Production and Characterization of a Single-Chain Fv Antibody–Alkaline Phosphatase Fusion Protein Specific for Clenbuterol

The production and characterization of an anti-clenbuterol single-chain Fv antibody (CBLscFv)–bacterial alkaline phosphatase (AP) fusion protein are described. The CBLscFv and the phoA gene of Escherichia coli strain K12 chromosomal DNA were cloned by PCR and sequentially inserted into the expression vector pBV220 to express the CBLscFv–AP fusion protein in E. coli strain BL21(DE3)pLysS. SDS–PAGE and western blot analyses revealed that the fusion protein showed a molecular weight of 73 kDa and bound with the antibacterial AP monoclonal antibody. Determination of enzymatic activity indicated that k _cat and K _m values of the fusion protein were 113.60 s⁻¹ and 29.82 μM, respectively. Competitive direct enzyme-linked immunosorbent assay based on the obtained fusion protein indicated that the average concentration required for 50% inhibition of binding (IC₅₀) and the limit of detection for CBL were 4.74 ± 0.003 (n = 3) and 0.54 ± 0.004 (n = 3) μg/l, respectively, and the linear response range extended from 1.13 to 69.68 μg/l. Cross-reactivity studies showed that the fusion protein did not cross-react with CBL analogs. The present findings indicate that the production of the CBLscFv–AP fusion protein in E. coli strain BL21(DE3)pLysS is feasible and suggest that it could be further used to develop a one-step ELISA for the specific detection of CBL.     

Proline Rich Polypeptide (PRP-1) Increases the Superoxide-Producing and Ferrihemoglobin Reducing Activities of Cytochrome B<Subscript>558</Subscript> Isoforms from Human Lymphosarcoma Tissue Cells

The two cytochromes (cyt) b₅₅₈ of acidic nature, one—95–100 kDa and another one, 60–70 kDa were isolated for the first time from the human’s lymphosarcoma tissue cells using gel filtration and ion exchange chromatography. These hemoproteins possess NADPH dependent O₂ ⁻-producing and ferrihemoglobin-reducing activities. The incubation of neuropeptide PRP-1 (5 μg) with cytochrome b₅₅₈, caused elevation of these activities. The gel filtration results indicated possible binding of PRP-1 to these cytochromes b₅₅₈. PRP-1 activated both NADPH dependent O₂ ⁻-producing and ferriHb-reducing activities of the cyt b¹ ₅₅₈ and cyt b² ₅₅₈, obtained from human lymphosarcoma tissue cells. One can assume that PRP-1 associated with cyt b₅₅₈ on the surface of the tumor cells by increasing both NADPH dependent O₂ ⁻-producing and ferriHb-reducing activities of cyt b₅₅₈, increases the oxidation- reduction status. Changing the oxidation–reduction status and oxygen homeostasis of the tumor cells by PRP-1 can serve as one of the possible explanation of antitumorigenic effect of this cytokine.     

Extracellular production of <Emphasis Type="Italic">Streptomyces exfoliatus</Emphasis> poly(3-hydroxybutyrate) depolymerase in <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. T104: determination of optimal biocatalyst conditions

The phaZ _Sex gene encoding poly(3-hydroxybutyrate) depolymerase from Streptomyces exfoliatus has been successfully cloned and expressed in Rhodococcus sp. T104 for the first time. Likewise, the recombinant enzyme was efficiently produced as an extracellular active form and purified to homogeneity by two hydrophobic chromatographic steps. MALDI-TOF analysis showed that the native enzyme is a monomer. Circular dichroism studies have revealed a secondary structure showing 25.6% α-helix, 21.4% β-sheet, 17.1% β-turns, and 35.2% random coil, with a midpoint transition temperature (T _m) of 55.8 °C. Magnesium and calcium ions enhanced the enzyme activity, whereas manganese inhibited it. EDTA moderately decreased the activity, and the enzyme was completely deactivated at 3 M NaCl. Chemical modification studies indicated the presence of the catalytic triad serine–histidine–carboxylic acid in the active site. High-performance liquid chromatography (HPLC)–mass spectrometry (MS) analysis of PHB products of enzymatic hydrolysis showed monomers and dimers of 3-hydroxybutyric acid, demonstrating that PHB depolymerase is an exo-hydrolase. Addition of methyl-β-cyclodextrin simultaneously increased the activity as well as preserved the enzyme during lyophilization. Finally, thermoinactivation studies showed that the enzyme is highly stable at 40 °C. All these features support the potential industrial application of this recombinant enzyme in the production of (R)-3-hydroxyalkanoic acid derivatives as well as in the degradation of bioplastics.     

Characterization of a compound bioflocculant produced by mixed culture of <Emphasis Type="Italic">Rhizobium radiobacter</Emphasis> F2 and <Emphasis Type="Italic">Bacillus sphaeicus</Emphasis> F6

A compound bioflocculant CBF-F26, produced by mixed culture of Rhizobium radiobacter F2 and Bacillus sphaeicus F6, was investigated with regard to its physicochemical and flocculating properties. It was identified as a polysaccharide bioflocculant composed of rhamnose, mannose, glucose, and galactose, respectively, in a 1.3: 2.1: 10.0: 1.0 molar ratio. The average molecular weight was determined as 4.79 × 10⁵ Da by gel-permeation chromatography. Infrared spectrum and X-ray photoelectron spectroscopy revealed the presence of carboxyl, hydroxyl and amino groups in its structure. Thermostability test suggested that CBF-F26 was thermostable and high flocculating activity was maintained. Thermogravimetric property, intrinsic viscosity and surface morphology of CBF-F26 were also studied. CBF-F26 was effective under neutral and weak alkaline conditions (pH 7.0–9.0), and flocculating activities of higher than 90% were obtained in the concentration range of 8–24 mg l⁻¹ at pH 8.0. The flocculation could be stimulated by cations Ca²⁺, Zn²⁺, Fe²⁺, Al³⁺, and Fe³⁺. In addition, the probable flocculation mechanisms were proposed.     

Over-Expression in <Emphasis Type="Italic">E. coli</Emphasis> and Purification of the Human OCTN2 Transport Protein

The OCTN2 cDNA amplified from human skin fibroblast was cloned in pET-41a(+) carrying the glutathione S-transferase (GST) gene. The construct pET-41a(+)–hOCTN2 was used to express the GST–hOCTN2 fusion protein in Escherichia coli Rosetta(DE3)pLysS. The best over-expression was obtained after 6 h of induction with IPTG at 28°C. The GST–hOCTN2 polypeptide was collected in the inclusion bodies and showed an apparent molecular mass on SDS-PAGE of 85 kDa. After solubilization with a buffer containing 0.8% sarkosyl and 3 M urea, the fusion protein was applied onto a Ni²⁺-chelating chromatography column. The purified GST–hOCTN2 was treated with thrombin, and the hOCTN2 was separated from the GST by size exclusion chromatography. After the whole procedure, a yield of about 0.2 mg purified protein per liter of cell culture was obtained. To improve the protein yield, hOCTN2 cDNA was subjected to codon bias. The second codon CGG was substituted with AAA; the substitution led to the mutation R2K in the hOCTN2 protein. hOCTN2(R2K) cDNA was cloned in pET-21a(+) carrying a C-terminal 6His tag. The resulting protein was expressed in E. coli Rosetta(DE3)pLysS and purified by Ni²⁺-chelating chromatography. A yield of about 3.5 mg purified protein per liter of cell culture was obtained with this procedure.     

Characterization and Reconstitute of a [Fe<Subscript>4</Subscript>S<Subscript>4</Subscript>] Adenosine 5′-Phosphosulfate Reductase from <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis>

Adenosine 5′-phosphosulfate (APS) reductase is a key enzyme involved in the pathways of sulfate reduction and sulfide oxidation in the biological sulfur cycle. In this study, the gene of APS reductase from Acidithiobacillus ferrooxidans was cloned and expressed in Escherichia coli, the soluble protein was purified by one-step affinity chromatography to apparent homogeneity. The molecular mass of the recombinant APS reductase was determined to be 28 kDa using SDS-PAGE. According to optical and EPR spectra results of the recombinant protein confirmed that the iron–sulfur cluster inserted into the active site of the protein. Site-directed mutation for the enzyme revealed that Cys110, Cys111, Cys193, and Cys196 were in ligation with the iron–sulfur cluster. The [Fe₄S₄] cluster could be assembled in vitro, and exhibited electron transport and redox catalysis properties. As we know so far, this is the first report of expression in E. coli of APS reductase from A. ferrooxidans.     

Effects of temperature,photosynthetic photon flux density,photoperiod and O<Subscript>2</Subscript> and CO<Subscript>2</Subscript> concentrations on growth rates of the symbiotic dinoflagellate, <Emphasis Type="Italic">Amphidinium</Emphasis> sp.

Symbiotic dinoflagellates of the species Amphidinium are expected to be pharmaceutically useful microalgae because they produce antitumor macrolides. A microalgae production system with a large number of cells at a high density has been developed for the efficient production of macrolide compounds. In the present study, the effects of culture conditions on the cellular growth rate of dinoflagellates were investigated to determine the optimum culture conditions for obtaining high yields of microalgae. Amphidinium species was cultured under conditions with six temperature levels (21–35°C), six levels of photosynthetic photon flux density (15–70 μmol photons m⁻² s⁻¹), three levels of CO₂ concentration (0.02–0.1%), and three levels of O₂ concentration (0.2–21%). The number of cells cultured in a certain volume of solution was monitored microscopically and the cellular growth rate was expressed as the specific growth rate. The maximum specific growth rate was 0.022 h⁻¹ at a temperature of 26°C and O₂ concentration of 5%, and the specific growth rate was saturated at a CO₂ concentration of 0.05%, a photosynthetic photon flux density of 35 μmol photons m⁻² s⁻¹ and a photoperiod of 12 h day⁻¹ upon increasing each environmental parameter. The results demonstrate that Amphidinium species can multiply efficiently under conditions of relatively low light intensity and low O₂ concentration.     

Effects of solar ultraviolet radiation on photochemical efficiency of <Emphasis Type="Italic">Chaetoceros curvisetus</Emphasis> (Bacillariophyceae)

To assess the short- and long-term impacts of Ultraviolet radiation (UVR, 280–400 nm) on the red tide alga Chaetoceros curvisetus, we exposed cells to three different solar radiation treatments–PAB:280–700 nm, PA:320–700 nm, and P:400–700 nm under 20°C incubated temperature. Short-term exposures were investigated: the photochemical efficiency (Φ_PSII) versus irradiance curves under six levels of solar radiation by covering the incubators with a variable number of neutral density screens (the irradiance thus varied from 100 to 3%) lasting 1 h, and long-term exposures were designed to assess how the cells acclimate to solar radiation (the growth, UV_abc and ratio of repair to damage rates of D1 protein were detected). A significant decrease in the photochemical efficiency (Φ_PSII) at high irradiance (100% of incident solar radiation, 261.6 Wm⁻²) was observed in short-term exposure (1 h). UVR-induced photoinhibition was reduced to 7% in 3% solar radiation (4.08 Wm⁻²), compared with 66% in 100% solar radiation (261.6 Wm⁻²). In long-term experiments (11 days) using batch cultures, cell densities during the first 6 days were relatively constant for treatment P, and decreased slightly under PA and PAB treaments, reflecting a change in the irradiance experienced in the laboratory to that of incident solar irradiance. Thereafter, cell density increased and UV-induced photoinhibition decreased with the following days, indicating acclimation to solar UV. At the end of experiment, cells were found to exhibit both higher ratios of repair to UV-related damage and increased concentrations of UV-absorbing compounds, whose maximum absorption was found to be at 329 nm. Our data indicate that C. curvisetus is sensitive to ultraviolet radiation, but was able to acclimate relatively rapidly (ca. 6 days) by synthesizing UV-absorbing compounds and by increasing the rates of repair processes of D1 protein in PSII.     

Characterization and flocculating properties of a novel bioflocculant produced by <Emphasis Type="Italic">Bacillus circulans</Emphasis>

We studied a novel bioflocculant, PX, that is produced from Bacillus Bacillus circulans X3, and has excellent flocculating activity with regard to its characterization and flocculating properties. The bioflocculant was purified from supernatant by ethanol precipitation, dialysis and gel permeation chromatography (GPC). The major component of PX was an acid polysaccharide including uronic (19.8%), pyruvic (6.5%) and acetic acids (0.7%). It consisted of galactose, mannose, xylitol, rhamnose and galacturonic acid in an approximate molar ration of 5:4.1:3:2:1.2. The molecular weight of PX was about 4.85 × 10⁴ Da as determined by GPC. The infrared spectrum of the bioflocculant indicated the presence of carboxyl, hydroxyl, amino and methoxyl groups. Studies of the flocculating properties revealed that it was stable at 60–100°C and pH 4–10. Moreover, it could flocculate a kaolin suspension over a wide range of pH and temperature in the presence of CaCl₂.     

Efficient isolation of high quality nucleic acids from different tissues of <Emphasis Type="Italic">Taxus baccata</Emphasis> L.

Improved and efficient methods were developed for isolating high quality DNA and RNA from different sources of Iranian Yew (Taxus baccata L.). The methods were based on CTAB extraction buffer added with high levels of polyvinylpyrrolidone (PVP) and β-mercaptoethanol to properly remove polysaccharides and prevent oxidation of phenolics. The pellets obtained by ethanol precipitation were washed only with Chloroform: isoamyl alcohol (24:1). So, we could successfully eliminate the dangerous phenol/chloroform extraction steps from the isolation procedure. Both spectrophotometric (A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios) and agarose electrophoresis analysis of isolated nucleic acids (DNA and RNA) indicated good results. DNA with the average yield of 100–300 μg/g leaf and stem tissue and total RNA with an average yield of 20–30 μg/g cell culture and 80–100 μg/g leaf and stem tissue of Iranian yew could be obtained. Successful amplification of pam and pds by PCR and RT-PCR, showed the integrity of isolated DNA and RNA, respectively.     

Penduliflorain I: A Cysteine Protease Isolated from <Emphasis Type="Italic">Hohenbergia penduliflora</Emphasis> (A.Rich.) Mez (<Emphasis Type="Italic">Bromeliaceae</Emphasis>)

Penduliflorain I, a new plant endopeptidase, was isolated and characterized from Hohenbergia penduliflora. Crude extract was obtained from stems. A partially purified enzyme preparation was obtained by ethanol precipitation. This preparation showed maximum activity between pH 7.5 and 8.5, was stable at ionic strength (20% decrease in proteolytic activity could be detected after 2 h in 0.4 M sodium chloride solution), and exhibited high thermal stability (inactivation required heating for 20 min at 75 °C). Inhibition and activation assays indicated the cysteine nature of the enzymatic preparation. Penduliflorain I was purified by anion exchange chromatography (Q-Sepharose HP) by FPLC system. Homogeneity was confirmed by mass spectroscopy. Molecular mass of the enzyme was 23 412.847 Da (MALDI-TOF–MS). Kinetic parameters were determined for PFLNA (K _m = 0.3227 mM and k _cat = 4.27 s⁻¹). The N-terminal sequence (AVPQSIDWRDYGAVTTDKNQ) of isolated protease showed considerable similarity to other cysteine proteases obtained from stems or fruits of different Bromeliaceae species.     

Biosynthesis of Poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-3-hydroxyvalerate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) terpolymer by <Emphasis Type="Italic">Cupriavidus</Emphasis> sp. USMAA2-4 through two-step cultivation process

A locally isolated Gram-negative bacterium, Cupriavidus sp. USMAA2-4 was found capable of producing terpolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)] using γ-butyrolactone or 1,4-butanediol with either valeric acid or 1-pentanol as the carbon source. The present of 3HB, 3HV and 4HB monomers were confirmed by gas chromatography (GC) and nuclear magnetic resonance (NMR) analysis. PHA concentration of 1.9 g/l was the highest value obtained using the combination of 1,4-butanediol and 1-pentanol through one-step cultivation process. PHA concentration obtained through two-step cultivation process was higher for all the combinations and the highest value achieved was 2.5 g/l using γ-butyrolactone and 1-pentanol as carbon source. Various molar fractions of 4HB and 3HV ranging from 6 to 14 mol% and 39 to 87 mol%, respectively were produced through two-step cultivation process by manipulating the concentration of γ-butyrolactone. As the culture aeration was reduced, the molar fraction of 3HV and 4HB increased from 40 to 67 mol% and 10 to 24 mol%, respectively while the dry cell weight and PHA content decreased. The terpolymer produced was characterized using gel permeation chromatography (GPC) and differential scanning calorimetry (DSC). The number-average molecular weight (M _n) and the melting temperature (T _m)) of the terpolymer were in the range of 177–484 kDa and 160–164°C, respectively.     

Effect of nitrate and glucose addition on denitrification and nitric oxide reductase (<Emphasis Type="Italic">cnorB</Emphasis>) gene abundance and mRNA levels in <Emphasis Type="Italic">Pseudomonas mandelii</Emphasis> inoculated into anoxic soil

The effect of glucose addition (0 and 500 μg C g⁻¹ soil) and nitrate (NO₃) addition (0, 10, 50 and 500 μg NO₃–N g⁻¹ soil) on nitric oxide reductase (cnorB) gene abundance and mRNA levels, and cumulative denitrification were quantified over 48 h in anoxic soils inoculated with Pseudomonas mandelii. Addition of glucose-C significantly increased cnorB _p (P. mandelii and related species) mRNA levels and abundance compared with soil with no glucose added, averaged over time and NO₃ addition treatments. Without glucose addition, cnorB _p mRNA levels were higher when 500 μg NO₃–N g⁻¹ soil was added compared with other NO₃ additions. In treatments with glucose added, addition of 50 μg NO₃–N g⁻¹ soil resulted in higher cnorB _p mRNA levels than soil without NO₃ but was not different from the 10 and 500 μg NO₃–N g⁻¹ treatments. cnorB _p abundance in soils without glucose addition was significantly higher in soils with 500 μg NO₃–N g⁻¹ soil compared to lower N-treated soils. Conversely, addition of 500 μg NO₃–N g⁻¹ soil resulted in lower cnorB _p abundance compared with soil without N-addition. Over 48 h, cumulative denitrification in soils with 500 μg glucose-C g⁻¹ soil, and 50 or 500 μg NO₃–N g⁻¹ was higher than all other treatments. There was a positive correlation between cnorB _p abundance and cumulative denitrification, but only in soils without glucose addition. Glucose-treated soils generally had higher cnorB _p abundance and mRNA levels than soils without glucose added, however response of cnorB _p abundance and mRNA levels to NO₃ supply depended on carbon availability.     

Screening of human vascular endothelial growth factor (VEGF) receptor Flt-1 domain and study on its biological activity

Four human vascular endothelial growth factor receptor Flt-1 cDNA fragments containing extracellular domain loops 2, 1–2, 2–3 and 1–3 respectively were amplified from human placental cDNA library by PCR and used for screening ligand binding domains by yeast two-hybrid system. The result showed that, not only loop 1–3, but also the smaller fragment loop 2–3 could bind to hVEGF₁₆₅. Recombinant expression plasmids pPIC9K/Flt-1(1–3) and pPIC9K/Flt-1(2–3) were constructed and transformed toPichia. pastoris host strain GS115, cultured in flasks, and expressed under the induction of 1% methanol. The expressed product existed in supernatant in the form of soluble molecules and contained more than 60% of total protein after being induced for 4d. After being purified by CM-Sepharose FF and Sephacryl S-100 chromatography, its purity reached above 90%. Biological assayin vitro showed that the binding capacity of expressed soluble Flt-1 (2–3) to hVEGF₁₆₅ and its inhibiting effect on the proliferation of human umbilical veins endothelial cells (HUVEC) stimulated with hVEGF₁₆₅ were close to those of sFlt-1(1–3). Animal test showed that sFlt-1(2–3) could inhibit the formation of regenerate blood vessels stimulated with hVEGF₁₆₅ significantly.     

Degradation of methamidophos by <Emphasis Type="Italic">Hyphomicrobium</Emphasis> species MAP-1 and the biochemical degradation pathway

Methamidophos is one of the most widely used organophosphorus insecticides usually detectable in the environment. A facultative methylotroph, Hyphomicrobium sp. MAP-1, capable of high efficiently degrading methamidophos, was isolated from methamidophos-contaminated soil in China. It was found that the addition of methanol significantly promoted the growth of strain MAP-1 and enhanced its degradation of methamidophos. Further, this strain could utilize methamidophos as its sole carbon, nitrogen and phosphorus source for growth and could completely degrade 3,000 mg l⁻¹ methamidophos in 84 h under optimal conditions (pH 7.0, 30°C). The enzyme responsible for methamidophos degradation was mainly located on the cell inner membrane (90.4%). During methamidophos degradation, three metabolites were detected and identified based on tandem mass spectrometry (MS/MS) and gas chromatography-mass spectrometry (GC–MS) analysis. Using this information, a biochemical degradation pathway of methamidophos by Hyphomicrobium sp. MAP-1 was proposed for the first time. Methamidophos is first cleaved at the P–N bond to form O,S-dimethyl hydrogen thiophosphate and NH₃. Subsequently, O,S-dimethyl hydrogen thiophosphate is hydrolyzed at the P–O bond to release –OCH₃ and form S-methyl dihydrogen thiophosphate. O,S-dimethyl hydrogen thiophosphate can also be hydrolyzed at the P–S bond to release –SCH₃ and form methyl dihydrogen phosphate. Finally, S-methyl dihydrogen thiophosphate and methyl dihydrogen phosphate are likely transformed into phosphoric acid.     

The influence of either no fluid or carbohydrate-electrolyte fluid ingestion and the environment (thermoneutral versus hot and humid) on running economy after prolonged, high-intensity exercise

This study investigated the effects on running economy (RE) of ingesting either no fluid or an electrolyte solution with or without 6% carbohydrate (counterbalanced design) during 60-min running bouts at 80% maximal oxygen consumption (V˙O_2max). Tests were undertaken in either a thermoneutral (22–23°C; 56–62% relative humidity, RH) or a hot and humid natural environment (Singapore: 25–35°C; 66–77% RH). The subjects were 15 young adult male Singaporeans [V˙O_2max = 55.5 (4.4 SD) ml kg⁻¹ min⁻¹]. The RE was measured at 3 m s⁻¹ [65 (6)% V˙O_2max] before (RE1) and after each prolonged run (RE2). Fluids were administered every 2 min, at an individual rate determined from prior tests, to maintain body mass (group mean = 17.4 ml min⁻¹). The V˙O₂ during RE2 was higher (P < 0.05) than that during the RE1 test for all treatments, with no differences between treatments (ANOVA). The mean increase in V˙O₂ from RE1 to RE2 ranged from 3.4 to 4.7 ml kg⁻¹ min⁻¹ across treatments. In conclusion, the deterioration in RE at 3 m s⁻¹ (65% V˙O_2max) after 60 min of running at 80% V˙O_2max appears to occur independently of whether fluid is ingested and regardless of whether the fluid contains carbohydrates or electrolytes, in both a thermoneutral and in a hot, humid environment. Accepted: 30 October 1997     

A novel one-step strategy for the preparation of HLA/HBc<Subscript>18–27</Subscript> and HLA/CEA<Subscript>694–702</Subscript> complexes with ion exchange chromatography

The heavy chain protein of HLA-peptide complexes (HLA/HBc_18–27 and HLA/CEA_694–702) immobilized onto an ion exchange chromatography column and then the dilution-refolded HBc_18–27-fused or CEA_694–702-fused β2m protein was able to pass through the column. Using this method, HLA/peptide complexes were prepared within 30 h with a refolding yield of at least 20% (w/w) and purity of over 80% (w/w). This strategy refolds, concentrates, and purifies HLA/peptide complexes in a single integrated step and offers a potential tool to refold multiple-subunit proteins other than the major histocompatibility complex (MHC)/peptide complexes.     

Plastid DNA variation in highly fragmented populations of <Emphasis Type="Italic">Microbiota decussata</Emphasis> Kom. (Cupressaceae), an endemic to Sikhote Alin Mountains

Microbiota decussata Kom. (Cupressaceae) is a subalpine species endemic to the Sikhote Alin Mountains with populations scattered throughout the range. We used sequence data for four noncoding regions of chloroplast DNA to characterize the genetic diversity in populations sampled from different parts of M. decussata natural range. No variation was observed in the trnT–trnF region, whereas the trnH–psbA, trnS–trnfM, and trnS–trnG regions showed polymorphisms. At the species level, we found a low nucleotide diversity (π = 0.0009) and high haplotype diversity (h = 0.981) as well as high differentiation (Φ_ST = 0.420). N _ST and G _ST values suggested the existence of a phylogeographic structure in M. decussata. The observed patterns of diversity could be explained in part by ecological features of the species and its long-term persistence throughout the range with population expansion, successive fragmentation and isolation.