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1.  High genetic diversity with moderate differentiation in Juniperus excelsa from Lebanon and the eastern Mediterranean region 
AoB Plants  2011;2011:plr003.
Juniperus excelsa constitutes a precious woody species of high ecological value able to grow up to Mountain treeline around the Mediterranean. Nuclear microsatellites were used to shed light on genetic diversity and differentiation of different Mediterranean populations. This information is essential in planning conservation strategies and reforestation programs.
Background and aims
Juniperus excelsa is an important woody species in the high mountain ecosystems of the eastern Mediterranean Basin where it constitutes the only coniferous species found at the tree line. The genetic diversity within and among J. excelsa populations of the eastern Mediterranean Basin is studied in the light of their historical fragmentation.
Methodology
Nuclear microsatellites originally developed for Juniperus communis and J. przewalskii were tested on 320 individuals from 12 different populations originating from Lebanon, Turkey, Cyprus, Greece and the Ukraine.
Principal results
Among the 31 nuclear microsatellite primers tested, only three produced specific amplification products, with orthology confirmed by sequence analysis. They were then used for genetic diversity studies. The mean number of alleles and the expected heterozygosity means were Na=8.78 and He=0.76, respectively. The fixation index showed a significant deviation from Hardy–Weinberg equilibrium and an excess of homozygotes (FIS=0.27–0.56). A moderate level of genetic differentiation was observed among the populations (FST=0.075, P<0.001). The most differentiated populations corresponded to old vestigial stands found at the tree line (>2000 m) in Lebanon. These populations were differentiated from the other populations that are grouped into three sub-clusters.
Conclusions
High levels of genetic diversity were observed at species and population levels. The high level of differentiation in the high-mountain Lebanese populations reflects a long period of isolation or possibly a different origin. The admixture observed in other populations from Lebanon suggests a more recent separation from the Turkish–southeastern European populations.
doi:10.1093/aobpla/plr003
PMCID: PMC3064508  PMID: 22476474
2.  Chemical composition and antimicrobial activity of essential oils of Juniperus excelsa Bieb. (Cupressaceae) grown in R. Macedonia 
Pharmacognosy Research  2015;7(1):74-80.
Background:
There are no information of the yield, chemical composition and antimicrobial activity of essential oils of berries (EOB) or leaves (EOL) of Juniperus excelsa Bieb. (Cupressaceae) growing wild in R. Macedonia.
Materials and Methods:
Plant material was collected from two localities during two seasons. Essential oil composition was analyzed by gas chromatography/flame ionization detector/mass spectrometry (GC/FID/MS) and antimicrobial screening was made by disc diffusion and broth dilution method.
Results and Discussion:
EOB yield ranged from 1.6-9.4 ml/kg and from 8.9-13.9 ml/kg for EOL. Two chemotypes of essential oil were differentiated, α-pinene-type (with 70.81% α-pinene in EOB and 33.83% in EOL), also containing limonene, β-pinene and β-myrcene while the sabinene-type (with 58.85-62.58% sabinene in EOB and 28.52-29.49% in EOL), was rich in α-pinene, β-myrcene, limonene, cis-thujone, terpinolene and α-thujene. The most sensitive bacteria to the antimicrobial activity of EOB was Haemophilus influenzae (MIC = 31 μl/ml). EOL have showed high activity towards: Staphylococcus aureus, Streptococcus pyogenes and Haemophilus influenzae (MIC = 125 μl/ml). The pinene-type of essential oil showed moderate activity against Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pyogenes, Corynebacterium spp. and Campylobacter jejuni (MIC >50%). The sabinene-type of the oil showed moderate activity to Streptococcus pyogenes, Haemopilus influenzae, Campylobacter jejuni and Escherichia coli (MIC >50%). No activity was observed toward Candida albicans.
Conclusion:
The analysis of EOB and EOL revealed two chemotypes (α-pinene and sabinene type) clearly depended on the geographical origin of the Macedonian Juniperus excelsa which also affected the antimicrobial activity of these oils.
doi:10.4103/0974-8490.147212
PMCID: PMC4285653  PMID: 25598638
Antimicrobial activity; essential oil; gas chromatography/flame ionization detector/mass spectrometry analysis; juniper berries and leaves; Juniperus excelsa
3.  Antioxidant Activity of the Essential Oils of Different Parts of Juniperus excelsa M. Bieb. subsp. excelsa and J. excelsa M. Bieb. subsp. polycarpos (K. Koch) Takhtajan (Cupressaceae) 
The essential oils of branchlets and fruits of Juniperus excelsa subsp. excelsa and Juniperus excelsa subsp. polycarpos were examined for their antioxidant activity. The compositions of the essential oils were studied by GC and GC-MS. To evaluation the antioxidants activity of the volatile oils, pure components and positive controls at different concentrations, thin-layer chromatography (TLC) screening methods, diphenylpicrylhydrazyl (DPPH) assay, deoxyribose degradation test and modified deoxyribose degradation test were employed. The results of the present study demonstrate some antioxidant activity for the tested essential oils obtained from various parts of both plants. It indicates that the use of these essential oils, in very low concentrations, may be useful as a natural preservative. However before any final conclusion, it is suggested that the antioxidant activity of these oils should also be evaluated by using lipid solvent system methods.
PMCID: PMC3813057  PMID: 24250416
Juniperus excelsa subsp. Excels; Juniperus excelsa subsp. Polycarpos; Cupressaceae; Essential oils; Antioxidant activity
4.  Anthropogenic Landscape in Southeastern Amazonia: Contemporary Impacts of Low-Intensity Harvesting and Dispersal of Brazil Nuts by the Kayapó Indigenous People 
PLoS ONE  2014;9(7):e102187.
Brazil nut, the Bertholletia excelsa seed, is one of the most important non-timber forest products in the Amazon Forest and the livelihoods of thousands of traditional Amazonian families depend on its commercialization. B. excelsa has been frequently cited as an indicator of anthropogenic forests and there is strong evidence that past human management has significantly contributed to its present distribution across the Amazon, suggesting that low levels of harvesting may play a positive role in B. excelsa recruitment. Here, we evaluate the effects of Brazil nut harvesting by the Kayapó Indigenous people of southeastern Amazonia on seedling recruitment in 20 B. excelsa groves subjected to different harvesting intensities, and investigated if management by harvesters influences patterns of B. excelsa distribution. The number of years of low-intensity Brazil nut harvesting by the Kayapó over the past two decades was positively related to B. excelsa seedling density in groves. One of the mechanisms behind the higher seedling density in harvested sites seems to be seed dispersal by harvesters along trails. The Kayapó also intentionally plant B. excelsa seeds and seedlings across their territories. Our results show not only that low-intensity Brazil nut harvesting by the Kayapó people does not reduce recruitment of seedlings, but that harvesting and/or associated activities conducted by traditional harvesters may benefit B. excelsa beyond grove borders. Our study supports the hypothesis that B. excelsa dispersal throughout the Amazon was, at least in part, influenced by indigenous groups, and strongly suggests that current human management contributes to the maintenance and formation of B. excelsa groves. We suggest that changes in Brazil nut management practices by traditional people to prevent harvesting impacts may be unnecessary and even counterproductive in many areas, and should be carefully evaluated before implementation.
doi:10.1371/journal.pone.0102187
PMCID: PMC4100818  PMID: 25029191
5.  Essential oil composition and nutrient analysis of selected medicinal plants in Sultanate of Oman 
Objective
To evaluate the nutrients and essential oils of five medicinal plants, Juniperus excelsa (J. excelsa), Dodonaea viscosa, Euryops pinifolius, Teucrium polium (T. polium), and Helianthemum lippii that were collected from Jabal Al Akhdar, Oman.
Methods
Proximate parameters (moisture, dry matter, ash, crude fats, proteins, fibers, nitrogen, carbohydrates, and energy values) and nutrient analysis (K, Na, Ca, Fe, P, Mg etc.) were evaluated in the five medicinal plants using standard techniques. On the basis of these analysis, T. polium and J. excels were selected for essential oil analysis using a rapid solvent-free microwave extraction method and GC-MS.
Results
The results showed that leaves of J. excelsa had highest proportion of crude fats, fibers and energy value while ash was highest in T. polium. J. excelsa was also rich in essential minerals such as calcium, magnesium, potassium and iron while the trace elements and heavy metals composition was marginal. A rapid solvent-free microwave extraction method to extract oil from medicinal plants species showed that only T. polium and J. excelsa yielded oil. The chemical composition of essential oils showed higher proportion of delta-3-carene, limonene, β-eudesmol, ledeneoxide (II), α-trans-bergamatene, linalyl acetate and germacrene.
Conclusions
J. excelsa and T. polium are a good source of proximate, minerals and essential oils, which can be considered for healthy life besides their medicinal values.
doi:10.1016/S2222-1808(13)60095-X
PMCID: PMC4027336
Proximate parameters; Essential oils; Omani medicinal plants; GC-MS
6.  Antioxidant Activity of Leaves and Fruits of Iranian Conifers 
Cupressus semipervirens var. horizontalis, Cupressus semipervirens var. semipervirens, Cupressus semipervirens cv. Cereifeormis, Juniperus communis subsp. hemisphaerica, Juniperus excelsa subsp. excelsa, Juniperus excelsa subsp. polycarpos, Juniperus foetidissima, Juniperus oblonga, Juniperus sabina, Platycladus orientalis and Taxus baccata are Iranian conifers. The antioxidant activity of leaves and fruits of these 11 different taxons were evaluated. The leaves of both male and female, and fruits of these plants were collected from different areas of the country. Methanol extract of leaves and fruits of these taxons were prepared. Antioxidant activity of each extracts was measured using two different tests of the ferric thiocyanate method and thiobarbituric acid. Results indicated that the methanol extracts of leaves, of male and female, and fruits of all these species (27 samples) possessed antioxidant activity when tested with both methods. The antioxidant activity was then compared with those of α-tocopherol (a natural antioxidant) and butylated hydroxytoluene (a synthetic antioxidant). Methanol extract of fruits of C. semipervirens cv. Cereifeormis showed the highest antioxidant activity while the methanol extract of leaves of C. semipervirens var. semipervirens possessed the lowest antioxidant activity. However, our finding showed that most of the tested extracts were showing strong antioxidant activity even higher than α-tocopherol.
doi:10.1093/ecam/nem011
PMCID: PMC1978238  PMID: 17965761
antioxidative activity; conifers, Cupressus; ferric thiocyanate test; Juniperus; medicinal plant; Platycladus orientalis; radical scavenging; thiobarbituric acid test
7.  In-vitro Antimicrobial Activities of Some Iranian Conifers 
Male and female leaves and fruits of eleven different taxons of Iranian conifers (Cupressus sempervirens var. horizontalis, C. sempervirens var. sempervirens, C. sempervirens cv. Cereifeormis, Juniperus communis subsp. hemisphaerica, J. excelsa subsp. excelsa, J. excelsa subsp. polycarpos, J. foetidissima, J. oblonga, J. sabina, Platycladus orientalis and Taxus baccata) were collected from different localities of Iran, dried and extracted with methanol. The extracts were tested for their antimicrobial activity against Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Candida albicans. The extracts were screened qualitatively using four different methods, the disc diffusion, hole plate, cylinder agar diffusion and agar dilution methods, whereas the minimum inhibitory concentrations (MIC) of each extract were determined by the agar dilution method. The best result was obtained by means of hole plate method in qualitative determination of antimicrobial activities of extracts and the greatest activity was found against S. aureus in all tested methods.
PMCID: PMC3813213  PMID: 24250573
Antimicrobial activity; Cupressus; Cupressaceae; Juniperus; Platycladus; Taxaceae; Taxus
8.  Gibberella xylarioides Sensu Lato from Coffea canephora: a New Mating Population in the Gibberella fujikuroi Species Complex 
Applied and Environmental Microbiology  2005;71(12):8466-8471.
Gibberella xylarioides Heim & Saccas (presumed anamorph, Fusarium xylarioides Steyaert) is the causal agent of coffee wilt disease, an economically important tracheomycosis in Africa. In vitro crosses carried out with Congolese, Ugandan, and Tanzanian single-ascospore/conidial isolates originating from diseased Coffea canephora/excelsa demonstrated a heterothallic mating system, controlled by a single locus with two alleles, MAT-1 and MAT-2. Compatible isolates produced fertile perithecia within 2 to 8 weeks after mating. Mating type (MAT) was characterized by PCR with primer pairs previously developed for the Gibberella fujikuroi species complex (GFC) and for Fusarium oxysporum. All strains analyzed were morphologically identical and corresponded to Booth's description of the “female” F. xylarioides strain. Based on crossing results and MAT-2/translation elongation 1-α (tef) sequence data, G. xylarioides, as currently understood, is demonstrated to encompass at least three “groups”: G. xylarioides sensu strictu Ia, defined hitherto by two “historical” West African strains originating from the severe 1930s to 1950s epidemic (CBS 25852 and CBS 74979); G. xylarioides sensu strictu Ib, defined by two “historical” Central African lowland strains (DSMZ 62457 and ATCC 15664); and G. xylarioides sensu lato II, containing Congolese, Ugandan, and Tanzanian C. canephora/excelsa isolates. Infertility of crosses between the coffee wilt pathogen and known GFC mating populations demonstrates that G. xylarioides sensu lato constitutes a new biological species within the G. fujikuroi complex. MUCL 44532/MUCL 43887 and MUCL 35223/MUCL 44549 are proposed as G. xylarioides sensu lato II MAT-1/MAT-2 reference mating type tester strains.
doi:10.1128/AEM.71.12.8466-8471.2005
PMCID: PMC1317314  PMID: 16332836
9.  Brazil nuts intake improves lipid profile, oxidative stress and microvascular function in obese adolescents: a randomized controlled trial 
Background
Obesity is a chronic disease associated to an inflammatory process resulting in oxidative stress that leads to morpho-functional microvascular damage that could be improved by some dietary interventions. In this study, the intake of Brazil nuts (Bertholletia excelsa), composed of bioactive substances like selenium, α- e γ- tocopherol, folate and polyunsaturated fatty acids, have been investigated on antioxidant capacity, lipid and metabolic profiles and nutritive skin microcirculation in obese adolescents.
Methods
Obese female adolescents (n = 17), 15.4 ± 2.0 years and BMI of 35.6 ± 3.3 kg/m2, were randomized 1:1 in two groups with the diet supplemented either with Brazil nuts [BNG, n = 08, 15-25 g/day (equivalent to 3 to 5 units/day)] or placebo [PG (lactose), n = 09, one capsule/day] and followed for 16 weeks. Anthropometry, metabolic-lipid profiles, oxidative stress and morphological (capillary diameters) and functional [functional capillary density, red blood cell velocity (RBCV) at baseline and peak (RBCVmax) and time (TRBCVmax) to reach it during post-occlusive reactive hyperemia, after 1 min arterial occlusion] microvascular variables were assessed by nailfold videocapillaroscopy at baseline (T0) and after intervention (T1).
Results
T0 characteristics were similar between groups. At T1, BNG (intra-group variation) had increased selenium levels (p = 0.02), RBCV (p = 0.03) and RBCVmax (p = 0.03) and reduced total (TC) (p = 0.02) and LDL-cholesterol (p = 0.02). Compared to PG, Brazil nuts intake reduced TC (p = 0.003), triglycerides (p = 0.05) and LDL-ox (p = 0.02) and increased RBCV (p = 0.03).
Conclusion
Brazil nuts intake improved the lipid profile and microvascular function in obese adolescents, possibly due to its high level of unsaturated fatty acids and bioactive substances.
Trial Registration
Clinical Trials.gov NCT00937599
doi:10.1186/1743-7075-8-32
PMCID: PMC3123174  PMID: 21619692
microcirculation; obesity; Brazil nuts; adolescents; oxidative stress; lipid profile
10.  Antihistaminic activity of aqueous extract of stem bark of Ailanthus excelsa Roxb. 
Pharmacognosy Research  2011;3(3):220-224.
Background:
Biologically active compounds from natural sources are of interest as possible new drugs for different diseases. Over many centuries humans have been mining the bounties of nature for discovering natural products that have been used for the treatment of all human diseases. Ailanthus excelsa Roxb. (Simaroubaceae) is widely used in the Indian system of medicine as an antiasthmatic, antispasmodic, bronchodilator, anticolic pain, anticancer, antidiabetic etc. The plant was also reported for its antiasthmatic, bronchodilatory, antiallegic and many more such activities.
Objective:
To evaluate the antihistaminic activity of aqueous extract of stem bark of Ailanthus excelsa Roxb.
Materials and Methods:
We have studied the effect of aqueous extract of stem barks of A. excelsa Roxb. at a doses 100 μg/mL in the isolated goat tracheal chain preparation in vitro and 100, 200, 400 mg/kg doses orally in passive paw anaphylaxis in rat, clonidine-induced catalepsy in mice models in vivo for its antihistaminic activity.
Results:
Aqueous extract of stem barks of A. excelsa Roxb. significantly (***P<0.001) inhibits the percentage contraction at concentration of 100 μg/mL in goat tracheal chain preparation. A. excelsa Roxb. extract (100, 200, and 400 mg/kg oral) and dexamethasone (0.5 mg/kg, i.p.) also significantly reduced (**P<0.01) the paw volume at fourth hour and the percentage inhibition was found to be 13.98%, 28.49%, 42.47% and 46.77% respectively. The aqueous extract of stem barks of A. excelsa Roxb. (100, 200, 400 mg/kg, p.o.) and chlorpheniramine maleate (10 mg/kg, i.p.) significantly inhibited (*P<0.05, **P< 0.01) clonidine-induced catalepsy in mice at 150 min after the administration of clonidine.
Conclusion:
The aqueous extract of stem bark of A. excelsa Roxb. possess significant antihistaminic activity (H1-antagonist) and can be attributed to bronchodilating, anti-inflammatory, adaptogenic activity etc. Hence detailed study needs to be conducted to evaluate the phytoconstituent responsible for the above mentioned results and their clinical efficacy in the treatment of related diseases.
doi:10.4103/0974-8490.85014
PMCID: PMC3193625  PMID: 22022173
Ailanthus excelsa Roxb; antihistaminic activity; passive paw anaphylaxis
11.  Genetic structure and seed-mediated dispersal rates of an endangered shrub in a fragmented landscape: a case study for Juniperus communis in northwestern Europe 
BMC Genetics  2011;12:73.
Background
Population extinction risk in a fragmented landscape is related to the differential ability of the species to spread its genes across the landscape. The impact of landscape fragmentation on plant population dynamics will therefore vary across different spatial scales. We quantified successful seed-mediated dispersal of the dioecious shrub Juniperus communis in a fragmented landscape across northwestern Europe by using amplified fragment length polymorphism (AFLP) markers. Furthermore we investigated the genetic diversity and structure on two spatial scales: across northwestern Europe and across Flanders (northern Belgium). We also studied whether seed viability and populations size were correlated with genetic diversity.
Results
Unexpectedly, estimated seed-mediated dispersal rates were quite high and ranged between 3% and 14%. No population differentiation and no spatial genetic structure were detected on the local, Flemish scale. A significant low to moderate genetic differentiation between populations was detected at the regional, northwest European scale (PhiPT = 0.10). In general, geographically nearby populations were also genetically related. High levels of within-population genetic diversity were detected but no correlation was found between any genetic diversity parameter and population size or seed viability.
Conclusions
In northwestern Europe, landscape fragmentation has lead to a weak isolation-by-distance pattern but not to genetic impoverishment of common juniper. Substantial rates of successful migration by seed-mediated gene flow indicate a high dispersal ability which could enable Juniperus communis to naturally colonize suitable habitats. However, it is not clear whether the observed levels of migration will suffice to counterbalance the effects of genetic drift in small populations on the long run.
doi:10.1186/1471-2156-12-73
PMCID: PMC3176195  PMID: 21859457
12.  Micropropagation of Araucaria excelsa R. Br. var. glauca Carrière from orthotropic stem explants 
The objectives of the present work were in vitro propagation of Araucaria excelsa R. Br. var. glauca Carrière (Norfolk Island pine) with focus on the evaluation of the mean number of shoots per explant (MNS/E) and mean length of shoots per explants (MLS/E) produced by different parts of the orthotropic stem of A. excelsa R. Br. var. glauca in response to plant growth regulators. Norfolk Island pine axillary meristems responded very well to the 2-iso-pentenyl adenine (2iP) and thidiazuron (TDZ) levels. Explants taken from stem upper segments in the media containing 2iP had a higher MNS/E (3.47) and MLS/E (6.27 mm) in comparison to those taken from stem lower segments, which were 0.71 and 0.51 mm, respectively. Using 0.045 μM TDZ in the MS medium not only resulted in 4.60 MNS/E with 7.08 mm MLS/E but proliferated shoots showed a good performance as well. Investigating the best position of stem explant on mother plant as well as the best concentrations of growth regulators were performed which were useful for efficient micropropagation of this plant. Thirty three percent of explants were rooted in the MS medium containing 3 % sucrose, supplemented with 7.5 μM of both NAA and IBA for 2 weeks before transferring to a half strength MS medium without any growth regulator. Plantlets obtained were acclimatized and transferred to the greenhouse with less than 20 % mortality. This procedure considered the first successful report for regeneration and acclimatization of A. excelsa R. Br. var. glauca plantlet through main stem explants.
doi:10.1007/s12298-012-0115-9
PMCID: PMC3550516  PMID: 23814441
Abnormality; Araucaria excelsa R. Br. var. glauca; Axillary meristems; Orthotropic stems; Proliferation; Topophysis
13.  Genetic Variation in Five Mediterranean Populations of Juniperus phoenicea as Revealed by Inter-Simple Sequence Repeat (ISSR) Markers 
Annals of Botany  2006;97(2):299-304.
• Background and Aims The assessment of the genetic variability and the identification of isolated populations within a given species represent important information to plan conservation strategies on a genetic basis. In this work, the genetic variability in five natural populations of Juniperus phoenicea, three from Sardinia, one from Cyprus and the last one in the Maritime Alps was analysed by means of ISSRs, on the hypothesis that the latter could have been a refugial one during the last glaciation.
• Methods ISSRs were chosen because of their ability to detect variation without any prior sequence information. The use of three primers yielded 45 reproducible, polymorphic bands, which were utilized to estimate the basic parameters of genetic variability and diversity.
• Key Results All of the populations analysed harboured an adequate amount of genetic variability, with HS = 0·1299. The proportion of genetic diversity between populations has been estimated by GST = 0·12. The three Sardinian populations are separated, as tested by AMOVA, from the Cyprus and the continental ones.
• Conclusions The results indicate that geographical isolation has represented a major barrier to gene flow in Juniperus phoenicea. This work represents a first step towards a full genetic characterization of a conifer from the Mediterranean, a world biodiversity hotspot confronted with climate change, and thus contributes towards the planning of genetics-informed conservation strategies.
doi:10.1093/aob/mcj024
PMCID: PMC2803359  PMID: 16311272
Juniperus phoenicea L; genetic variation; ISSR; conservation; Cupressaceae
14.  Comparative Phylogeography in Rainforest Trees from Lower Guinea, Africa 
PLoS ONE  2014;9(1):e84307.
Comparative phylogeography is an effective approach to assess the evolutionary history of biological communities. We used comparative phylogeography in fourteen tree taxa from Lower Guinea (Atlantic Equatorial Africa) to test for congruence with two simple evolutionary scenarios based on physio-climatic features 1) the W-E environmental gradient and 2) the N-S seasonal inversion, which determine climatic and seasonality differences in the region. We sequenced the trnC-ycf6 plastid DNA region using a dual sampling strategy: fourteen taxa with small sample sizes (dataset 1, mean n = 16/taxon), to assess whether a strong general pattern of allele endemism and genetic differentiation emerged; and four taxonomically well-studied species with larger sample sizes (dataset 2, mean n = 109/species) to detect the presence of particular shared phylogeographic patterns. When grouping the samples into two alternative sets of two populations, W and E, vs. N and S, neither dataset exhibited a strong pattern of allelic endemism, suggesting that none of the considered regions consistently harboured older populations. Differentiation in dataset 1 was similarly strong between W and E as between N and S, with 3–5 significant FST tests out of 14 tests in each scenario. Coalescent simulations indicated that, given the power of the data, this result probably reflects idiosyncratic histories of the taxa, or a weak common differentiation pattern (possibly with population substructure) undetectable across taxa in dataset 1. Dataset 2 identified a common genetic break separating the northern and southern populations of Greenwayodendron suaveolens subsp. suaveolens var. suaveolens, Milicia excelsa, Symphonia globulifera and Trichoscypha acuminata in Lower Guinea, in agreement with differentiation across the N–S seasonal inversion. Our work suggests that currently recognized tree taxa or suspected species complexes can contain strongly differentiated genetic lineages, which could lead to misinterpretation of phylogeographic patterns. Therefore the evolutionary processes of such taxa require further study in African tropical rainforests.
doi:10.1371/journal.pone.0084307
PMCID: PMC3885573  PMID: 24416215
15.  A Novel Triterpenoid Isolated from the Root Bark of Ailanthus excelsa Roxb (Tree of Heaven), AECHL-1 as a Potential Anti-Cancer Agent 
PLoS ONE  2009;4(4):e5365.
Background
We report here the isolation and characterization of a new compound Ailanthus excelsa chloroform extract-1 (AECHL-1) (C29H36O10; molecular weight 543.8) from the root bark of Ailanthus excelsa Roxb. The compound possesses anti-cancer activity against a variety of cancer cell lines of different origin.
Principal Findings
AECHL-1 treatment for 12 to 48 hr inhibited cell proliferation and induced death in B16F10, MDA-MB-231, MCF-7, and PC3 cells with minimum growth inhibition in normal HEK 293. The antitumor effect of AECHL-1 was comparable with that of the conventional antitumor drugs paclitaxel and cisplatin. AECHL-1-induced growth inhibition was associated with S/G2-M arrests in MDA-MB-231, MCF-7, and PC3 cells and a G1 arrest in B16F10 cells. We observed microtubule disruption in MCF-7 cells treated with AECHL-1 in vitro. Compared with control, subcutaneous injection of AECHL-1 to the sites of tumor of mouse melanoma B16F10 implanted in C57BL/6 mice and human breast cancer MCF-7 cells in athymic nude mice resulted in significant decrease in tumor volume. In B16F10 tumors, AECHL-1 at 50 µg/mouse/day dose for 15 days resulted in increased expression of tumor suppressor proteins P53/p21, reduction in the expression of the oncogene c-Myc, and downregulation of cyclin D1 and cdk4. Additionally, AECHL-1 treatment resulted in the phosphorylation of p53 at serine 15 in B16F10 tumors, which seems to exhibit p53-dependent growth inhibitory responses.
Conclusions
The present data demonstrate the activity of a triterpenoid AECHL-1 which possess a broad spectrum of activity against cancer cells. We propose here that AECHL-1 is a futuristic anti-cancer drug whose therapeutic potential needs to be widely explored for chemotherapy against cancer.
doi:10.1371/journal.pone.0005365
PMCID: PMC2671403  PMID: 19399188
16.  Multilocus Microsatellite Typing (MLMT) of Strains from Turkey and Cyprus Reveals a Novel Monophyletic L. donovani Sensu Lato Group 
Background
New foci of human CL caused by strains of the Leishmania donovani (L. donovani) complex have been recently described in Cyprus and the Çukurova region in Turkey (L. infantum) situated 150 km north of Cyprus. Cypriot strains were typed by Multilocus Enzyme Electrophoresis (MLEE) using the Montpellier (MON) system as L. donovani zymodeme MON-37. However, multilocus microsatellite typing (MLMT) has shown that this zymodeme is paraphyletic; composed of distantly related genetic subgroups of different geographical origin. Consequently the origin of the Cypriot strains remained enigmatic.
Methodology/Principal Findings
The Cypriot strains were compared with a set of Turkish isolates obtained from a CL patient and sand fly vectors in south-east Turkey (Çukurova region; CUK strains) and from a VL patient in the south-west (Kuşadasi; EP59 strain). These Turkish strains were initially analyzed using the K26-PCR assay that discriminates MON-1 strains by their amplicon size. In line with previous DNA-based data, the strains were inferred to the L. donovani complex and characterized as non MON-1. For these strains MLEE typing revealed two novel zymodemes; L. donovani MON-309 (CUK strains) and MON-308 (EP59). A population genetic analysis of the Turkish isolates was performed using 14 hyper-variable microsatellite loci. The genotypic profiles of 68 previously analyzed L. donovani complex strains from major endemic regions were included for comparison. Population structures were inferred by combination of Bayesian model-based and distance-based approaches. MLMT placed the Turkish and Cypriot strains in a subclade of a newly discovered, genetically distinct L. infantum monophyletic group, suggesting that the Cypriot strains may originate from Turkey.
Conclusion
The discovery of a genetically distinct L. infantum monophyletic group in the south-eastern Mediterranean stresses the importance of species genetic characterization towards better understanding, monitoring and controlling the spread of leishmaniasis in this region.
Author Summary
In eastern Mediterranean, leishmaniasis represents a major public health problem with considerable impact on morbidity and potential to spread. Cutaneous leishmaniasis (CL) caused by L. major or L. tropica accounts for most cases in this region although visceral leishmaniasis (VL) caused by L. infantum is also common. New foci of human CL caused by L. donovani complex strains were recently described in Cyprus and Turkey. Herein we analyzed Turkish strains from human CL foci in Çukurova region (north of Cyprus) and a human VL case in Kuşadasi. These were compared to Cypriot strains that were previously typed by Multilocus Enzyme Electrophoresis (MLEE) as L. donovani MON-37. Nevertheless, they were found genetically distinct from MON-37 strains of other regions and therefore their origin remained enigmatic. A population study was performed by Multilocus Microsatellite Typing (MLMT) and the profile of the Turkish strains was compared to previously analyzed L. donovani complex strains. Our results revealed close genetic relationship between Turkish and Cypriot strains, which form a genetically distinct L. infantum monophyletic group, suggesting that Cypriot strains may originate from Turkey. Our analysis indicates that the epidemiology of leishmaniasis in this region is more complicated than originally thought.
doi:10.1371/journal.pntd.0001507
PMCID: PMC3279343  PMID: 22348162
17.  Analysis of antimicrobial, antifungal and antioxidant activities of Juniperus excelsa M. B subsp. Polycarpos (K. Koch) Takhtajan essential oil 
Pharmacognosy Research  2010;2(3):128-131.
Juniperus excelsa M.B subsp. Polycarpos (K.Koch), collected from south of Iran, was subjected to hydrodistillation using clevenger apparatus to obtain essential oil. The essential was analyzed by gas chromatography/mass spectrometry (GC/MS) and studied for antimicrobial, antifungal and antioxidant activities. The results indicated α-pinene (67.71%) as the major compound and α-cedral (11.5%), δ3-carene (5.19%) and limonene (4.41%) in moderate amounts. Antimicrobial tests were carried out using disk diffusion method, followed by the measurement of minimum inhibitory concentration (MIC). All the Gram positive and Gram negative bacteria were susceptible to essential oil. The oil showed radical scavenging and antioxidant effects.
doi:10.4103/0974-8490.65505
PMCID: PMC3141302  PMID: 21808554
Antimicrobial activity; antioxidant activity; essential oil; Juniperus excelsa; thin layer chromatography autographic assay
18.  Genetic structure and systematic relationships within the Ophrys fuciflora aggregate (Orchidaceae: Orchidinae): high diversity in Kent and a wind-induced discontinuity bisecting the Adriatic 
Annals of Botany  2009;104(3):483-495.
Background and Aims
A recent phylogenetic study based on multiple datasets is used as the framework for a more detailed examination of one of the ten molecularly circumscribed groups identified, the Ophrys fuciflora aggregate. The group is highly morphologically variable, prone to phenotypic convergence, shows low levels of sequence divergence and contains an unusually large proportion of threatened taxa, including the rarest Ophrys species in the UK. The aims of this study were to (a) circumscribe minimum resolvable genetically distinct entities within the O. fuciflora aggregate, and (b) assess the likelihood of gene flow between genetically and geographically distinct entities at the species and population levels.
Methods
Fifty-five accessions sampled in Europe and Asia Minor from the O. fuciflora aggregate were studied using the AFLP genetic fingerprinting technique to evaluate levels of infraspecific and interspecific genetic variation and to assess genetic relationships between UK populations of O. fuciflora s.s. in Kent and in their continental European and Mediterranean counterparts.
Key Results
The two genetically and geographically distinct groups recovered, one located in England and central Europe and one in south-eastern Europe, are incongruent with current species delimitation within the aggregate as a whole and also within O. fuciflora s.s. Genetic diversity is higher in Kent than in the rest of western and central Europe.
Conclusions
Gene flow is more likely to occur between populations in closer geographical proximity than those that are morphologically more similar. Little if any gene flow occurs between populations located in the south-eastern Mediterranean and those dispersed throughout the remainder of the distribution, revealing a genetic discontinuity that runs north–south through the Adriatic. This discontinuity is also evident in other clades of Ophrys and is tentatively attributed to the long-term influence of prevailing winds on the long-distance distribution of pollinia and especially seeds. A cline of gene flow connects populations from Kent and central and southern Europe; these individuals should therefore be considered part of an extensive meta-population. Gene flow is also evident among populations from Kent, which appear to constitute a single metapopulation. They show some evidence of hybridization, and possibly also introgression, with O. apifera.
doi:10.1093/aob/mcp039
PMCID: PMC2720644  PMID: 19251716
AFLP; conservation; gene flow; Kent; migration; Ophrys fuciflora; prevailing winds; pseudocopulation; speciation; species delimitation
19.  Genetic rescue of remnant tropical trees by an alien pollinator. 
Habitat fragmentation is thought to lower the viability of tropical trees by disrupting their mutualisms with native pollinators. However, in this study, Dinizia excelsa (Fabaceae), a canopy-emergent tree, was found to thrive in Amazonian pastures and forest fragments even in the absence of native pollinators. Canopy observations indicated that African honeybees (Apis mellifera scutellata) were the predominant floral visitors in fragmented habitats and replaced native insects in isolated pasture trees. Trees in habitat fragments produced, on average, over three times as many seeds as trees in continuous forest, and microsatellite assays of seed arrays showed that genetic diversity was maintained across habitats. A paternity analysis further revealed gene flow over as much as 3.2 km of pasture, the most distant pollination precisely recorded for any plant species. Usually considered only as dangerous exotics, African honeybees have become important pollinators in degraded tropical forests, and may alter the genetic structure of remnant populations through frequent long-distance gene flow.
doi:10.1098/rspb.2001.1781
PMCID: PMC1088891  PMID: 11703880
20.  Masting in ponderosa pine: comparisons of pollen and seed over space and time 
Oecologia  2010;165(3):651-661.
Many plant species exhibit variable and synchronized reproduction, or masting, but less is known of the spatial scale of synchrony, effects of climate, or differences between patterns of pollen and seed production. We monitored pollen and seed cone production for seven Pinus ponderosa populations (607 trees) separated by up to 28 km and 1,350 m in elevation in Boulder County, Colorado, USA for periods of 4–31 years for a mean per site of 8.7 years for pollen and 12.1 for seed cone production. We also analyzed climate data and a published dataset on 21 years of seed production for an eighth population (Manitou) 100 km away. Individual trees showed high inter-annual variation in reproduction. Synchrony was high within populations, but quickly became asynchronous among populations with a combination of increasing distance and elevational difference. Inter-annual variation in temperature and precipitation had differing influences on seed production for Boulder County and Manitou. We speculate that geographically variable effects of climate on reproduction arise from environmental heterogeneity and population genetic differentiation, which in turn result in localized synchrony. Although individual pines produce pollen and seed, only one-third of the covariation within trees was shared. As compared to seed cones, pollen had lower inter-annual variation at the level of the individual tree and was more synchronous. However, pollen and seed production were similar with respect to inter-annual variation at the population level, spatial scales of synchrony and associations with climate. Our results show that strong masting can occur at a localized scale, and that reproductive patterns can differ between pollen and seed cone production in a hermaphroditic plant.
doi:10.1007/s00442-010-1742-x
PMCID: PMC3038219  PMID: 20706849
Climate forcing; Masting; Spatial autocorrelation; Resource pulse; Wind pollination
21.  Purification, crystallization and initial crystallographic characterization of brazil-nut allergen Ber e 2 
The crystallization of the brazil nut allergen Ber e 2 is reported.
Peanut and tree-nut allergies have attracted considerable attention because of their frequency and their lifelong persistence. Brazil-nut (Bertholletia excelsa) allergies have been well documented and the 11S legumin-like seed storage protein Ber e 2 (excelsin) is one of the two known brazil-nut allergens. In this study, Ber e 2 was extracted from brazil-nut kernels and purified to high purity by crystalline precipitation and gel-filtration chromatography. Well diffracting single crystals were obtained using the hanging-drop vapour-diffusion method. A molecular-replacement structural solution has been obtained. Refinement of the structure is currently under way.
doi:10.1107/S1744309107051445
PMCID: PMC2339761  PMID: 18007055
brazil nut allergy; Ber e 2; cupin superfamily; allergenicity
22.  Broad geographic covariation between floral traits and the mating system in Camissoniopsis cheiranthifolia (Onagraceae): multiple stable mixed mating systems across the species' range? 
Annals of Botany  2011;109(3):599-611.
Background and Aims
Plants vary widely in the extent to which seeds are produced via self-fertilization vs. outcrossing, and evolutionary change in the mating system is thought to be accompanied by genetic differentiation in a syndrome of floral traits. We quantified the pattern of variation and covariation in floral traits and the proportion of seeds outcrossed (t) to better understand the evolutionary processes involved in mating system differentiation among and within populations of the short-lived Pacific coastal dune endemic Camissoniopsis cheiranthifolia across its geographic range in western North America.
Methods
We quantified corolla width and herkogamy, two traits expected to influence the mating system, for 48 populations sampled in the field and for a sub-sample of 29 populations grown from seed in a glasshouse. We also measured several other floral traits for 9–19 populations, estimated t for 16 populations using seven allozyme polymorphisms, and measured the strength of self-incompatibility for nine populations.
Key Results
Floral morphology and self-incompatibility varied widely but non-randomly, such that populations could be assigned to three phenotypically and geographically divergent groups. Populations spanned the full range of outcrossing (t = 0·001–0·992), which covaried with corolla width, herkogamy and floral life span. Outcrossing also correlated with floral morphology within two populations that exhibited exceptional floral variation.
Conclusions
Populations of C. cheiranthifolia seem to have differentiated into three modal mating systems: (1) predominant outcrossing associated with self-incompatibility and large flowers; (2) moderate selfing associated with large but self-compatible flowers; and (3) higher but not complete selfing associated with small, autogamous, self-compatible flowers. The transition to complete selfing has not occurred even though the species appears to possess the required genetic capacity. We hypothesize that outcrossing populations in this species have evolved to different stable states of mixed mating.
doi:10.1093/aob/mcr266
PMCID: PMC3278294  PMID: 22028462
Allozymes; Camissoniopsis cheiranthifolia; corolla width; flower size; geographic variation; herkogamy; mating system; Onagraceae; outcrossing; self-fertilization; self-incompatibility
23.  An ecological genetic delineation of local seed-source provenance for ecological restoration 
Ecology and Evolution  2013;3(7):2138-2149.
An increasingly important practical application of the analysis of spatial genetic structure within plant species is to help define the extent of local provenance seed collection zones that minimize negative impacts in ecological restoration programs. Here, we derive seed sourcing guidelines from a novel range-wide assessment of spatial genetic structure of 24 populations of Banksia menziesii (Proteaceae), a widely distributed Western Australian tree of significance in local ecological restoration programs. An analysis of molecular variance (AMOVA) of 100 amplified fragment length polymorphism (AFLP) markers revealed significant genetic differentiation among populations (ΦPT = 0.18). Pairwise population genetic dissimilarity was correlated with geographic distance, but not environmental distance derived from 15 climate variables, suggesting overall neutrality of these markers with regard to these climate variables. Nevertheless, Bayesian outlier analysis identified four markers potentially under selection, although these were not correlated with the climate variables. We calculated a global R-statistic using analysis of similarities (ANOSIM) to test the statistical significance of population differentiation and to infer a threshold seed collection zone distance of ∼60 km (all markers) and 100 km (outlier markers) when genetic distance was regressed against geographic distance. Population pairs separated by >60 km were, on average, twice as likely to be significantly genetically differentiated than population pairs separated by <60 km, suggesting that habitat-matched sites within a 30-km radius around a restoration site genetically defines a local provenance seed collection zone for B. menziesii. Our approach is a novel probability-based practical solution for the delineation of a local seed collection zone to minimize negative genetic impacts in ecological restoration.
doi:10.1002/ece3.595
PMCID: PMC3728953  PMID: 23919158
AMOVA; AFLP; ANOSIM; BayeScan; Banksia; Proteaceae
24.  X-chromosome SNP analyses in 11 human Mediterranean populations show a high overall genetic homogeneity except in North-west Africans (Moroccans) 
Background
Due to its history, with a high number of migration events, the Mediterranean basin represents a challenging area for population genetic studies. A large number of genetic studies have been carried out in the Mediterranean area using different markers but no consensus has been reached on the genetic landscape of the Mediterranean populations. In order to further investigate the genetics of the human Mediterranean populations, we typed 894 individuals from 11 Mediterranean populations with 25 single-nucleotide polymorphisms (SNPs) located on the X-chromosome.
Results
A high overall homogeneity was found among the Mediterranean populations except for the population from Morocco, which seemed to differ genetically from the rest of the populations in the Mediterranean area. A very low genetic distance was found between populations in the Middle East and most of the western part of the Mediterranean Sea.
A higher migration rate in females versus males was observed by comparing data from X-chromosome, mt-DNA and Y-chromosome SNPs both in the Mediterranean and a wider geographic area.
Multilocus association was observed among the 25 SNPs on the X-chromosome in the populations from Ibiza and Cosenza.
Conclusion
Our results support both the hypothesis of (1) a reduced impact of the Neolithic Wave and more recent migration movements in NW-Africa, and (2) the importance of the Strait of Gibraltar as a geographic barrier. In contrast, the high genetic homogeneity observed in the Mediterranean area could be interpreted as the result of the Neolithic wave caused by a large demic diffusion and/or more recent migration events. A differentiated contribution of males and females to the genetic landscape of the Mediterranean area was observed with a higher migration rate in females than in males. A certain level of background linkage disequilibrium in populations in Ibiza and Cosenza could be attributed to their demographic background.
doi:10.1186/1471-2148-8-75
PMCID: PMC2315647  PMID: 18312628
25.  Colonization of Abandoned Land by Juniperus thurifera Is Mediated by the Interaction of a Diverse Dispersal Assemblage and Environmental Heterogeneity 
PLoS ONE  2012;7(10):e46993.
Land abandonment is one of the most powerful global change drivers in developed countries where recent rural exodus has been the norm. Abandonment of traditional land use practices has permitted the colonization of these areas by shrub and tree species. For fleshy fruited species the colonization of new areas is determined by the dispersal assemblage composition and abundance. In this study we showed how the relative contribution to the dispersal process by each animal species is modulated by the environmental heterogeneity and ecosystem structure. This complex interaction caused differential patterns on the seed dispersal in both, landscape patches in which the process of colonization is acting nowadays and mature woodlands of Juniperus thurifera, a relict tree distributed in the western Mediterranean Basin. Thrushes (Turdus spp) and carnivores (red fox and stone marten) dispersed a high amount of seeds while rabbits and sheeps only a tiny fraction. Thrushes dispersed a significant amount of seeds in new colonization areas, however they were limited by the presence of high perches with big crop size. While carnivores dispersed seeds to all studied habitats, even in those patches where no trees of J. thurifera were present, turning out to be critical for primary colonization. The presence of Pinus and Quercus was related to a reduced consumption of J. thurifera seeds while the presence of fleshy fruited shrubs was related with higher content of J. thurifera seeds in dispersers’ faeces. Therefore environmental heterogeneity and ecosystem structure had a great influence on dispersers feeding behaviour, and should be considered in order to accurately describe the role of seed dispersal in ecological process, such as regeneration and colonization. J. thurifera expansion is not seed limited thanks to its diverse dispersal community, hence the conservation of all dispersers in an ecosystem enhance ecosystems services and resilience.
doi:10.1371/journal.pone.0046993
PMCID: PMC3468541  PMID: 23071692

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