Journal of Applied Phycology 10: 539–546, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.
539
A new isolation procedure and further characterisation of the lectin from the red marine alga Ptilota serrata Alexandre H. Sampaio1,∗ , David J. Rogers2 , Clive J. Barwell2 , Silvana Saker-Sampaio1 , Francisco H. F. Costa1 & M´arcio V. Ramos3 1 Department
of Fishing Engineering, BioMol Lab, Federal University of Cear´a, P.O. Box 6033, 60451-970 – Fortaleza, Cear´a, Brazil 2 School of Pharmacy and Biomedical Sciences, University of Portsmouth, St Michael’s Building, White Swan Road, Portsmouth, PO1 2DT, U.K. 3 Department of Biology, BioMol Lab, Federal University of Cear´ a, Fortaleza, Cear´a, Brazil (∗ Author for correspondence; email
[email protected]) Received 6 September 1998; revised and accepted 15 September 1998
Key words: Ptilota serrata, red marine alga, lectin, agglutinin, affinity chromatography, guar gum, sugar inhibition
Abstract Ptilota serrata has been shown previously to contain a lectin (PSL) which is non-specific for human blood groups. We report here a new isolation procedure for PSL using a single step affinity chromatography technique on cross-linked guar gum and further characterisation studies. PSL was inhibited by galactose and its derivatives. The carbohydrates o-nitrophenyl-N-acetyl-α-D-galactoside, p-nitrophenyl-N-acetyl-β-D-galactoside and lactose were strong inhibitors. The glycoproteins porcine stomach mucin, asialo bovine mucin and asialofetuin were also inhibitory. The Mr of PSL, determined by gel filtration, was 55,470. SDS-PAGE revealed one single protein band with Mr of 18,390, indicating the native protein to be a trimer of apparently identical subunits. PSL was shown to contain large amounts of acidic and hydroxyl amino acids but low in basic amino acids.
Introduction Lectins have been isolated and characterised from various biological sources, mainly land plants. There is only a limited amount of information about algal lectins in comparison with those of higher plants and invertebrate. However, considering their particular characteristics, marine algal lectins appear to be a potential tool for biochemical and biomedical applications. The lectin from the red marine alga Ptilota serrata (PSL) was first isolated and partially characterised by Fish (1989) using affinity chromatography on porcine stomach mucin coupled to Sepharose 4B. The purification process exhibited low lectin recovery and the molecular weight was found to be 35,000 or 55,590 by gel filtration on BioGel P-100 or HPLC on Waters Protein Pack 300 SW column, respectively. Fur-
ther investigations were carried out by Rogers et al. (1990). The lectin was purified by a combination of ion-exchange chromatography in DEAE-Sephacel and size exclusion chromatography on Bio Gel P-100. PSL was inhibited by galactose and its derivatives and by the glycoproteins porcine stomach mucin and bovine submaxillary gland mucin. The apparent molecular weight of PSL determined by HPLC was 64,500. As part of our continuing studies on lectins from the genus Ptilota, and due to the conflicting reports concerning the molecular characteristics of PSL, we report here a new isolation procedure by affinity chromatography on cross-linked guar gum and further characterisation of PSL.
Article: japh 587 Pips nr. 192352 GSB: 702057 (japhkap:bio2fam) v.1.1 japh587.tex; 16/03/1999; 23:18; p.1
540 Materials and methods Algal collection and preparation of extracts Ptilota serrata was collected from Bay of Fundy, Newfoundland, Canada, and freeze-dried. The freeze dried alga was left in contact with 0.17 M phosphate buffered saline (PBS), pH 7.3, 1:20 (w/v) for 18 h at 4 ◦ C. The rehydrated material was strained through nylon tissue and ground to a fine powder in liquid nitrogen. The powder was combined with the buffer again and stirred for 18 h at 4 ◦ C. Particulate matter was removed by straining through nylon tissue. This step was repeated twice. The combined filtrates were centrifuged at 15,000 × g for 30 min at 4 ◦ C. The supernatant liquid was removed and used for further investigations. Purification of P. serrata lectin Dialysed precipitate in PBS from ammonium sulphate precipitation (0–75%) of the extract was loaded on a column (2.8 × 8 cm) of cross-linked guar gum, prepared as described by Appucathan et al. (1977), which had been equilibrated with the same buffer. The column was then washed with PBS at a flow rate of 30 mL h−1 until the column effluent showed absorbances of less than 0.05 at 280 nm. The adsorbed proteins were eluted by the addition of 50 mL 0.1 M D-galactose (Sigma) in PBS. All fractions were tested for haemagglutinating activity using blood group O papain-treated erythrocytes and absorbance at 280 nm was recorded. The active fractions were pooled, dialysed extensively against distilled water and concentrated by ultrafiltration (Amicon, Ltd-membrane size exclusion 10,000 Da). Finally the samples were freeze-dried and stored at −30 ◦ C until required.
(24,000), soybean trypsin inhibitor (20,100) and αlactalbumin (14,200). Molecular mass (Mr ) of the lectin was determined by molecular exclusion chromatography on a Bio Gel P-100 column in PBS (60 × 1.6 cm), calibrated with BSA (66,000), olvalbumin (45,000), carbonic anhydrase (29,000), myoglobin (18,800) and cytochrome C (12,400). Preparation of human red blood cells Human red blood cells were obtained from the Wessex Regional Transfusion Centre, Southampton, UK. Native erythrocytes were prepared by washing the red cells three times with PBS, then resuspending at a final concentration of 5% in PBS. Enzyme-treated erythrocytes were prepared by first washing native cells three times with PBS. Washed packed red cells were suspended in an equal vol of papain 0.1%; (w/v), left at 37 ◦ C for 30 min and then washed again three times with PBS and adjusted to a 5% suspension in PBS. Haemagglutination and haemagglutination inhibition tests Haemagglutination tests were performed by standard methods using 5% native or papain treated erythrocytes. Inhibition studies were performed using 4 haemagglutinating units of lectin with a range of simple sugars and glycoprotein using established methods (Sampaio et al., 1998a). Effect of EDTA, heat and pH on lectin stability The purified lectin was tested for the effect of EDTA, heat and pH on the haemagglutinating activity following standard methodology as described previously (Sampaio et al., 1996).
Homogeneity and molecular mass determination
Amino acid and N-terminal amino acid analysis
Flat bed gel electrophoresis (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis-SDS- PAGE) was carried out on the purified lectin at pH 7.0 using LKB Multiphor II electrophoresis equipment with a gel containing 10% (w:v) acrylamide. Samples and standards (Sigma) were prepared in 20% SDS and 1% 2-mercaptoethanol and heated at 100 ◦ C for 2 min. A standard picrate-Coomassie-blue method was used for staining the gel following electrophoresis. Protein markers used were BSA (66,000), ovalbumin (45,000), carbonic anhydrase (29,000), trypsinogen
The amino acid analysis of the purified lectin was carried out on a Applied Byosystems 420H Amino Acid Analyser with automatic hydrolysis and derivatization, employing a C18 reverse phase narrow bore cartridge. The N-terminal of P. serrata lectin was analysed on a Applied Biosystems ABI 477A Protein Sequencer. Protein determination Protein was determined by the method of Bradford (Bradford, 1976) using bovine serum albumin (Sigma)
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541 Table 1. Purification of the lectin from Ptilota serrata. Data from 30 g freeze-dried alga. HU = haemagglutinating units. MAC = Minimum Agglutination Capacity (minimum amount of protein that is still able to agglutinate erythrocytes). Blood group O papain-treated erythrocytes were used as indicator cells. Fraction
Protein total (mg)
HU mL−1
Specific activity HU mg P−1 Total
Yield (%)
Purification (fold)
MAC µg mL−1
Aqueous extract F 0/75% Affinity column
1230.0 85.0 1.58
128 2,048 32,768
1,040 1,917 153,121
100 128 189
1 1.8 147
0.96 0.52 0.0014
127,920 163,840 241,913
Figure 1. Affinity chromatography on cross-linked guar gum of PSL. Ammonium sulphate fraction (0/75%) was applied to the column (2.8 × 8 cm), equilibrated and eluted with PBS, at a flow rate of 30 mL h−1 . The absorbed lectin was eluted with 50 mL 0.1 M D-galactose in PBS. Fractions of 5 mL were collected and assayed for haemagglutinating activity using blood group O papain-treated erythrocytes. HU = haemagglutinating units. (•–•) absorbance at 280 nm, (◦–◦) haemagglutinating activity. Table 2. Titration values produced by PSL with human erythrocytes. Blood group of erythrocytes Native cells A B O Papain-treated cells A B O
HU mL−1 Crude extract
Pure lectin
4 4 8
128 128 256
128 128 256
4096 8192 8192
as standard. Eluates of the columns were monitored spectrophotometrically at 280 nm.
Results
Aqueous extract of P. serrata were used for precipitation with 75% saturated ammonium sulphate. This step let to a purification of lectin activity of 1.8 fold, with specific activity of 1,917 (Table 1). The 0–75% ammonium sulphate fraction was subject to affinity chromatography in column packed with cross-linked guar gum. All haemagglutinating activity present in the 0–75% fraction was bound to the gel and was eluted with addition of 0.1 M D-galactose (Figure 1). The material eluted from the affinity resin achieved 147 fold purification with an increment on the specific activity up to 153,121. The amount of homogeneous PSL recovered by extraction, fractionation and lyophilization was approximately 1.6 mg from 30 g of freeze-dried alga. The summary of purification is presented in Table 1.
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542 Table 3. Substances inhibitory to the lectin from Ptilota serrata. Substance
Minimum inhibitory concentration∗ mM µg mL−1
o-Nitrophenyl-N-acetyl-α-D-galactoside p-Nitrophenyl-N-acetyl-β-D-galactoside Lactose o-Nitrophenyl-N-acetyl-β-D-galactoside p-Nitrophenyl-N-acetyl-α-D-galactoside p-Nitrophenyl-β-D-fucoside o-Nitrophenyl-β-D-fucoside N-acetyl-galactosamine Methyl-α-D-galactoside Methyl-β-D-galactoside D-Galactose Melibiose D-Fucose o-Nitrophenyl-α-D-galactoside p-Nitrophenyl-β-D-galactoside Galactosamine-HCl Raffinose Lactulose o-Nitrophenyl-β-D-galactoside p-Nitrophenyl-α-D-galactoside 2-Deoxy-D-galactose Fucoidan Porcine stomach mucin Asaialo bovine mucin Asialofetuin Bovine submaxillary gland mucin
0.39 0.78 0.78 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.56 2.50 3.12 3.12 3.12 3.12 6.25 6.25 6.25 313 < 4.8 19 39 1250
Minimum concentrations required for inhibition of 4 haemagglutinating units of the lectin. Blood group O papain-treated erythrocytes were used as indicator cells. D-Arabinose, L-fucose, galactose-6-phosphate, D-galacturonic acid, D-glucose, N-acetyl-glucosamine, 2-deoxy-D-glucose, glucuronic acid, glucosamine-HCl, pNitrophenyl-α-D-glucoside, p-Nitrophenyl-β-D-glucoside, mannose, N-acetyl-βmannosamine, muramic acid, N-acetyl-neuraminic acid, rhamnose, trehalose were not inhibitory at concentrations up to 50 mM. Egg albumin, fetuin, α-acid glycoprotein, lactoferrin, ovomucoid, thyroglobulin, apo-transferrin were not inhibitory at concentrations up to 2.5 mg mL−1 .
PSL was able to agglutinate all the native and papainised human erythrocytes tested to almost the same degree (Table 2). Papainised cells from all groups gave higher titration values than those obtained with equivalent native cells. The studied on the effect of EDTA and the requirement for divalent cationsby PSL showed that the presence of 5 mM EDTA in the reaction medium decreased the initial activity from 256 to 4 HU mL−1 . Addition of 10 mM CaCl2 , MnSO4 or MgCl2 at 10 mM concentration restored almost the total haemagglutinating activity at initial level.
PSL was active at pH values of 6 to 8. Outside this range, however, the haemagglutinating capacity was reduced to 50% of the control value when exposed to pH 5, 9, 10 or 11. Exposure of the lectin to pH 2, 3, 4 or 12 reduced the agglutinating activity to 25% of the original value. In addition, the haemagglutinating capacity of PSL was not affected by exposure to a temperature of 40 ◦ C and remained still active at temperatures up to 80 ◦ C. The results of sugar inhibition tests using a large number of simple sugars and glycoproteins are shown in Table 3. PSL was inhibited by D-galactose and some of its derivatives and glycoproteins. The most
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543 potent inhibitory simple sugar was o-nitrophenyl-Nacetyl-α-D-galactoside at a concentration of 0.39 mM. Of the glycoproteins tested, porcine stomach mucin was the most powerful inhibitor, inhibiting the lectin at the minimum concentration tested (4.8 µg mL−1 ). SDS-polyacrylamide gel electrophoresis, in the presence of 2-mercaptoethanol revealed one single band of protein corresponding to a molecular weight of 18,390 ± 560 (n = 4) (Figure 2). The homogeneity of PSL was also observed by a single symmetrical peak obtained by size exclusion chromatography on Bio Gel P-100 in PBS (not shown), with apparent molecular weight of the native lectin of 55,470 ± 570 (n = 5). Mass spectrometry analysis of PSL, exhibited a MW ranging between 17,727 and 17,992 (unpublished results). The amino acid composition of PSL is shown in Table 4. The lectin is rich in alanine, valine, leucine and glutamic acid and has a low content of histidine and cystine. Tryptophan was not detected.
Discussion
Figure 2. SDS-PAGE of PSL. Lanes 2 and 3 purified P. serrata lectin. Lane 1 corresponds to molecular weight markers: bovine serum albumin (66 kDa), ovalbumin (45 kDa), glyceraldehyde-3-phosphate-dehydrogenase (36 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa), trypsin inhibitor (20 kDa), α-lactalbumin (14.2 kDa).
Table 4. Amino acid composition of PSL. ND, not detected. Amino acid
Mol%
Amino acid
Mol%
Asx Glx Ser Gly His Arg Thr Ala Pro
6.7 9.8 2.0 5.6 0.8 4.6 5.7 14.2 5.4
Tyr Val Met Cys Ile Leu Phe Trp Lys
3.8 14.2 3.7 0.8 4.1 10.6 6.3 ND 1.7
Affinity chromatography is a standard procedure for isolation of lectins. The affinity purified lectin preparations obtained in this way are frequently homogeneous when examined for a variety of criteria and do not require further purification. Guar gum is a galactomannan composed of a chain of β-(1→4)linked D-mannopyranosyl residues having attached α-D-galactopyranosyl unit linked α-(1→6) as single unit side chains, to approximately one half of the β-D-mannosyl residues (Lonngren et al., 1976; Appukuttan et al., 1977). Therefore, cross linked guar gum has been used successfully as an affinity support for D-galactose-binding agglutinins, such as Erythrina velutina lectin (Moraes et al., 1996), and for another Ptilota species P. filicina PFL (Sampaio et al., 1998a). The lectin in the ammonium sulphate fraction obtained from P. serrata was completely bound to the cross-linked guar gum and was eluted from the affinity column in an apparently pure form by addition of 0.1 M D-galactose in buffer. The resulting lectin have a single band on SDS-PAGE and represented 147 fold purification, compared to the lectin activity present in the original crude extract. Fish (1989), in attempts to isolate PSL, tested porcine stomach mucin coupled to Sepharose 4B as a support for affinity chromatography. Very low recovery of lectin was obtained using three different
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544 compositions of eluant. Elution of the lectin from the porcine mucin using a decrease in pH recovered only 1% of the total lectin activity with a purification of 0.08 fold. The use of borate in the elution buffer which is believed to break up lectin-sugar complexes (Tyagi & Gupta, 1993) recovered only 0.7% of the total activity with 0.28 fold purification. The best results were achieved by decrease in pH and addition of 200 mM lactose to the buffer. Yet, only 14% of the total activity was recovered with a purification of 9.9. In all three assays the presence of unbound lectin eluted before the addition of elution buffer was observed, which represented 28, 1.7 and 1.6 % of total activity. Probably, porcine mucin, even though it is a strong inhibitor of PSL, is not the best affinity support for the purification of this lectin. Therefore, the use of a galactose-affinity resin such as guar gum described here represents a much better approach to isolate the lectin from P. serrata as observed for the purification of the lectin from the closely related species P. filicina (Sampaio et al., 1998a). Some of the characteristics of PSL have already been reported (Fish, 1989; Rogers et al., 1990) using semi-purified material. All characterisation studies presented here were carried out using purified lectin. PSL was found to agglutinate human native or papaintreated erythrocytes of blood group A, B or O to almost the same degree. This pattern was observed with both, crude extract or purified lectin. PSL agglutinated enzyme-treated cells more strongly than native cells as was found by Rogers et al. (1980). In contrast to many other marine algal lectins (Sampaio, 1997), the haemagglutinating activity of PSL was virtually abolished when 10 mM EDTA was incorporated in the reaction medium. Since EDTA is a strong chelating reagent and the lectin had its agglutination capacity restored on the addition of divalent cations to the reaction medium, the results indicate that the lectin is dependent on these ions for its activity. All three divalent cations tested, Ca2+ , Mn2+ or Mg2+ , were shown to restore the haemagglutinating activity to the same level. PSL was found to be active over a wide range of pH (6 to 8), and stable when exposed to various temperatures up to 80 ◦ C. These results for pH and heat stability of PSL are in general agreement with those found for the great majority of marine algal lectins which have been isolated. Although not common in marine algae (Sampaio, 1997), the haemagglutination produced by PSL was strongly inhibited by simple sugars such as galactose
and its derivatives. The results show that PSL does not differentiate between α- or -β-D-galactosides, since these substances, in general, produce very similar levels of inhibition. Methyl-α-D-galactoside had the same inhibitory effect as methyl-β-D-galactoside as well as D-raffinose (α-anomeric linkage) and lactulose (β-anomeric linkage). Like some land plant lectins, PSL seems to possess a hydrophobic region in the vicinity of its carbohydrate binding site, since the inhibition shown by nitrophenyl-galactosides was stronger than D-galactose (Mo & Goldstein, 1994). The importance of a C-6 hydroxymethyl group was indicated by the failure of galacturonic acid and galactose-6phosphate to act as inhibitors, as has been observed with peanut lectin (Lotan et al., 1975). Also, the configuration of C-4 of the pyranose ring is important, since neither glucose nor glucose-derived compounds showed any inhibitory activity. The presence of substituents at C-1 and C-2, in substances such as nitrophenyl or nitrophenyl-N-acetyl derivatives of galactose, N-acetyl-D-galactosamine (GalNAc) and galactosamine hydrochloride, does not abolish the inhibitory capacity of these molecules and, therefore, are probably not important in the binding of the sugars to PSL. Furthermore, replacement of the free hydroxyl group at C-2 in the nitrophenylgalactosides with an acetamide group (Nitrophenyl-Nacetyl-galactosaminides) strongly improves the sugar binding of the lectin. D-fucose (6-deoxy-D-galactose) has a very similar configuration to D-galactose and the result show that these sugars have a similar grade of inhibition. The presence of a nitrophenyl group at C-1 in both increased the binding of these sugars to the lectin. All this evidence indicates the presence of a hydrophobic pocket in or near the sugar combining site of PSL. Remarkable, PSL showed no inhibition by Lfucose but was inhibited by fucoidan, a polysaccharide composed of sulphated L-fucose. Similar results are described for the lectins from the closely related species P. filicina lectin (PFL) (Sampaio et al., 1998a) and for the lectins from Ulva laetevirens (Sampaio et al., 1996) and U. lactuca (Sampaio et al., 1998b). Possibly, these lectins react with more extended structures than monosaccharides. Rini (1995), reported that larger, more complex polysaccharides, interact with secondary sites on the lectin surface as well as with the primary binding site. From the glycoproteins tested, porcine stomach mucin, an O-linked glycoprotein mainly composed of galactose, GalNAc and fucose residues, (Slomiany &
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545 Mayer, 1972), exhibited the most inhibitory effect at the minimum concentration tested of 4.8 µg mL−1 , whereas bovine submaxillary gland mucin, a glycoprotein having N-acetyl neuraminic acid (NeuNAc) as a terminal residue linked to GalNAc, was 260 times less inhibitory (1,250 µg mL−1 ). Although some plant lectins are specific for free sialic acid and even some galactose-specific lectins recognise it, sialated glycoproteins are usually weak receptors for lectins. In fact, elimination of sialic acid residues from bovine submaxillary gland mucin, rendered this molecule 65 times more inhibitory than the parental glycoprotein. The same pattern was observed with fetuin (non inhibitory) and asialofetuin (39 µg mL−1 ), which possesses triantennary N-linked glycans containing terminal non reducing sialic acid and galactose, respectively. However, the presence of O-linked glycans in both glycoproteins renders many possible interactions between PSL and these two glycoproteins. Ovomucoid which possesses highly complex N-linked glycans, ovalbumin exhibiting mainly heterogeneous N-linked glycans and thyroglobulin which possesses both, high mannose and N-acetyllactosamine type N-glycans did not interact with PSL. These results suggest that PSL possesses a well-defined monosaccharide binding-site which may recognise specific arrangement in more complex glycans where galactose occurs and that minor differences between related structures may completely abolish the interaction. The lectin was purified to homogeneity as determined by SDS-PAGE. An molecular weight of 18,390 was obtained for the lectin by this method, while molecular exclusion chromatography gave a native molecular weight of 55,470. The native molecular weight of PSL has been already reported. First, Fish (1989) found by gel filtration on Bio Gel P-100 a value of 35,600 and by HPLC on Waters protein Pack 300 SW column a value of 55,590. Later, Rogers et al. (1990) reported a molecular weight of 64,500 by HPLC in a Water’s column. The variation in the molecular weights observed previously may possibly be due to different levels of purity of the lectin by the use of preparations still containing contaminating proteins and proteinaceous pigments. Mass spectrometry analysis of PSL, exhibited a MW ranging between 17,727 and 17,992 (unpublished results). These results indicate that PSL is probably composed of three identical or similar subunits, therefore, suggesting that native PSL is a trimer. Although homotrimeric lectins are unusual, there are some reports of trimeric lectins, such as, the lectin purified from the land plant Sarotham-
mus welwitschii (Sampietro & Vattuone, 1994). The haemagglutinin glycoprotein of influenza virus is also a trimer (Wilson et al., 1981). We have also found that the lectins from the closely related species P. filicina – PFL (Sampaio et al., 1998a) and P. plumosa – PPL (Sampaio, 1997), to be trimeric lectins, with subunits of 19,320 and 17,440 respectively. This property of lectins from species of same tribe, family or genus showing very close similarity with regard to their physical characteristics, is well established. Plant lectins from the sub-tribe Diocleinae exhibit similar primary structures (Cavada et al., 1993). The lectins from the marine red algae Bryothamnion seaforthii and B. triquetrum (Ainouz et al., 1995) share similar characteristics, as do the lectins from various species of the marine green alga Codium (Rogers et al., 1994). The amino acid composition of PSL is similar to the composition of other marine algal lectins, with high amounts of acidic amino acids and low amounts of basic amino acids (Sampaio et al., 1998a, 1998b). Attempts were made to determine the sequence of the N-terminal amino acids of PSL. The lectin was shown to have blocking groups in the N-terminal primary structure since no amino acid could be detected. Such behaviour is not uncommon in proteins and therefore with lectins. Two GalNAc-specific lectins from the mushroom Phaeolepiota aurea (Kawagishi et al., 1996) were reported to have their N-terminal amino acids blocked. Also, the lectin from the closely related species P. plumosa has been shown to have blocking groups (Sampaio, 1997). However, the N-terminal sequence of the first ten amino acids from the lectin of P. filicina – PFL has been reported by Sampaio et al., (1998a). Quite clearly there is much work still to be done on the structure of PSL and to compare it with other lectins from the same genus. Further work in the attempt to elucidate the complete primary structure of PSL is in progress. This would contribute to understanding the differences in sugar inhibition and blood group specificity for the lectins from P. plumosa, P. serrata and P. filicina.
Acknowledgements We express our gratitude to Dr Bob Hooper of the Biology Department, Memorial University of Newfoundland, for his kind supply of P. serrata samples. This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq),
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546 Fundação Cearense de Amparo à Pesquisa (FUNCAP), BIOTOOLS – BioTools Ecological Foundation.
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