Species boundaries and genetic and phytochemical diversity of Cistus creticus L.

Cistus creticus, Cretan rockrose, is a purple-flowering shrub widespread in the Mediterranean. The essential oil of C. creticus is rich in labdane-type diterpenes, an interesting group of plant compounds that exhibit diverse health-affecting activities. Among them there are significant cell-toxic and tumor-inhibiting properties that could be of use for diverse medicinal applications. Therefore, the interest in C. creticus has increased. However, C. creticus is a complex species that is difficult to determine. High morphological and essential oil variability, the fact that different scientific names (e. g. C. incanus, C. villosus) have been synonymously used and its therewith inconsistent naming in field guides, scientific literature/publications and databases make it difficult to obtain a comprehensive view. Due to the intra-specific variability some botanists have introduced subspecies. Species boundaries and relationship to related species are difficult to define. All these ambiguities hamper the trade of unambiguously designated, high-quality raw material for pharmaceutical production and impede progress in systematic investigations of the medicinal potential of C. creticus. The aim of this project is a comprehensive evaluation of the C. creticus complex by linking data from comparative analyses of morphological, genetic and chemical plant characteristics. Our basic assumptions are: 1) the different scientific plant names that have been synonymously used all refer to the same plant group (species) that can be clearly distinguished from all other Cistus species. 2) The striking intra-specific variability is rather due to continuous variation over the species distribution area than to the presence of distinct subgroups (-species). 3) Labdane diterpenes are not ubiquitously distributed in C. creticus and a characteristic of the more oil-rich plants of the Eastern Mediterranean. To proof these assumptions a high number of plants of different geographical origin will be collected and analyzed with respect to certain morphological, genetic (comparative analysis of composition of certain DNA- segments and DNA sequence changes (mutations)) and chemical (comparative analysis of plant extracts via gas chromatography, high-pressure liquid chromatography and metabolomic fingerprinting) characteristics. The integration of metabolomic data is an innovative aspect that will provide a vast of data giving deep insights into compositional differences of C. creticus plants and providing a broad basis for discussions about the suitability of different species concepts. A better knowledge of morphological, chemical and genetic characteristics of C. creticus will serve as a sound basis for an adapted species concept of this cryptic plant group and will provide also insights in e. g. plant migration movement, genetic exchange with related species and genetic diversity. The results will also find their way into pharmacognosy, phytomedicine and pharmaceutical production where authenticity and secondary compound composition of raw plant material is a primary focus.

Cistus creticus (Cretan Rockrose) is a purple-flowering shrub widespread in the Mediterranean region. The species is rich in valuable secondary compounds (polyphenols, labdane-type diterpenes) that exhibit diverse health-affecting activities. Amongst others, antioxidative, antimicrobial or antiviral activities but also e.g., cell-toxic and tumor-inhibiting properties were described. However, C. creticus is a complex species that is difficult to determine. High intraspecific variability and the therefore introduced subspecies and the fact that different scientific names (e. g. also C. incanus or C. villosus) have been used in field guides, scientific literature and databases make it difficult to obtain a comprehensive view. The aim of this project was a comprehensive evaluation of the C. creticus complex by linking data from morphological, genetic, and phytochemical analyses. The main aims were to proof (1) whether the synonymously used names refer to the same species that can be clearly distinguished from all other Cistus species, (2) whether the striking intraspecific variability is rather due to continuous variation over the species distribution than to the presence of distinct subspecies, (3) whether valuable secondary compounds are ubiquitously distributed and (4) how results of different laboratory methods can contribute to a clarification of these questions. The analysed plant material was collected from wild populations of Portugal, Spain, France, Italy, Croatia, Albania, Greece, Turkey, and Cyprus and was complemented by plant material from greenhouse populations that were raised from seeds of diverse geographical origin. Our results confirmed the heterogeneous character of C. creticus. Traits in different DNA-sequences suggested the presence of two lineages with clearly different evolutionary histories. The heterogeneous 'Western Mediterranean clade' comprises the C. creticus populations from Spain, Italy, Croatia, Albania, and Greece. The more homogeneous 'Eastern Mediterranean clade' comprises the populations from Cyprus, Turkey, Lebanon, Israel and Jordan and shows a close relationship to C. albidus, a species that is distributed from Morocco/Spain to Italy. Our results would argue for the introduction of two C. creticus subspecies, subspecies hesperius ('Western Mediterranean clade) and subspecies orientalis ('Eastern Mediterranean clade'). The two lineages seem also to differ in their polyphenolic compound spectrum. Plants of the 'Western Mediterranean clade' are generally speaking rich in flavonols and punicalagin derivatives whereas punicalagin derivatives seem to be of lesser or no importance in plant material of the 'Eastern Mediterranean clade'. Within the 'Western Mediterranean clade', plant material of Italian and Croatian populations could be distinguished from all other C. creticus populations based on metabolomic data (allows a comprehensive chemical fingerprint of analysed plants). Within both evolutionary lineages, populations rich in essential oils and volatile-poor populations exhibiting dense whitish hairiness seem to be present. We suppose them to be ecological variants representing different strategies to cope with the harsh Mediterranean climate conditions.