding latency and incidence. Cystus® tea mouthwash solution is tolerated well and can be applied in addition to intensive oral care and hygiene along with the application of fluorides.

Reconstruction of the spatio-temporal diversification and ecological niche evolution of Helianthemum (Cistaceae) in the Canary Islands using genotyping-by-sequencing data.

BACKGROUND AND AIMS: Several biogeographical models have been proposed to explain the colonization and diversification patterns of Macaronesian lineages. In this study, we calculated the diversification rates and explored what model best explains the current distribution of the 15 species endemic to the Canary Islands belonging to Helianthemum sect. Helianthemum (Cistaceae). METHODS: We performed robust phylogenetic reconstructions based on genotyping-by-sequencing data and analysed the timing, biogeographical history and ecological niche conservatism of this endemic Canarian clade. KEY RESULTS: Our phylogenetic analyses provided strong support for the monophyly of this clade, and retrieved five lineages not currently restricted to a single island. The pristine colonization event took place in the Pleistocene (~1.82 Ma) via dispersal to Tenerife by a Mediterranean ancestor. CONCLUSIONS: The rapid and abundant diversification (0.75-1.85 species per million years) undergone by this Canarian clade seems the result of complex inter-island dispersal events followed by allopatric speciation driven mostly by niche conservatism, i.e. inter-island dispersal towards niches featuring similar environmental conditions. Nevertheless, significant instances of ecological niche shifts have also been observed in some lineages, making an important contribution to the overall diversification history of this clade.

In vitro Propagation to Conserve the Local Endemic and Endangered Medicinal Plant Helianthemum germanicopolitanum Bornm

Abstract In this study, in vitro propagation and acclimatization of Helianthemum germanicopolitanum Bornm. plant, a local endemic in Çankırı Province (Turkey) with arid and semi-arid lands, and an endangered species taking part among medicinal and aromatic plants were accomplished, which is under-researched. In this study, three basal media [a) Murashige and Skoog b) Gamborg's B5, and c) Nitsch & Nitsch], two gelling agents (agar 7 g/L, and gelrite 2.1 g/L), eight cytokinins and eight auxin doses of plant growth regulators [a) 6-benzyladenin, b) Kinetin-(0, 0.5, 1, and 2 mg/L), c) Indole-3-butyric acid, d) α-napthaleneacetic acid-(0, 0.25, 0.5, and 1 mg/L)] prepared in 64 different combinations with 30 g/L sucrose was added to the basal media and adjusted to pH 5.7 for in vitro propagation of H. germanicopolitanum. During in vitro propagation of the plant, external and internal infections were frequently encountered and this was solved by the developed protocol. The best shoot growth (1.141 cm) and shoot length (0.572 cm) were obtained in the Gamborg's B5 medium in combination with Kinetin (0.5 mg/L)+Indole-3-butyric acid (0.5 mg/L)+gelrite. The maximum number of shoots (19.50) and the best multiplication rate (94%) were obtained in the media containing benzyladenin (1 mg/L)+Indole-3-butyric acid (0.5 mg/L) plant growth regulator in Murashige and Skoog medium solidified with agar. At the rooting stage, the maximum number of roots (30) was reached in the Murashige and Skoog medium containing gelrite and the best rooting rate (92%) with agar. A hundred plants representing the best shoot and root growth were taken to acclimatization stage, and 32 of these plants adapted to external conditions.

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Helianthemum songaricum is a second-class protected plant in China, threatened by decreased population size and fragmentation of distribution area. It is a new record species in Ningxia. We analyzed population structure, spatial distribution, and relationship of different age classes of H. songaricum living in sandy and gravel habitats. The results showed that majority of H. songaricum individuals were belonged to age-class Ⅲ. The age structure of the population was inverted triangle. The population was at a declining stage. The spatial pattern and spatial association between different age class of H. songaricum were greatly affected by habitat condition, with strong dependence on spatial scale. Compared with sandy habitat, H. songaricum population in gravel habitat tended to be clustered distribution, with a more obvious positive correlation among different age classes. The difference between two habitats was related to environmental condition and plant community structure. H. songaricum from age-class Ⅰ and Ⅱ had mutually dependent ecological relationships.

Agrobacterium tumefaciens-Mediated Genetic Transformation of the Ect-endomycorrhizal Fungus Terfezia boudieri.

Mycorrhizal desert truffles such as Terfezia boudieri, Tirmania nivea, and Terfezia claveryi, form mycorrhizal associations with plants of the Cistaceae family. These valued truffles are still collected from the wild and not cultivated under intensive farming due to the lack of basic knowledge about their biology at all levels. Recently, several genomes of desert truffles have been decoded, enabling researchers to attempt genetic manipulations to enable cultivation. To execute such manipulations, the development of molecular tools for genes transformation into truffles is needed. We developed an Agrobacterium tumefaciens-mediated genetic transformation system in T. boudieri. This system was optimized for the developmental stage of the mycelia explants, bacterial optical density, infection and co-cultivation durations, and concentrations of the selection antibiotics. The pFPL-Rh plasmid harboring hph gene conferring hygromycin resistance as a selection marker and the red fluorescent protein gene were used as visual reporters. The optimal conditions were incubation with 200 µM of acetosyringone, attaining a bacterial optical density of 0.3 OD600; transfer time of 45 min; and co-cultivation for 3 days. This is the first report on a transformation system for T. boudieri, and the proposed protocol can be adapted for the transformation of other important desert truffles as well as ectomycorrhizal species.

Elevated atmospheric CO2 modifies responses to water-stress and flowering of Mediterranean desert truffle mycorrhizal shrubs.

Predicted increases in atmospheric concentration of carbon dioxide (CO2 ) coupled with increased temperatures and drought are expected to strongly influence the development of most of the plant species in the world, especially in areas with high risk of desertification like the Mediterranean basin. Helianthemum almeriense is an ecologically important Mediterranean shrub with an added interest because it serves as the host for the Terfezia claveryi mycorrhizal fungus, which is a desert truffle with increasingly commercial interest. Although both plant and fungi are known to be well adapted to dry conditions, it is still uncertain how the increase in atmospheric CO2 will influence them. In this article we have addressed the physiological responses of H. almeriense × T. claveryi mycorrhizal plants to increases in atmospheric CO2 coupled with drought and high vapor pressure deficit. This work reports one of the few estimations of mesophyll conductance in a drought deciduous Mediterranean shrub and evaluates its role in photosynthesis limitation. High atmospheric CO2 concentrations help desert truffle mycorrhizal plants to cope with the adverse effects of progressive drought during Mediterranean springs by improving carbon net assimilation, intrinsic water use efficiency and dispersal of the species through increased flowering events.

Phytochemical and Biological Investigation of Helianthemum nummularium, a High-Altitude Growing Alpine Plant Overrepresented in Ungulates Diets.

Helianthemum nummularium is a European shrub growing at high altitude where it copes with a high level of stress. It was found to be overexpressed in ungulates diets compared to more abundant surrounding plants. These elements combined with the fact that H. nummularium from the Alps has never been investigated prompted us to study the phytochemical composition of its aerial parts. The analysis of the polar extract allowed for the isolation of eight compounds: p-hydroxybenzoic acid, tiliroside, kaempferol, astragalin, quercetin, plantainoside B, quercetin-3-O-glucoside, and quercetin-3-O-glucuronide. We investigated the effect of the polar extract and isolated compounds on nuclear factor erythroid 2-related factor 2 transcription factor, which regulates the expression of a wide variety of cytoprotective genes. We found that the ethanolic extract activates the expression of nuclear factor erythroid 2-related factor 2 in a dose-dependent manner, whereas the pure compounds were much less active. The activation of the nuclear factor erythroid 2-related factor 2 pathway by the plant extract could pave the way for studies to promote healthy aging through protection of cells against oxidative stress. Moreover, the isolated compounds could be investigated alone or in combination in the perspective of making the link between the ungulate's preference for this plant and possible use of it for self-medication.

Production of Encecalin in Cell Cultures and Hairy Roots of Helianthella quinquenervis (Hook.) A. Gray.

Plant cell and organ cultures of Helianthella quinquenervis, a medicinal plant whose roots are used by the Tarahumara Indians of Chihuahua, Mexico, to relieve several ailments, were established to identify and quantify some chromenes with biological activity, such as encecalin, and to evaluate their potential for biotechnological production. Gas chromatography-mass spectrometry (GC-MS) analysis corroborated the presence of quantifiable amounts of encecalin in H. quinquenervis cell cultures (callus and cell suspensions). In addition, hairy roots were obtained through three transformation protocols (prick, 45-s sonication and co-culture), using wild type Agrobacterium rhizogenes A4. After three months, cocultivation achieved the highest percentage of transformation (66%), and a comparable production (FW) of encecalin (110 µg/g) than the sonication assay (120 µg/g), both giving far higher yields than the prick assay (19 µg/g). Stable integration of rolC and aux1 genes in the transformed roots was confirmed by polymerase chain reaction (PCR). Hairy roots from cocultivation (six months-old) accumulated as much as 1086 µg/g (FW) of encecalin, over three times higher than the cell suspension cultures. The production of encecalin varied with growth kinetics, being higher at the stationary phase. This is the first report of encecalin production in hairy roots of H. quinquenervis, demonstrating the potential for a future biotechnological production of chromenes.

Spring stomatal response to vapor pressure deficit as a marker for desert truffle fruiting.

The cultivation of desert truffle Terfezia claveryi using Helianthemum almeriense as a host plant has recently become a solid alternative crop in the Mediterranean region due to its adaptation to arid and semiarid ecosystems, which are expected to increase during the following years because of climate change. However, management models are still being developed in order to improve and stabilize the production, which varies greatly from one year to another. According to gatherers and farmers, one of the key factors for desert truffle production is the plant phenology in spring, which, in turn, depends on environmental conditions. In this manuscript, we have characterized the physiological, morphological, and molecular responses of the mycorrhizal plants in spring, coinciding with the fructification period of the plant and fungal species. Thanks to this characterization, a sigmoidal relationship between stomatal conductance and vapor pressure deficit (VPD) was found, which can be used as a marker of plant phenological switch. In order to confirm that this phenology status is related to desert truffle fructification, this marker has been successfully correlated to total truffle production. The results of this manuscript suppose a big step forward that will help to develop management models for the desert truffle crop.

Differentiation between Cistus L. (Sub-) Species (Cistaceae) Using NMR Metabolic Fingerprinting.

The genus Cistus is taxonomically complex, as taxonomic classification of individual species based on morphological criteria is often difficult and ambiguous. However, specific species contain valuable natural products, especially terpenoids and polyphenols, which exert various biological effects and might therefore be used for treatment of a broad array of disorders. Hence, a fast and reliable method for clear identification of different Cistus (sub-) species is required. Approaches for analysis of secondary metabolite profiles, e.g., with NMR, might remedy the challenging classification of Cistus (sub-) species and help to identify specific markers for differentiation between them. In the present study, 678 samples from wild-growing Cistus populations, including 7 species and 6 subspecies/varieties thereof, were collected in 3 years from populations in 11 countries all over the Mediterranean basin. Samples were extracted with buffered aqueous methanol and analysed with NMR. From the resulting 1D-1H-NOESY and J-Res profile spectra, marker signals or spectral regions for the individual (sub-) species were identified with multivariate statistical tools. By examining the NMR profiles of these extracts, we were able to identify discriminators and specific markers for the investigated Cistus (sub-) species. Various influencing factors, like (sub-) species, wild harvestings of different populations from several countries, numerous collection sites, different years, and cultivation in greenhouses have been considered in this work. As the here identified markers are independent from these influencing factors, the results can be considered a robust model and might be used for future differentiation between Cistus (sub-) species.