Tolerance and bioaccumulation of heavy metals in five populations of Cistus ladanifer L. subsp. ladanifer

The effects of heavy metals on the growth, mineral composition (P, K, Fe and Mn) and metal accumulation of five populations of Cistus ladanifer subsp. ladanifer from NE Portugal were investigated in hydroponic experiments. Plants were exposed to increasing concentrations (0–2000 M) of one of eight heavy metals: Cd, Co, Cr, Cu, Mn, Ni, Pb or Zn. Populations of C. ladanifer, whose origin was ultramafic soils (S and UB) or soils developed on basic rocks (B), showed a higher tolerance to the metals Cd, Co, Cr, and Mn, and a considerable degree of tolerance to Ni. In contrast, populations originating on acid-rock soils (M and SC) showed higher tolerance to the metals Cu and Zn. Populations showed different patterns of metal accumulation and distribution in the plant parts, suggesting different mechanisms of metal tolerance are used. The more Cd-, Co- and Mn-tolerant populations (S, UB, B and SC (Cd)) showed accumulation of these three metals in the shoots (shoot:root metal concentration ratios (S:R) > 1). Shoot concentrations of up to 309 g Cd g^–1, 2667 g Co g^–1 and 6214 g Mn g^–1 were found in these populations. The populations, UB and M, showed considerable tolerance to Ni and Zn, respectively. These populations accumulated up to 4164 g Ni g^–1 and 7695 g Zn g^–1 in their shoot tissues, and these metals were efficiently transported from the roots to aerial parts (S:R > 3 (Ni), S:R > 1 (Zn)). In contrast, the S and SC populations maintained higher growth rates in the presence of Ni and Zn, respectively, but showed exclusion mechanisms of metal tolerance: reduced Ni and Zn transport to shoots (S:R < 1). Cistus ladanifer was not able to efficiently transport Cr, Cu or Pb from its roots to its aerial parts (S:R ranged from 0–0.4). The more Cu-tolerant populations, M and SC, showed a greater restriction of Cu transport to the shoots than the ultramafic- or basic-rock populations. Significant changes in the plant mineral composition were found, however, concentrations were generally above mineral deficiency levels. Based on these preliminary results the possible usefulness of this plant for phytoremediation technologies is discussed. However, further investigations are necessary to evaluate its growth and metal accumulation under soil and field conditions.     

Interpopulational variation in the flavonoid composition of Cistus ladanifer L. exudate

A study was made in different populations of Cistus ladanifer of the quantitative and qualitative variation of the exudate of the leaves and stems, including the exudate's aglycone flavonoid content. Two groups of populations were established according to different climate conditions, and the results showed the existence of interpopulational variability both quantitatively and qualitatively. The populations subject to the greatest thermal stress and moisture stress (Group I) synthesized more exudate and total flavonoids. The amounts of the different flavonoids synthesized also differed between the two groups of populations: Group I secreted more 3-O-methylkaempferol, 3,4′-di-O-methylkaempferol and 3,7-di-O-methylkaempferol and Group II more 4′-O-methylapigenin. Quantifying the proportion of each apigenin and kaempferol relative to their totals, it was observed that the synthesis of position 7 methylated flavonoids are enhanced in Group I, and position 4′ methylated flavonoids in Group II. The indication is that the climatic conditions to which a population of C. ladanifer is subject may lead to diversification in the flavonoid composition of the leaf and stem exudate, with the implication that flavonoids could have various ecological functions in this species.     

Autotoxicity Against Germination and Seedling Emergence in Cistus ladanifer L

Autotoxic species are those which adversely affect their own seeds’ germination and/or seedling development. Cistus ladanifer L (labdanum or jara) has been shown to have a pattern of allelopathic behaviour against the herbs that share its habitat. The present work studied whether an autotoxic effect also exists. The aqueous solution obtained from washing jara leaves was found by itself to inhibit germination and cotyledon emergence of the species’ seeds. When these same trials were carried out in soils, autotoxicity was observed only from leaves and soils collected in winter. This was so both in soils collected away from the influence of the jaral to which was added the greatest concentration of aqueous extract prepared from the leaves, and in soils collected within the jaral, except that in the latter group of soils germination was inhibited with or without the addition of C. ladanifer extracts. This autotoxic behaviour could be involved in the species’ own population control, and would explain the scant self-regeneration within established C. ladanifer stands.     

Persistence of flavonoids in Cistus ladanifer soils

Tuber spp. are ectomycorrizal fungi that produce edible ascocarps termed truffles. The most valuable species, T. magnatum, is perhaps the most selective among these for soil environment. Since T. magnatum is often found in fluvial plains, the present study focused on the characterization of the soil physical characteristics suitable for this ectomycorrhizal fungus, and the relationship of these characteristics with fluvial landforms. The investigation was carried out in a truffle-producing area which encompassed the most representative landforms of a fluvial landscape characterized by the presence of Populus deltoides symbionts in both productive and unproductive locations. Soil physical characteristics were determined in productive and unproductive transects according to a stratified random design. Truffle-producing locations displayed a coarser texture, smaller aggregates, a less stable aggregation and a larger volume of soil occupied by highly interconnected macropores. The spatial distribution of this combination of characteristics was then investigated by using a geostatistical approach utilizing clay content and a soil structure indicator as markers. Spatial analysis connected T. magnatum habitats with the soil environment present in river levees, where scattered and chaotic redeposition of solid particles creates very porous, highly aerated surface strata.     

Identification of apple cultivars using RAPD markers

Summary Eleven apple cultivars were differentiated using randomly amplified polymorphic DNA (RAPD) markers obtained by the polymerase chain reaction (PCR). The variability of the technique and of the origin of the DNA extract was analyzed. A set of bands consistent in their presence or absence was chosen to create a differentiating band pattern. A key is proposed by which one can differentiate apple cultivars using commercially available prime.     

Allelopathic effect of Cistus ladanifer on seed germination

1. The allelopathic effect of the exudate secreted by Cistus ladanifer leaves was tested on different plant species. Cynodon dactylon and Rumex crispus , species absent from C. ladanifer scrub (jarales), were clearly inhibited by the exudate.
2. Species present in the jarales, Medicago polymorpha and Lolium rigidum , showed no direct inhibition of germination, but the process this was delayed, and cotyledon and root size was diminished. This implies inhibition of seedling growth, and may explain the low abundance of these species inside the jarales.
3. The active constituents in the inhibition of germination are low molecular weight phenolic compounds. The flavonoids appear to exert no direct effect on germination of the species tested, but do provoke a reduction in cotyledon and root size, and could thereby inhibit subsequent seedling development.     

Identification of white truffle species using RAPD markers

Morphologically very similar species of white truffle were analyzed by the random amplified polymorphic DNA (RAPD) technique. Species-specific RAPD fragments were selected and pairs of primers were designed on their sequences. Sequence-characterized amplified regions were developed and applied to identify these species throughout their entire life cycle: fruit body, mycelium, ectomycorrhiza. This procedure provides an unambiguous and rapid tool for typing species whose morphology is very similar.     

Identification of cultivars and validation of genetic relationships in Mangifera indica L. using RAPD markers

Twenty-five accessions of mango were examined for random amplified polymorphic DNA (RAPD) genetic markers with 80 10-mer random primers. Of the 80 primers screened, 33 did not amplify, 19 were monomorphic, and 28 gave reproducible, polymorphic DNA amplification patterns. Eleven primers were selected from the 28 for the study. The number of bands generated was primer- and genotype-dependent, and ranged from 1 to 10. No primer gave unique banding patterns for each of the 25 accessions; however, ten different combinations of 2 primer banding patterns produced unique fingerprints for each accession. A maternal half-sib (MHS) family was included among the 25 accessions to see if genetic relationships could be detected. RAPD data were used to generate simple matching coefficients, which were analyzed phenetically and by means of principal coordinate analysis (PCA). The MHS clustered together in both the phenetic and the PCA while the randomly selected accessions were scattered with no apparent pattern. The uses of RAPD analysis for Mangifera germ plasm classification and clonal identification are discussed.     

Estimation of heterozygosity in Ribes nigrum L. using RAPD markers

Heterozygosity in three cultivars of the blackcurrant (Ribes nigrum L.) was estimated. A selfed population of each cultivar was screened for Random Amplified Polymorphic DNA markers (RAPDs) and heterozygous loci were identified by band segregation in contrast with the non-segregation of homozygous loci. On average, 21% of the loci scored in each cultivar were heterozygous. The implications for mapping studies in Ribes nigrum are discussed.     

Germination of Cistus ladanifer seeds in relation to parent material

Variability in seed germination behaviour of Cistus ladanifer L. (rockrose), a Cistaceous species widely distributed in the Mediterranean Basin, was studied in a central Spanish population under controlled conditions. No correlation between seed moisture content and germination was found. Great variability in germination was found among seeds of the population studied, not only between seeds belonging to different mother plants, but also between those collected from different capsules on the same plant. In most cases, seeds preheated at 100 °C for 30 minutes showed a significant increase in germination. This germination behaviour is related to fire regimes as this plant is a typical shrubby element of the mediterranean shrublands. Percent germination did not vary significantly after several months of seed storage at room temperature. In the same way, no difference was found in final germination percentage of seeds stored under room temperature vs. seeds stored under cold conditions.     

Chloroplast microsatellites reveal that metallicolous populations of the Mediterranean shrub Cistus ladanifer L have multiple origins

Cistus ladanifer L. (Cistaceae) is a Mediterranean shrub covering different kinds of soils in the Western Mediterranean area. This species has colonised several metalliferous areas (serpentine outcrops as well as human-polluted sites) throughout its distribution range, and is therefore an interesting species to study the possible effects on genetic diversity and differentiation produced by the colonisation of areas polluted with heavy metals. The genetic structure of 33 natural populations distributed across its entire natural distribution range (Morocco, Portugal and Spain) and growing on either metalliferous or non-metalliferous soils was investigated using chloroplast microsatellites. Population genetic parameters were estimated and genetic groups were identified using Bayesian inference. In addition, we compared the genetic diversity and differentiation among metallicolous and non-metallicolous populations within each Bayesian-defined group. The cpSSR data suggested that metallicolous populations of Cistus ladanifer have arisen through multiple independent evolutionary origins within two different chloroplast lineages. Evidence that the soil type provoked genetic bottlenecks in metallicolous populations or genetic differentiation among metallicolous and non-metallicolous populations was not observed. Historical factors are the main cause of the present genetic structure of C. ladanifer. The nature of tolerance to heavy metals as a species-wide trait in this shrub is discussed.     

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.     

Tolerance assessment of Cistus ladanifer to serpentine soils by developmental stability analysis

Developmental instability is the result of random environmental perturbations during development. Its absence (developmental stability) depends on an organism's ability to buffer environmental disturbances. Both genotype and environment influence the phenotypic expression of developmental instability and it is susceptible to selection pressure. We studied developmental instability (as indicated by increased within-individual asymmetry of repeated traits) in vegetative and reproductive structures of three populations of Cistus ladanifer L. living in different soil substrates (serpentine, siliceous and contact zone) to detect tolerance to serpentine soils. Serpentine soils, characterized by high concentrations of heavy metals (Ni, Cr, and Co), low levels of Ca/Mg ratio and high water deficit, can adversely affect plant performance. In this study we demonstrated that asymmetry and within-plant variance were higher in the contact zone population than either the silica or serpentine populations, proving the adaptation of C. ladanifer to serpentine soils. Within-population estimates of developmental instability were concordant for both vegetative and reproductive traits. There was little or no within-individual correlation among estimates of developmental instability based on different structures, i.e., plants that had highly asymmetric leaves always had high developmental instability in translational symmetry. Radial asymmetry of petals was negatively correlated with petal size, especially in silica soil plants, providing evidence of selection for symmetric and large petals. While leaf size was positively correlated with absolute fluctuating asymmetry, suggesting selection for small or intermediate size leaves. Serpentine soils presented the largest foliar and floral traits, as well as shoot elongation, while silica soil plants had the smallest scores. On the contrary, aboveground plant biomass was larger in silica soil plants, while the contact zone plants had the lowest biomass.     

Identification of RAPD markers linked to A and B genome sequences in Musa L.

Plantains and bananas (Musa spp. sect. eumusa) originated from intra- and interspecific hybridization between two wild diploid species, M. acuminata Colla. and M. balbisiana Colla., which contributed the A and B genomes, respectively. Polyploidy and hybridization have given rise to a number of diploid, triploid, and tetraploid clones with different permutations of the A and B genomes. Thus, dessert and highland bananas are classified mainly as AAA, plantains are AAB, and cooking bananas are ABB. Classification of Musa into genomic groups has been based on morphological characteristics. This study aimed to identify RAPD (random amplified polymorphic DNA) markers for the A and B genomes. Eighty 10-mer Operon primers were used to amplify DNA from M. acuminata subsp. burmannicoides clone 'Calcutta 4' (AA genomes) and M. balbisiana clone 'Honduras' (BB genomes). Three primers (A17, A18, and D10) that produced unique genome-specific fragments in the two species were identified. These primers were tested in a sample of 40 genotypes representing various genome combinations. The RAPD markers were able to elucidate the genome composition of all the genotypes. The results showed that RAPD analysis can provide a quick and reliable system for genome identification in Musa that could facilitate genome characterization and manipulations in breeding lines.     

Characterization and identification of field ectomycorrhizae of Boletus edulis and Cistus ladanifer

Field ectomycorrhizae sampled under Boletus edulis and Cistus ladanifer have been characterized and described in detail based on standard morphological and anatomical characters. The described ectomycorrhiza has traits typical of Boletales: whitish with three differentiated plectenchymatous layers in the mantle in plan view forming ring-like structures and rhizomorphs with highly differentiated hyphae. The inflated, smooth cystidia-like clavate end cells on the surface of the rhizomorphs and their slightly twisted external hyphae are additional characterizing features. The Hartig net occupies 1 1/2 rows of cortical cells, partly reaching the endodermis. Not all hyphae have clamps. The identification of the fungal symbiont as B. edulis was confirmed by ITS rDNA sequence comparison between mycorrhizas and sporocarps. The singularity of this symbiotic association, as well as its ecological and practical implications, are discussed.     

Identification of RAPD markers linked to common bacterial blight resistance genes in Phaseolus vulgaris L.

Seven hundred and fifty-six random primers were screened with bulks of genomic DNA from common bacterial blight (CBB) resistant and susceptible bean plants. The plants were from a breeding population derived from an interspecific cross between Phaseolus acutifolius and Phaseolus vulgaris. Four RAPD markers, named R7313, RE416, RE49, and R4865, were found to be significantly associated with CBB resistance in this population. Forty-nine molecular markers segregating in the population were clustered into 8 linkage groups by a MAPMAKER linkage analysis. The largest linkage group was 140 cM long and contained 25 marker loci, including marker R4865. Markers R7313, RE416, and RE49 were clustered on another linkage group. A regression analysis indicated that the markers in these two groups together accounted for 81% of the variation in CBB resistance in the population. The addition of another marker, M56810, which was not individually associated with CBB resistance, increased the total contribution to the trait to 87%.     

Identification of sex in Simmondsia chinensis (Jojoba) using RAPD markers

Simmondsia chinensis (Link) Schneider, a multipurpose dioecious shrub of arid zones, has emerged as a cash crop. It is being cultivated for its seeds which store liquid wax whose properties are similar to spermaceti (Sperm whale oil), a substitute for petro products and precious high-priced lubricants. Jojoba is a slow-growing desert shrub having a male biased (5:1; male:female ratio) population. Since there is no method available to determine the sex at the seedling stage, current investigations have been carried out to generate a sex-specific random amplified polymorphic DNA (RAPD) marker in jojoba which is based on the PCR amplification of random locations in the genome of plant. Of the 72 primers tested, only one random decamer primer, OPG-5, produced a unique ∼1,400 base pairs fragment in male DNA. To validate this observation, this primer was re-tested with the individuals of male and female samples of four cultivars. The unique ∼1,400 bp fragment was present in male individuals of all the four cultivars and completely absent in respective female individuals tested. To the best of our knowledge, this is the first report to ascertain the sex of jojoba plants at an early stage of development of the taxon.     

Identification of RAPD markers for percent hull in oat.

Percent hull is an important physical parameter of oat grain quality, but it is affected by environment. Multiple time-consuming evaluations are required to obtain a correct determination of phenotype. The application of marker-assisted selection for the genes involved would greatly simplify the identification of desirable oat genotypes. Bulked segregant analysis, with selected progeny lines derived from a cross between Cascade and AC Marie (30 and 23% hull, respectively), was used to identify randomly amplified polymorphic DNA markers linked to genetic factors controlling primary kernel hull percentage in oat. Twelve polymorphisms, identified between bulks, were tested for linkage to genetic factors controlling hull percentage by genotyping 80 randomly selected F2-derived F8 lines from the progeny population. Three markers showed significant test statistics for quantitative trait locus effects, when tested with primary kernel percent hull data from two environments. Together, the unlinked marker loci OPC13800, OPD20600, and OPK71300 explained approximately 41% of the genetic variance in primary kernel percent hull, after accounting for the main effect of environment.     

The effects of plant density upon pollination success, reproductive effort and fruit parasitism in Cistus ladanifer L. (Cistaceae)

Human activity often causes a decline in the local density of plant populations. Below some critical lower density, populations may suffer a progressive decline in reproductive success because of the difficulties associated with finding suitable mates. Therefore, to conserve endangered plant species it is necessary to understand in greater detail how changes in population density affect different determinants of plant reproductive success. We simultaneously recorded individual plant pollination success, reproductive effort and fruit parasitism in three populations of Cistus ladanifer L. in eastern Portugal. Pollination success declined significantly as distance to the nearest conspecific increased (p<0.001). However, reproductive effort and fruit parasitism showed the opposite pattern (both p<0.001). On average, plants farther than 1 m from their nearest conspecific suffered from three times more fruit parasitism compared to plants closer to a conspecific. Overall, net female reproductive output decreased as nearest neighbor distance rose (p<0.001). Thus, isolated plants were able to compensate only partially for reduced pollination success through increased reproductive effort. We conclude that management plans for plant populations should recognize that reproductive success is the accumulated result of several different processes, which may each respond to plant density in different ways.     

Effect of allelopathic compounds produced by Cistus ladanifer on germination of 20 Mediterranean taxa

In order to analyze the extent to which allelopathic action of Cistus ladanifer may influence the successional progression towards Mediterranean sub-climacic shrublands and the geographical distribution of other species, the inhibitory effect of Cistus ladanifer extracts on the germination of 20 Mediterranean species was analyzed. Five of the species tested were characteristic of maquis sub-climacic shrublands: Arbutus unedo, Adenocarpus argyrophyllus, Phillyrea angustifolia, Phillyrea latifolia, and Rhamnus alaternus. Tests were also carried out on 6 Cistaceae species in order to evaluate the auto-toxicity rate of allelopathic extracts: Halimium umbellatum subsp. viscosum, Halimium ocymoides, Cistus ladanifer, Cistus salvifolius, Cistus monspeliensis, and Cistus populifolius. Nine herbaceous species sharing the natural habitat with C. ladanifer were also examined. Results confirmed a clear inhibitory and delaying effect of aqueous C. ladanifer-leaf extracts on the germination of P. angustifolia, P. latifolia, R. alaternus, H. ocymoides, C. populifolius, Erysimum lagascae, Brassica barrelieri, Silene tridentata, and Moricandia moricandioides. Assays with soil collected below the canopy of the C. ladanifer community showed more pronounced inhibitory effect on sub-climacic shrub species than the aqueous extracts. In contrast, the opposite pattern was detected when analyzing the allelopathic effect of natural soil on Cistaceae and herbaceous species. Allelopathic compounds produced by C. ladanifer showed little auto-toxicity. The inhibitory effect of phytotoxic compounds accumulated in the jaral soil upon germination of A. unedo, B.␣barrelieri, and M. moricandioides was eliminated by heating soil at 150 °C for 10 min. Phenolic compounds (i.e. ferulic, p-hydroxybenzoic, vanillic, p-coumaric, and caffeic acids) and terpenes (i.e. α and β-pinene) with allelopathic documented effect were detected in the aqueous extracts and soil samples used in the analyses. We suggest that the allelopathic effect of C. ladanifer may influence the composition and structure of Mediterranean communities where the species is present, since it hinders the establishment of some sub-climacic species and may reduce the area occupied by numerous herbaceous species.