Chemical and transcriptomic analyses of leaf trichomes from Cistus creticus subsp. creticus reveal the biosynthetic pathways of certain labdane-type diterpenoids and their acetylated forms.

Labdane-related diterpenoids (LRDs), a subgroup of terpenoids, exhibit structural diversity and significant commercial and pharmacological potential. LRDs share the characteristic decalin-labdanic core structure that derives from the cycloisomerization of geranylgeranyl diphosphate (GGPP). Labdanes derive their name from the oleoresin known as 'Labdanum', 'Ladano', or 'Aladano', used since ancient Greek times. Acetylated labdanes, rarely identified in plants, are associated with enhanced biological activities. Chemical analysis of Cistus creticus subsp. creticus revealed labda-7,13(E)-dien-15-yl acetate and labda-7,13(E)-dien-15-ol as major constituents. In addition, novel labdanes such as cis-abienol, neoabienol, ent-copalol, and one as yet unidentified labdane-type diterpenoid were detected for the first time. These compounds exhibit developmental regulation, with higher accumulation observed in young leaves. Using RNA-sequencing (RNA-seq) analysis of young leaf trichomes, it was possible to identify, clone, and eventually functionally characterize labdane-type diterpenoid synthase (diTPS) genes, encoding proteins responsible for the production of labda-7,13(E)-dien-15-yl diphosphate (endo-7,13-CPP), labda-7,13(E)-dien-15-yl acetate, and labda-13(E)-ene-8α-ol-15-yl acetate. Moreover, the reconstitution of labda-7,13(E)-dien-15-yl acetate and labda-13(E)-ene-8α-ol-15-yl acetate production in yeast is presented. Finally, the accumulation of LRDs in different plant tissues showed a correlation with the expression profiles of the corresponding genes.

Heterologous production of labdane-type diterpenes in the green alga Chlamydomonas reinhardtii.

Labdane diterpenes (LDs), and especially sclareol, are important feedstocks for the pharmaceutical and cosmetic industries, and therefore several lines of research have led to their heterologous production in non-photosynthetic microbes and higher plants. The potential of microalgae as bioreactors of natural products has been established for a variety of bioactive metabolites, including terpenes. In this work, a codon optimized sequence encoding a key plant labdane-type diterpene (LD) cyclase, copal-8-ol diphosphate synthase from Cistus creticus (CcCLS), was introduced into the chloroplast genome of Chlamydomonas reinhardtii. Of 49 transplastomic algal lines, 12 produced variable amounts of four LD compounds, namely ent-manoyl oxide, sclareol, labda-13-ene-8α,15-diol and ent-13-epi-manoyl oxide. The total LD concentrations measured in the transplastomic lines reached 1.172 ±â€¯0.05 µg/mg cell DW for the highest overall producer, while the highest yield for sclareol was 0.038 ±â€¯0.001 µg/mg cell DW. Thus, transplastomic expression of a key plant labdane diterpene cyclase in the C. reinhardtii chloroplast genome enabled the production of important plant-specific LD compounds.

Dissecting Adaptation Mechanisms to Contrasting Solar Irradiance in the Mediterranean Shrub Cistus incanus.

Molecular mechanisms that are the base of the strategies adopted by Mediterranean plants to cope with the challenges imposed by limited or excessive solar radiation during the summer season have received limited attention. In our study, conducted on C. incanus plants growing in the shade or in full sunlight, we performed measurements of relevant physiological traits, such as leaf water potential, gas exchange and PSII photochemistry, RNA-Seq with de-novo assembly, and the analysis of differentially expressed genes. We also identified and quantified photosynthetic pigments, abscisic acid, and flavonoids. Here, we show major mechanisms regulating light perception and signaling which, in turn, sustain the shade avoidance syndrome displayed by the 'sun loving' C. incanus. We offer clear evidence of the detrimental effects of excessive light on both the assembly and the stability of PSII, and the activation of a suite of both repair and effective antioxidant mechanisms in sun-adapted leaves. For instance, our study supports the view of major antioxidant functions of zeaxanthin in sunny plants concomitantly challenged by severe drought stress. Finally, our study confirms the multiple functions served by flavonoids, both flavonols and flavanols, in the adaptive mechanisms of plants to the environmental pressures associated to Mediterranean climate.

No genetic adaptation of the Mediterranean keystone shrub Cistus ladanifer in response to experimental fire and extreme drought.

In Mediterranean ecosystems, climate change is projected to increase fire danger and summer drought, thus reducing post-fire recruitment of obligate seeder species, and possibly affecting the population genetic structure. We performed a genome-wide genetic marker study, using AFLP markers, on individuals from one Central Spain population of the obligate post-fire seeder Cistus ladanifer L. that established after experimental fire and survived during four subsequent years under simulated drought implemented with a rainout shelter system. We explored the effects of the treatments on marker diversity, spatial genetic structure and presence of outlier loci suggestive of selection. We found no effect of fire or drought on any of the genetic diversity metrics. Analysis of Molecular Variance showed very low genetic differentiation among treatments. Neither fire nor drought altered the small-scale spatial genetic structure of the population. Only one locus was significantly associated with the fire treatment, but inconsistently across outlier detection methods. Neither fire nor drought are likely to affect the genetic makeup of emerging C. ladanifer, despite reduced recruitment caused by drought. The lack of genetic change suggests that reduced recruitment is a random, non-selective process with no genome-wide consequences on this keystone, drought- and fire tolerant Mediterranean species.

Cistus ladanifer (Cistaceae): a natural resource in Mediterranean-type ecosystems.

MAIN CONCLUSION: Cistus ladanifer has a well-defined taxonomic identity. 2,2,6-trimethylcyclohexanone may be an authenticity and taxonomic marker. Its traits and applications make it a possible economic resource fitted for Mediterranean areas. Cistus ladanifer is a dominant shrub species endemic to the western Mediterranean region. Due to its dominant nature and its potential ecological, aromatic or pharmacological applications, C. ladanifer has been the object of numerous studies. In this review current knowledge on different aspects of this species is summarized, from its taxonomy to its chemical characterisation or its competitive traits. There are no doubts about the taxonomic entity of C. ladanifer, although the recognition of infraspecific taxa deserves more attention. Given that the fragrant exudate of C. ladanifer holds a very specific composition, one species specific carotenoid, 2,2,6-trimethylcyclohexanone, derivative is proposed as an authenticity marker for uses of C. ladanifer in pharmacological or aromatic industries. Evidence is also gathered on the extreme adaptation of C. ladanifer to stressful conditions in the Mediterranean region, such as the ability to survive in low hydric and high solar exposition conditions, presistence in poor and contaminated soils, and growth inhibition of several other plants through the release of allelochemicals. Thus, the finding of potential applications for this plant may contribute to enhance the economic dimension of derelict lands, such as mine tailings or poor agricultural Mediterranean areas.

Trichome patterning control involves TTG1 interaction with SPL transcription factors.

Epidermal cell differentiation is a paramount and conserved process among plants. In Arabidopsis, a ternary complex formed by MYB, bHLH transcription factors and TTG1 modulates unicellular trichome morphogenesis. The formation of multicellular glandular trichomes of the xerophytic shrub Cistus creticus that accumulate labdane-type diterpenes, has attained much attention renowned for its medicinal properties. Here, we show that C. creticus TTG1 (CcTTG1) interacts with the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPLA/B) proteins, putative homologs of AtSPL4/5 that in turn interact with AtTTG1. These interactions occur between proteins from evolutionarily distant species supporting the conserved function of TTG1-SPL complex. Overexpression of AtSPL4 and AtSPL5 decreased the expression of GLABRA2 (AtGL2), the major regulator of trichome morphogenesis, resulting in trichome reduction on the adaxial surface of cauline leaves, thereby illuminating the significance of TTG1-SPLs interactions in trichome formation control. AtGL2 and AtSPL4 have opposite expression patterns during early stages of leaf development. We postulate an antagonistic effect between SPLs and the heterogeneous MYB-bHLH factors binding to TTG1. Hence, the SPLs potentially rearrange the complex, attenuating its transcriptional activity to control trichome distribution.

Zeatin modulates flower bud development and tocopherol levels in Cistus albidus (L.) plants as they age.

In a previous study we showed that Cistus albidus (L.) experiences an age-dependent decay in flower vigour correlated with a decline in trans-zeatin (tZ) levels. In the present study we aimed to establish a causal relationship between these two phenomena. Exogenous tZ applied to plants grown under semi-controlled conditions did not rescue flower vigour; however, it accelerated flower development, but only in younger individuals. Older plants showed lower tocopherol levels in flower buds, which were restored by exogenous tZ, suggesting that a loss of antioxidant defences may underlie the age-dependent decay in flower vigour. We conclude that declining tZ levels may not be directly responsible for the age-associated loss of floral vigour; that tZ modulates the speed of flower development as plants age; and that flower buds alter their sensitivity to tZ as plants age.

Chloroplast DNA phylogeography of the shrub Cistus ladanifer L. (Cistaceae) in the highly diverse Western Mediterranean region.

This study investigated the phylogeographic structure of Cistus ladanifer, in order to locate its Quaternary refugia, reconstruct its recolonisation patterns and assess the role of geographical features (mountain ranges, rivers and the Strait of Gibraltar) as barriers to its seed flow and expansion through the Western Mediterranean. Thirty-eight populations were screened for length variation of polymorphic chloroplast simple sequence repeats (cpSSRs). Statistical analyses included estimation of haplotypic diversity, hierarchical analysis of molecular variation (amova) and fixation indices. Mantel tests, SAMOVA and BARRIER analyses were applied to evaluate the geographical partitioning of genetic diversity across the entire species range. Pollen data from bibliography were used to complement molecular inferences. Chlorotype diversity within populations was similar throughout the natural range of C. ladanifer (mean haplotypic diversity=0.32). High differentiation among populations was estimated (G(ST)=0.60). Our data suggest that the barriers of the Strait of Gibraltar and the Betic ranges may have favoured the divergence during glacial periods of four different lineages of populations inferred with SAMOVA. The main northward colonisation of in the Iberian Peninsula occurred from refugia in southwest Iberia. This process may have been influenced by human activities (forest clearance, livestock grazing and even commerce) in the Iberian Peninsula. In contrast, populations in the Betic area have conserved a specific haplotype.

Genetically depauperate in the continent but rich in oceanic islands: Cistus monspeliensis (Cistaceae) in the Canary Islands.

BACKGROUND: Population genetic theory holds that oceanic island populations are expected to have lower levels of genetic variation than their mainland counterparts, due to founder effect after island colonization from the continent. Cistus monspeliensis (Cistaceae) is distributed in both the Canary Islands and the Mediterranean region. Numerous phylogenetic results obtained in the last years allow performing further phylogeographic analyses in Cistus. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed sequences from multiple plastid DNA regions in 47 populations of Cistus monspeliensis from the Canary Islands (21 populations) and the Mediterranean basin (26 populations). The time-calibrated phylogeny and phylogeographic analyses yielded the following results: (1) a single, ancestral haplotype is distributed across the Mediterranean, whereas 10 haplotypes in the Canary Islands; (2) four haplotype lineages are present in the Canarian Islands; (3) multiple colonization events across the archipelago are inferred; (4) the earliest split of intraspecific lineages occurred in the Early to Middle Pleistocene (<930,000 years BP). CONCLUSIONS/SIGNIFICANCE: The contrasting pattern of cpDNA variation is best explained by genetic bottlenecks in the Mediterranean during Quaternary glaciations, while the Canarian archipelago acted as a refugium of high levels of genetic diversity. Active colonization across the Canarian islands is supported not only by the distribution of C. monspeliensis in five of the seven islands, but also by our phylogeographic reconstruction in which unrelated haplotypes are present on the same island. Widespread distribution of thermophilous habitats on every island, as those found throughout the Mediterranean, has likely been responsible for the successful colonization of C. monspeliensis, despite the absence of a long-distance dispersal mechanism. This is the first example of a plant species with higher genetic variation among oceanic island populations than among those of the continent.

A copal-8-ol diphosphate synthase from the angiosperm Cistus creticus subsp. creticus is a putative key enzyme for the formation of pharmacologically active, oxygen-containing labdane-type diterpenes.

The resin of Cistus creticus subsp. creticus, a plant native to Crete, is rich in labdane-type diterpenes with significant antimicrobial and cytotoxic activities. The full-length cDNA of a putative diterpene synthase was isolated from a C. creticus trichome cDNA library. The deduced amino acid sequence of this protein is highly similar (59%-70% identical) to type B diterpene synthases from other angiosperm species that catalyze a protonation-initiated cyclization. The affinity-purified recombinant Escherichia coli-expressed protein used geranylgeranyl diphosphate as substrate and catalyzed the formation of copal-8-ol diphosphate. This diterpene synthase, therefore, was named CcCLS (for C. creticus copal-8-ol diphosphate synthase). Copal-8-ol diphosphate is likely to be an intermediate in the biosynthesis of the oxygen-containing labdane-type diterpenes that are abundant in the resin of this plant. RNA gel-blot analysis revealed that CcCLS is preferentially expressed in the trichomes, with higher transcript levels found in glands on young leaves than on fully expanded leaves, while CcCLS transcript levels increased after mechanical wounding. Chemical analyses revealed that labdane-type diterpene production followed a similar pattern, with higher concentrations in trichomes of young leaves and increased accumulation upon wounding.

Stress and developmental responses of terpenoid biosynthetic genes in Cistus creticus subsp. creticus.

Plants, and specially species adapted in non-friendly environments, produce secondary metabolites that help them to cope with biotic or abiotic stresses. These metabolites could be of great pharmaceutical interest because several of those show cytotoxic, antibacterial or antioxidant activities. Leaves' trichomes of Cistus creticus ssp. creticus, a Mediterranean xerophytic shrub, excrete a resin rich in several labdane-type diterpenes with verified in vitro and in vivo cytotoxic and cytostatic activity against human cancer cell lines. Bearing in mind the properties and possible future exploitation of these natural products, it seemed interesting to study their biosynthesis and its regulation, initially at the molecular level. For this purpose, genes encoding enzymes participating in the early steps of the terpenoids biosynthetic pathways were isolated and their gene expression patterns were investigated in different organs and in response to various stresses and defence signals. The genes studied were the CcHMGR from the mevalonate pathway, CcDXS and CcDXR from the methylerythritol 4-phosphate pathway and the two geranylgeranyl diphosphate synthases (CcGGDPS1 and 2) previously characterized from this species. The present work indicates that the leaf trichomes are very active biosynthetically as far as it concerns terpenoids biosynthesis, and the terpenoid production from this tissue seems to be transcriptionally regulated. Moreover, the CcHMGR and CcDXS genes (the rate-limiting steps of the isoprenoids' pathways) showed an increase during mechanical wounding and application of defence signals (like meJA and SA), which is possible to reflect an increased need of the plant tissues for the corresponding metabolites.

Ecological rather than geographical isolation dominates Quaternary formation of Mediterranean Cistus species.

The lack of a comparative approach makes it impossible to determine the main factors influencing colonization and evolution in plants. Here we conducted the first comparative study of a characteristic Mediterranean lineage (white-flowered Cistus) taking advantage of its well-known phylogenetic relationships. A two-scale approach was applied to address the hypothesis of higher levels of isolation in mountain than in lowland species. First, a time-calibrated phylogeny using plastid sequences of Cistaceae suggested that the origin of Cistus species postdated both the refilling of the Mediterranean Sea (5.59-5.33 Ma) and the onset of the Mediterranean climate (3.2 Ma). Two hundred and sixty-three additional, plastid sequences from 111 populations showed different numbers of haplotypes in C. laurifolius (7), C. monspeliensis (2) and C. salviifolius (7). Although haplotype sharing among disjunct populations was observed in all species, phylogeographic analyses revealed haplotype lineages exclusive to Europe or Africa only in the mountain species (C. laurifolius). Isolation by either geographical distance or sea barriers was not significantly supported for the lowland species (C. monspeliensis; C. ladanifer from a previous study). The same is true for the less habitat-specific species of the lineage (C. salviifolius). Comparative phylogeography of the Cistus species leads us to interpret a general pattern of active colonization surpassing Mediterranean barriers. In contrast, ecological conditions (precipitation, temperature, soils) appear to have determined the distribution of the Cistus species of Mediterranean mountains. This study further provides molecular evidence for multiple colonization patterns in the course of successful adaptation of Cistus species to Mediterranean habitats.

Networks of spatial genetic variation across species.

Spatial patterns of genetic variation provide information central to many ecological, evolutionary, and conservation questions. This spatial variability has traditionally been analyzed through summary statistics between pairs of populations, therefore missing the simultaneous influence of all populations. More recently, a network approach has been advocated to overcome these limitations. This network approach has been applied to a few cases limited to a single species at a time. The question remains whether similar patterns of spatial genetic variation and similar functional roles for specific patches are obtained for different species. Here we study the networks of genetic variation of four Mediterranean woody plant species inhabiting the same habitat patches in a highly fragmented forest mosaic in Southern Spain. Three of the four species show a similar pattern of genetic variation with well-defined modules or groups of patches holding genetically similar populations. These modules can be thought of as the long-sought-after, evolutionarily significant units or management units. The importance of each patch for the cohesion of the entire network, though, is quite different across species. This variation creates a tremendous challenge for the prioritization of patches to conserve the genetic variation of multispecies assemblages.

Morphological, chemical and genetic differentiation of two subspecies of Cistus creticus L. (C. creticus subsp. eriocephalus and C. creticus subsp. corsicus).

Cistus creticus L., an aromatic species from the Mediterranean area, contains various diterpenes bearing the labdane skeleton. The production of essential oil from this species has potential economic value, but so far, it has not been optimized. In order to contribute to a better knowledge of this species and to its differentiation, the morphological characters, volatile chemical composition and genetic data of two subspecies (C. creticus subsp. eriocephalus and C. creticus subsp. corsicus) were investigated. The leaf trichomes were studied using scanning electron microscopy. The chemical composition of Corsican essential oil (C. creticus subsp. corsicus) has been reported using GC, GC/MS and 13C NMR; the main constituents were oxygenated labdane diterpenes (33.9%) such as 13-epi-manoyl oxide (18.5%). Using plant material (54 samples) collected from 18 geographically distinct areas of the islands of Corsica and Sardinia, the basis of variation in the headspace solid-phase microextraction volatile fraction and an inter-simple sequence repeat genetic analysis were also examined. It was shown that the two subspecies of C. creticus differed in morphology, essential oil production, volatile fraction composition and genetic data.

Physiological and molecular responses of the isoprenoid biosynthetic pathway in a drought-resistant Mediterranean shrub, Cistus creticus exposed to water deficit.

The goal of the present research was to obtain new insights into the mechanisms underlying drought stress resistance in plants. Specifically, we evaluated changes in the expression of genes encoding enzymes involved in isoprenoid biosynthesis, together with the levels of the corresponding metabolites (chlorophylls, carotenoids, tocopherols and abscisic acid), in a drought-resistant Mediterranean shrub, Cistus creticus grown under Mediterranean field conditions. Summer drought led to reductions in the relative leaf water content (RWC) by 25%, but did not alter the maximum efficiency of PSII, indicating the absence of damage to the photosynthetic apparatus. While the expression of genes encoding C. creticus chlorophyll a oxygenase/chlorophyll b synthase (CAO) and phytoene synthase (PSY) were not affected by water deficit, the genes encoding homogentisate phytyl-transferase (HPT) and 9-cis-epoxycarotenoid dioxygenase (NCED) were induced in water-stressed (WS) plants. Drought-induced changes in gene expression were observed at early stages of drought and were strongly correlated with levels of the corresponding metabolites, with simultaneous increases in abscisic acid and alpha-tocopherol levels of up to 4-fold and 62%, respectively. Furthermore, alpha-tocopherol levels were strongly positively correlated with abscisic acid contents, but not with the levels of jasmonic acid and salicylic acid. We conclude that the abscisic acid and tocopherol biosynthetic pathway may be regulated at the transcript level in WS C. creticus plants, and that the genes encoding HPT and NCED may play a key role in the drought stress resistance of this Mediterranean shrub by modulating abscisic acid and tocopherol biosynthesis.

Transcriptome analysis approaches for the isolation of trichome-specific genes from the medicinal plant Cistus creticus subsp. creticus.

Cistus creticus subsp. creticus is a plant of intrinsic scientific interest due to the distinctive pharmaceutical properties of its resin. Labdane-type diterpenes, the main constituents of the resin, exhibit considerable antibacterial and cytotoxic activities. In this study chemical analysis of isolated trichomes from different developmental stages revealed that young leaves of 1-2 cm length displayed the highest content of labdane-type diterpenes (80 mg/g fresh weight) whereas trichomes from older leaves (2-3 or 3-4 cm) exhibited gradual decreased concentrations. A cDNA library was constructed enriched in transcripts from trichomes isolated from young leaves, which are characterized by high levels of labdane-type diterpenes. Functional annotation of 2,022 expressed sequence tags (ESTs) from the trichome cDNA library based on homology to A. thaliana genes suggested that 8% of the putative identified sequences were secondary metabolism-related and involved primarily in flavonoid and terpenoid biosynthesis. A significant proportion of the ESTs (38%) displayed no significant similarity to any other DNA deposited in databases, indicating a yet unknown function. Custom DNA microarrays constructed with 1,248 individual clones from the cDNA library facilitated transcriptome comparisons between trichomes and trichome-free tissues. In addition, gene expression studies in various Cistus tissues and organs for one of the genes highlighted as the most differentially expressed by the microarray experiments revealed a putative sesquiterpene synthase with a trichome-specific expression pattern. Full length cDNA isolation and heterologous expression in E. coli followed by biochemical analysis, led to the characterization of the produced protein as germacrene B synthase.

Isolation and functional analysis of two Cistus creticus cDNAs encoding geranylgeranyl diphosphate synthase.

Cistus creticus ssp. creticus is an indigenous shrub of the Mediterranean area. The glandular trichomes covering its leaf surfaces secrete a resin called "ladanum", which among others contains a number of specific labdane-type diterpenes that exhibit antibacterial and antifungal action as well as in vitro and in vivo cytotoxic and cytostatic activity against human cancer cell lines. In view of the properties and possible future exploitation of these metabolites, it was deemed necessary to study the geranylgeranyl diphosphate synthase enzyme (GGDPS, EC 2.5.1.30), a short chain prenyltransferase responsible for the synthesis of the precursor molecule of all diterpenes. In this work, we present the cloning, functional characterisation and expression profile at the gene and protein levels of two differentially expressed C. creticus full-length cDNAs, CcGGDPS1 and CcGGDPS2. Heterologous yeast cell expression system showed that these cDNAs exhibited GGDPS enzyme activity. Gene and protein expression analyses suggest that this enzyme is developmentally and tissue-regulated showing maximum expression in trichomes and smallest leaves (0.5-1.0cm). This work is the first attempt to study the terpenoid biosynthesis at the molecular level in C. creticus ssp. creticus.

Systematics, character evolution, and biogeography of Cistus L. (Cistaceae) based on ITS, trnL-trnF, and matK sequences.

This paper presents the first phylogenetic hypotheses for the 20 species of Cistus based on plastid (trnL-trnF, matK) and nuclear (ITS) DNA sequence data. Phylogenetic relationships reveal that: (1) Halimium and Cistus form a cohesive, natural group; (2) two major lineages of purple-flowered and white-flowered species are defined, except for the purple-flowered C. parviflorus; (3) monophyly of conspecific populations is congruent with the circumscription of species. Topological congruence between nuclear and plastid phylogenies does not support a predominant reticulate system of evolution in Cistus. Reconstruction of character evolution suggests an increment of number of fruit valves in the Cistaceae from 3 to 12 in a unidirectional manner. In contrast, reproductive characters, such as sepal number, petal color, and style length, evolved multiple times in the course of evolution. A single colonization of Cistus into the Canary Islands appears to be responsible for a lineage of four species sharing a most recent common ancestor with five sepals, purple flowers, styles as long as stamens, and five fruit valves. Species diversity in Cistus (14) and Halimium (8), coupled with sister-group relationships and molecular divergence, lead us to suggest the western Mediterranean as a major center of present-day differentiation, but paleobotanical data indicate an earlier formation of the Cistus-Halimium assemblage in different areas.

Identification of metalliferous ecotypes of Cistus ladanifer L. using RAPD markers.

The genetic diversity of Cistus ladanifer ssp. ladanifer (Cistaceae) growing on ultramafic and non-ultramafic (basic and schists) soils in the NE of Portugal was studied in order to identify molecular markers that could distinguish the metal-tolerant ecotypes of this species. Random Amplified Polymorphic DNA (RAPD) markers were used in order to estimate genetic variation and differences between populations. The RAPD dataset was analysed by means of a cluster analysis and an analysis of molecular variance (AMOVA). Our results indicate a significant partitioning of molecular variance between ultramafic and non-ultramafic populations of Cistus ladanifer, although the highest percentage of this variance was found at the intra-population level. Mantel's test showed no relationship between inter-population genetic and geographic distances. A series of RAPD bands that could be related to heavy metal tolerance were observed. The identification of such markers will enable the use of Cistus ladanifer in phytoremediation procedures.