Gene flow among different taxonomic units: evidence from nuclear and cytoplasmic markers in Cedrus plantation forests

Hybridization and introgression are important natural evolutionary processes that can be successfully investigated using molecular markers and open- and controlled-pollinated progeny. In this study, we collected open-pollinated seeds from Cedrus atlantica, Cedrus libani and C. libani × C. atlantica hybrids from three French-plantation forests. We also used pollen from C. libani and Cedrus brevifolia to pollinate C. atlantica trees. The progeny were analyzed using three different types of molecular markers: RAPDs, AFLPs and cpSSRs. Chloroplast DNA was found to be paternally inherited in Cedrus from the progeny of controlled-crosses. Heteroplasmy, although possible, could not be undoubtedly detected. There was no indication of strong reproductive isolating barriers among the three Mediterranean Cedrus taxa. Gene flow between C. atlantica and C. libani accounted for 67 to 81% of viable open-pollinated seedlings in two plantation forests. We propose that Mediterranean Cedrus taxa should be considered as units of a single collective species comprising two regional groups, North Africa and the Middle East. We recommend the use of cpSSRs for monitoring gene flow between taxa in plantation forests, especially in areas where garden specimens of one species are planted in the vicinity of selected seed-stands and gene-conservation reserves of another species.Communicated by D.B. Neale     

Effects of soil and air drought on growth,plant water status and leaf gas exchange in three Mediterranean cedar species: <Emphasis Type="Italic">Cedrus atlantica</Emphasis>, <Emphasis Type="Italic">C. brevifolia</Emphasis> and <Emphasis Type="Italic">C. libani</Emphasis>

Three- and four-year-old potted, greenhouse-grown cedar seedlings were subjected to two different watering regimes: half received full water supply and the other half was submitted to moderate drought (50% of the full water supply). Height growth was the greatest for C. atlantica and the most-limited for C. brevifolia in the well-watered set. However, in the dry set, height growth was less affected by drought conditions for C. brevifolia than for C. atlantica. Cedrus libani gave intermediate results for both watering regimes. Moderate drought provoked a decrease in osmotic potential at full leaf turgor and a long-lasting osmotic adjustment. When irrigation was withheld completely to induce severe soil drying, gas exchange decreased and then stopped at predawn water potentials of −3.0 MPa for C. brevifolia, between −2.6 and −2.8 MPa for C. libani, and at −2.4 MPa for C. atlantica, irrespective of watering regime. For all species, the dry set showed lower net photosynthesis (A) and stomatal conductance (g _s) than the plants in the well-watered set. A and g _s responded to variations in atmospheric water-vapour pressure deficit (VPD). As VPD increased, A and g _s decreased, and this trend was proportionate to initial values at low VPD, but remained independent of previous watering treatments, plant water status or species. To conclude, C. brevifolia appears to be a species with limited growth potential but strong soil drought tolerance whereas C. atlantica has strong growth potential when an adequate water supply is available but is more sensitive to soil drought. C. libani shows an intermediate behaviour for growth and drought tolerance.     

Relationships among Cedrus libani, C. brevifolia and C. atlantica as revealed by the morphological and anatomical needle characters

The main aim of the present study was testing the value of the morphological and anatomical characteristics of the needles in distinguishing Cedrus atlantica, C. libani and C. brevifolia. Nine populations were sampled in their natural habit and 25 characters were used to describe the variation of the brachyblast needles and to analyze the differences between species. The results indicated that morphological and anatomical needle characters provide valuable tools in discrimination of the taxa. The scored differences were statistically significant, as revealed in the Tukey’s t test, discrimination analysis and hierarchical analysis of variation. The results support treating C. libani, C. atlantica and C. brevifolia as independent species.     

Geographical diversity and genetic relationships among Cedrus species estimated by AFLP

Genetic diversity was described in 17 cedar populations covering the geographical range of the four species of the genus Cedrus. The study was conducted using amplified fragment length polymorphism (AFLP) on haploid tissues (megagametophytes). Eleven selective AFLP primer pairs generated a total of 107 polymorphic amplification products. Correspondence and genetic distance analyses indicated that Cedrus deodara constitutes a separate gene pool from the Mediterranean cedars. Within Mediterranean cedars, we distinguished two groups: the first one is made of Cedrus atlantica, while the second one is made of Cedrus libani and Cedrus brevifolia, these latter two species being genetically similar despite important divergence previously observed for morphological and physiological traits. The lowest intrapopulation variability was found in the two C. deodara populations analyzed. Surprisingly, C. brevifolia, the endemic taxon from the island of Cyprus that is found in small and fragmented populations, showed one of the highest levels of diversity. This unexpected pattern of diversity and differentiation observed for C. brevifolia suggests a recent divergence rather than a relictual, declining population. Patterns of diversity within- and among-populations were used to test divergence and fragmentation hypotheses and to draw conclusions for the conservation of Cedrus gene pools.     

Karyotype analysis reveals interspecific differentiation in the genus Cedrus despite genome size and base composition constancy

The DNA content and GC% of the four true cedar (Cedrus) species, C. atlantica, C. brevifolia, C. deodara and C. libani, were assessed. Genome size was homogeneous among representative populations of the four species with an average of 32.6±0.6 pg per 2 C or 15.7×10⁹ base pairs per 1 C. The composition in GC was calculated to be 40.7%. A simple monosomatic haploid level was found in the megagametophyte, as compared to the diploid level of the corresponding embryo. Cytogenetic studies showed a diploid chromosome number of 2n=2x=24 in 11 populations sampled over the four species. The chromosome complements have similar morphology and symmetry. However, fluorochromes revealed specific banding patterns in each of the four cedar species. Eight GC-rich chromomycin A₃ bands were observed in Cedrus deodara chromosomes, six in both Cedrus libani and Cedrus brevifolia, and four bands in Cedrus atlantica chromosomes. Moreover, Hoechst 33258 fluorochrome revealed AT-rich sequences specifically located in the centromeric regions while the GC-rich sequences appeared negatively stained. These investigations provide a systematic characterisation of the Cedrus genus and should contribute towards clarification of the phylogenetic relationships among the four species. Received: 10 October 2000 / Accepted: 20 March 2001     

Assessments of somaclonal variation in micropropagated shoots of <Emphasis Type="Italic">Cedrus</Emphasis>: consequences of axillary bud breaking

Reversed-phase HPLC analysis and random amplified polymorphic DNA (RAPD) markers were used to monitor DNA methylation status and genetic stability of C. atlantica and C. libani shoots generated through axillary bud proliferation. Average DNA methylation in C. atlantica or C. libani seedlings and mature 200-year-old trees of C. libani was 19.8, 19.5 and 22.3%, respectively. These global amounts showed no significant variation after the in vitro establishment of seedling-originated shoot stocks. In contrast, in vitro culture caused a significant decrease in the amount of 5-methylcytosine in genomic DNA of the tissue culture (TC) progenies of one of the adult C. libani genotypes. This DNA demethylation event accompanied an enhancement of the regrowth capacity of this genotype. Detected RAPD variation between mother plants and their TC progenies was species-related, with C. libani TC progenies being genetically more stable than those of C. atlantica. Nevertheless, similarity indices ranged from 0.97 to 1 among mother plants and their TC progenies. Furthermore, the analyses of molecular variance (AMOVA) suggest that RAPD variation among the mother plants and their TC progenies might be considered as not significant. The application of various statistical approaches, including cluster-based genetic distance methods and AMOVA, demonstrates that RAPD markers discriminate C. atlantica and C. libani appropriately.     

Studies on the production of conjugated linoleic acid from linoleic and vaccenic acids by mixed rumen protozoa

The conversion of β-myrcene to the furanoid flavour compound perillene by Pleurotus ostreatus was investigated using trideutero β-myrcene, trideutero α-(Z)-acaridiol and non-labeled 1,2- and 3,10-epoxy-β-myrcene, α,α-acarilactol, and perillene as substrates. Myrcene diols were formed from the cleavage of myrcene epoxides, but only α-(Z)-acaridiol, a 1,4-butanediol derivative most likely generated through a base-catalysed epoxide opening, was a suitable precursor of perillene. Once formed, this key intermediate was rapidly oxidised and the resulting cyclic lactol was dehydrated to yield perillene. Bioconversion of the supplemented perillene to α,α-acariolide indicated that perillene was another intermediate of the pathway and prone to further oxidative degradation. The data suggest that the fungus converted the cytotoxic β-myrcene in its environment into a metabolically useable carbon source along this route.     

Changes in biochemical composition of needles of ornamental dwarf spruce (Picea glauca ‘Conica’) induced by spruce spider mite (Oligonychus ununguis Jacobi, Acari: Tetranychidae) feeding

The impact of spruce spider mite (SSM) (Oligonychus ununguis Jacobi, Acari: Tetranychidae) feeding on needle compounds of young dwarf white spruce (Picea glauca ‘Conica’), important in defence against pests, was determined. It was shown that the direction and intensity of changes in chemical composition of spruce needles was related to the density of spruce spider mite population. Relative to uninfested controls, needles of trees infested by 2–6 mites per 5 cm oftwig during 8 weeks contained markedly higher concentration of soluble proteins, total phenolics and essential oil volatiles (linalool, β-phellandrene, β-myrcene, δ-3-carene, p-cymene, limonene, α-pinene, β-pinene, borneol, methyl salicylate, geranyl acetate). Feeding of a three times larger population of mites (18 specimens per 5 cm of twig) caused either reduction of concentration of those compounds, or no significant difference in comparison to the control. The results of our study show that O. ununguis at relatively low density (not exceeding 2–6 specimens per 5 cm of twig per 8 weeks) stimulates even susceptible host-plant to alter metabolism, however induced responses are suppressed as mite density increases. Findings are discussed in relation to the importance of changes in the concentration of needle primary and secondary metabolites to white spruce defence against O. ununguis.     

Traditional Tar Production from <Emphasis Type="Italic">Cedrus libani</Emphasis> A. Rich on the Taurus Mountains in Southern Turkey

Traditional Tar Production from Cedrus libani A. Rich on the Taurus Mountains in Southern Turkey. Cedrus libani A. Rich is a magnificent and sacred tree species native to the eastern Mediterranean region. Today, traditional people in southern Turkey produce wood extract, called katran, from C. libani, and use it to protect wooden structures against insects and fungi, to fight parasites and bacteria, and to heal wounds and cure various diseases in humans and domestic animals, both internally and externally. Here we illustrate traditional methods of producing katran, its use by local people, and its chemical composition.     

Biotransformation of β-myrcene to geraniol by a strain of Rhodococcus erythropolis isolated by selective enrichment from hop plants

The biocatalytic generation of high-value chemicals from abundant, cheap and renewable feedstocks is an area of great contemporary interest. A strain of Rhodococcus erythropolis designated MLT1 was isolated by selective enrichment from the soil surrounding hop plants, using the abundant triene β-myrcene from hops as a sole carbon source for growth. Resting cells of the organism were challenged with β-myrcene, and the major product of biotransformation was determined by mass spectrometric analysis to be the monoterpene alcohol geraniol. Controls demonstrated that the product was biogenic and that an aerobic environment was required. The ability to transform β-myrcene was shown to be restricted to cells that had been grown on this substrate as sole carbon source. Pre-incubation of cells with the cytochrome P450 inhibitors metyrapone or 1-aminobenzotriazole reduced geraniol production by 23% and 73% respectively, but reduction in activity was found not to correlate with the inhibitor concentration. A comparative analysis of insoluble and soluble cell extracts derived from cells of MLT1 grown on either β-myrcene or glucose revealed at least four proteins that were clearly overproduced in response to growth on β-myrcene. Mass spectrometric analysis of tryptic digests of three of these protein bands suggested their identities as an aldehyde dehydrogenase, an acyl-CoA dehydrogenase and a chaperone-like protein, each of which has a precedented role in hydrocarbon metabolism clusters in Rhodococcus sp. and which may therefore participate in a β-myrcene degradation pathway in this organism.     

Chemical composition and antimicrobial activity of the essential oil of Cacalia tangutica (Maxim.) Hand.-Mazz

Essential oil of the subterranean part of Cacalia tangutica (Maxim.) Hand.-Mazz was analyzed by gas chromatography (GC)-mass spectrum (MS) technique in two different capillary columns of different polarities. Thirty-one components were identified in the oil and the main compounds were α-zingiberene (13.49%), germacrene D (10.76%), α-pinene (8.54%), caryophyllene(Z-) (6.36%), linalool (6.16%), β-myrcene (4.89%), β-ocimene (Z-) (4.40%)and ocimenone(Z-) (3.58%). The antimicrobial activity of the oil was evaluated against 2 fungi and 12 bacteria including 6 clinically isolated strains using the agar disc diffusion and broth microdilution methods. The results show that the oil presented a broad antimicrobial spectrum and had better antimicrobial activity against yeast and gram-positive bacteria. The minimum inhibitory concentration values were 0.16–5.00 g/L and minimum bactericidal concentration values were 0.16–5.00 g/L. __________ Translated from Journal of Wuhan University (Science Edition), 2007, 53 (2): 198–203 [译自: 武汉大学学报(理学版)]     

Putative glacial refugia of Cedrus atlantica deduced from Quaternary pollen records and modern genetic diversity

Aim To investigate the impact of past environmental changes on Cedrus atlantica and its current genetic diversity, and to predict its future distribution. Location Morocco, Algeria and Tunisia. Methods Eleven fossil pollen records from these three countries were used to locate putative glacial refugia and to reconstruct past climate changes. A mechanistic vegetation distribution model was used to simulate the distribution of C. atlantica in the year 2100. In addition, a genetic survey was carried out on modern Moroccan C. atlantica. Results Pollen records indicate that Cedrus was present during the last glacial period, probably in scattered refugia, in Tunisia, Algeria and Morocco. In the Tunisian and Algerian sites, cedar expanded during the late glacial and the early Holocene, then disappeared after c. 8000 yr bp . Reconstructed mean annual precipitation and January temperature show that the last glacial period in Morocco was cooler by 10–15°C and drier by c. 300–400 mm year^?1 than the climate today. Modern chloroplast microsatellites of 15 C. atlantica populations in Morocco confirm the presence of multiple refugia and indicate that cedar recolonized the Moroccan mountains fairly recently. Model simulation indicates that by the year 2100 the potential distribution of C. atlantica will be much restricted with a potential survival area located in the High Atlas. Main conclusions Environmental changes in northern Africa since the last glacial period have had an impact on the geographical distribution of C. atlantica and on its modern genetic diversity. It is possible that by the end of this century C. atlantica may be unable to survive in its present‐day locations. To preserve the species, we suggest that seedlings from modern C. atlantica populations located in the High Atlas mountains, where a high genetic diversity is found, be transplanted into the western High Atlas.     

A new fossil Cedrus species from the early Miocene of northwestern Turkey and its possible affinities

A new fossil species, Cedrus anatolica n. sp., is described from the early Miocene Hançili Formation of Turkey. All analyses were performed on the thin sections housed at Istanbul University – Cerrahpasa. The new species was interpreted as having the closest affinity with the modern Mediterranean species Cedrus atlantica (Endlicher) Manetti ex Carriere and Cedrus libani Richard. The evolutionary line shows some changes in wood anatomy. From the early Cretaceous to the early Miocene, the pits on the tangential walls of the tracheids gradually decreased, the height of rays increased and the number of epithelial cells in the traumatic resin canals increased slightly. These features are similar in three modern species; other wood anatomical features are also quite stable among the new fossil and modern species.     

In vitro storage of cedar shoot cultures under minimal growth conditions

We developed procedures for slow-growth storage of Cedrus atlantica and Cedrus libani microcuttings of juvenile and adult origin, noting factors favouring the extension of subculture intervals. Microcuttings could be stored effectively up to 6 months at 4°C and reduced light intensity, provided that they were grown on a diluted modified MS medium. The addition of 6% mannitol to the storage media affected negatively survival and multiplication capacity of the cultures. The slow-growth storage conditions used in our experiments did not induce remarkable effects on both RAPD variability and average DNA methylation in the species.     

Essential Oils in Mexican Bays (<Emphasis Type="Italic">Litsea</Emphasis> spp<Emphasis Type="Italic">.</Emphasis>, Lauraceae): Taxonomic Assortment and Ethnobotanical Implications<Superscript>1</Superscript>

Essential Oils in Mexican Bays ( Litsea spp . , Lauraceae): Taxonomic Assortment and Ethnobotanical Implications. The seven species of Litsea found in Mexico, all of them popularly known as “laurel,” were surveyed by gas chromatography-mass spectrometry for their foliar essential oils composition and related ethnobotanical applications. Litsea glaucescens is in high demand as a condiment, and is sold in rural and urban markets all over Mexico. However, four other species are also locally used for food seasoning. Litsea guatemalensis is the species most used in traditional medicine, especially to treat fever, chills, infectious diseases of the digestive system, and arthritis. No reports of culinary, medicinal, or other applications were located for L. muelleri, and L. pringlei. This is the first report on the essential oils for L. neesiana, L. muelleri, L. parvifolia, L. pringlei, and L. schaffneri. The terpenoids commonly found in all the Litsea species studied were 1,8-cineole, linalool, α-pinene, β-pinene, m-cymene, terpinen-4-ol, α-terpineol, caryophyllene, and caryophyllene oxide. Nevertheless, each species can be distinguished by its characteristic assortment of terpenoids. According to hierarchical cluster analysis, three groups of species were recognized: (1) 1,8-cineole group (C-10 terpenes), consisting of L. glaucescens, L. schaffnerii, L. pringlei, and L. muelleri; (2) limonene-rich group (C-10 oxygenated terpenes), including L. guatemalensis, and L. neesiana, and (3) oxygenated sesquiterpenes-rich group (C-15 oxygenated terpenes), comprising L. parvifolia. The chemical profiles of L. glaucescens and L. guatemalensis suggest a correlation with the culinary and medicinal uses of these species due to the known properties of their main constituents.     

Genotoxic activity of <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> essential oil in <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> diploid cells

Eucalyptus globulus essential oil was evaluated for its genotoxic potential using a somatic segregation assay and a diploid strain of the fungus Aspergillus nidulans, heterozygous for nutritional and conidia color markers. The main compounds of the current essential oil sample were eucalyptol (49.0 %), α-pinene (8.9), β-pinene (1.5), globulol (6.9), α-eudesmol (1.12), spathulenol (1.42), γ-cadinene (1.45), trans-β-elemenone (1.23) and aromandendrene (2.3), totaling 74 % of oil. Oil at 0.12 and 0.25 μL/mL was found to increase the mitotic instability of the original diploid strain and the number of diploid mitotic recombinants of A. nidulans. The genotoxicity of the oil was associated with the induction of mitotic crossing-over or with oil-broken chromosomes.     

Biotransformation of monoterpenes by immobilized microalgae

This paper reports the biotransformation of carvone, limonene, β-pinene, thymol, and linalool using whole-cell-immobilized microalgal strains isolated from paddy fields of Iran. The strains was recognized by morphological characterization and assigned according to amplified 16S/18S rRNA genes by PCR. Ten unialgal strains including Chlorella, Oocystis, Chlamydomonas, and Synechococcus were immobilized in calcium alginate beads. After a 24-h incubation with substrates, characterization and identification of biotransformation products were done by GC/MS. None of the isolated immobilized microalgae converted β-pinene. In contrast, most of these strains biotransformed carvone and limonene to the related compounds. Some strains only reduced the C = C double bond to yield the dihydrocarvone isomers while others reduced the ketone to give the dihydrocarveol. The transformation ratio showed that Oocystis sp. MCCS 033 and Synechococcus sp. MCCS 035 produced dihydrocarvone isomers with the highest efficiency. Furthermore, limonene was converted into a mixture of five corresponding products and the maximum yield was 52.1% for carvone, the bioconverted product. Only one strain, Synechococcus sp. MCCS 034, oxidized thymol, and the product obtained from thymol was thymoquinone. Also, linalooloxide isomers and dihydrolinalool were obtained from linalool, and finally dihydrolinalool was the main product. These results showed a novel conversion pathway of linalool-forming dihydrolinalool.     

Extracellular enzymatic activity in 11 Cryptococcus species

The extracellular enzymatic activity of 36 strains of yeast belonging to 11 species of the genus Cryptococcus, has been investigated, using the API-ZYM (BioMérieux, France) commercial system, with the objective of determining the differences in the enzymatic profiles of the various species. The strains studied were : 9 of C. neoformans, 7 of C. albidus, 6 of C. laurentii, 5 of C. uniguttulatus, 3 of C. humicolus, and 1 each of C. ater, C. curvatus, C. dimennae, C. hungaricus, C. infirmo-miniatus and C. magnus. All the strains showed enzymatic activity with positivity to Phosphatase alkaline, Esterase lipase C8, Leucine arylamidase, Phosphatase acid and Naphthol-AS-BI-phosphohydrolase, and negativity to Lipase C14, Trypsin, Chemotrypsin, β-galactosidase, β-glucuronidase and α-manosidase. Variable enzymatic activity was shown to Esterase C4, Valine arylamidase, Cystine arylamidase, α-galactosidase,α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase and α-fucosidase. This allowed 11 separate enzymatic patterns to be established. The species C. neoformans and C. laurentii each presented two distinct patterns; C. uniguttulatus, C. hungaricus andC. magnus shared the same pattern; C. albidus, C. ater, C. curvatus,C. dimennae, C. humicolus and C. infirmo-miniatus presented an individual enzymatic pattern. The results obtained suggest that the API-ZYM system could be useful for the identification of species of the genus Cryptococcus and for the differentiation of the enzymotypes for epidemiological purposes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Phylogenetic analysis of the genus Pistacia by AFLP markers

This study addresses the phylogenetic relationships among Pistacia species by amplified fragment length polymorphisms (AFLPs). A total of 31 wild Pistacia accessions belonging to P. eurycarpa, P. khinjuk, P. atlantica, P. mutica, P. integerrima, P. terebinthus, P. palaestina, P. mexicana, P. lentiscus species and to a hybrid between P. atlantica and P. integerrima and four P. vera cultivars were the plant materials of this study. Six AFLP primer combinations generated a total of 275 fragments, an average of 45.8 bands per primer pair, of which 254 (92.4) were polymorphic. Unweighted pair group method based on arithmetic average (UPGMA) and principle coordinates (PCo) analysis were performed using both mean character difference and Jaccard similarity matrices. According to the results, P. vera, P. khinjuk, P. eurycarpa, P. atlantica, P. mutica, P. integerrima and P. atlantica x P. integerrima hybrids were in the first cluster. UPGMA analysis using mean character difference clustered P. palaestina, P. terebinthus, P. mexicana and P. lentiscus in the second cluster, whereas UPGMA analysis using Jaccard coefficient separated P. palaestina and P. terebinthus from P. lentiscus and P. mexicana. The P. khinjuk accessions had closer relationships to P. eurycarpa and P. atlantica than to P. vera which led to mis-identification of P. khinjuk samples as P. eurycarpa in this tudy. P. atlantica – P. mutica and P. terebinthus – P. palaestina pairs were the closest species, and therefore P. mutica were classified as P. atlantica, and P. palaestina were as P. terebinthus. The resulted dendrograms and the PCo plots in this study did not support evergreen versus deciduous sectional division of Pistacia species and suggested classifying P. terebinthus in a separate group rather than being in the first cluster. Further study is inevitable including more evergreen species and accessions to clarify the position of P. terebinthus and the evergreen species.     

Cedar forest communities in Western Antalya (Taurus Mountains,Turkey)

Abstract

The most extensive and magnificent forests of Cedrus libani A. Rich. in the world are on the Taurus Mountains in Turkey. They cover an area of about 400,000 hectares. C. libani forms either pure stands or mixed stands with other tree species, such as Abies cilicica, Pinus nigra, P. brutia, Quercus species (Q. cerris, Q. infectoria, Q. libani etc.), Juniperus excelsa and J. foetidissima. In this work, the floristic composition of the cedar forests in Western Antalya has been investigated and the ecological factors have been measured. The main forest types have been established and correlated with the ecological factors. In total, 201 relevés were analyzed by numerical methods and classified into five groups of C. libani forests and one group of Q. coccifera scrublands. It was observed that the topographical gradient mainly determines the differentiation patterns of cedar forests in Western Antalya.