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Use of palmae wax hydrocarbon fractions as chemotaxonomical markers in Butia and Syagrus/Uso de fracoes de hidrocarbonetos de palmeiras como marcadores quimiotaxonomicos em Butia e Syagrus.

1. Introduction

Palmae is an extremely interesting botanical group, which represents one of the main evolutionary lines for Monocots. Their distribution is pan-tropical, with only a few species adapted to cooler climates (Tomlinson, 1961; Corner, 1966; Lotschert, 1985). The study of the group is difficult, as their collection involves extensive explorations and expeditions, while the preparation of herbarium samples is very complex. Studies have been generally carried out in specialized collections in arboreta, usually in botanical gardens located in tropical regions or established in glass-houses, which can be the largest buildings in these institutions. Although the Palmae have always attracted the interest of the most important botanists, these factors have reduced their scientific study.

The group has immense commercial and industrial importance, and many species are the origin of edible and industrial oils, waxes and fibres of multiple uses (Del Canizo, 1991). Certain species have their growth centres used as food, some have edible fruits or yield syrups used for fermented drinks (Jones, 1994). Thus, where Palms grow naturally or where they can adapt easily, palms are exploited commercially and can represent important economic benefits for the native inhabitants (Balick, 1988).

The taxonomic classification for this huge Family (2,500 to 3,500 described species) is traditionally based on the morphological characteristics of trunks, leaves, fruits and flowers, in the anatomical peculiarities of their organs, in the cytology of tissues, including the description of the pollen grains, studies on the present day geographical distribution, and of the evolutionary study of the Family and its genera (Glassman, 1972; Moore and Uhl, 1982; Henderson, 1988).

Approximately one fourth of all known Palmae are native to the Americas, and more than 60 of the genera described are present, and several are exclusive, to the New World. In the southern countries of South America there are over 70 native genera. Of these, 37 have been described for Brazil, 21 in Bolivia, 10 in Paraguay, 7 in Argentina, 3 in Uruguay and 1 in Chile (Glassman, 1972; Reitz, 1974; Henderson et al., 1995; Cerqueira and von Behr, 2004). The more common species in the Southeastern region are related to Butia and Syagrus genera, and represent interesting taxonomical problems, as their specific morphological (differences) traits are vague (Reitz, 1974; Cerqueira and von Behr, 2004), and having been exploited by humans from pre-Columbian times with the resulting artifactual overlapping of species, sub-species and hybrids (Lombardo, 1964; Iriarte et al., 2005).

[FIGURE 1 OMITTED]

Some secondary metabolites, such as flavonoids and anthocyanins (from the shikimate pathway) and certain epicuticular wax constituents have been shown to be excellent taxonomical markers in other botanical groups (Lombardo, 1964; Vieira et al., 2001). The epicuticular waxes that cover the surfaces of leaves, green twigs, fruits and flowers of plants behave as barriers between the individual plants and their environment, are principal factors in avoiding their dehydration and water permeation, resulting in a stable internal milieu (Kolattukudy, 1976).

As the epicuticular waxes (Figure 1) are external to plants, and thus not involved in their metabolic cycles, their compositions are quite stable, reacting only with a few environmental agents. More than one chemical marker can be enclosed in a wax, and their presence or absence reflects the specificities of the enzymes involved in the series of biosynthetic steps leading to the production of hydrocarbons, fatty acids, fatty alcohols or their esters, or on constituents that have their origin in squalene oxide, leading to steroids and/or pentacyclic triterpenes (Xu et al., 2004). Some have a valuable taxonomical nature, as is the case of pentacyclic triterpene methyl ethers, which are specific to Monocots, and are particularly important in Palmae (Garcia et al., 1995; Escalante-Erosa et al., 2002).

The stability or instability of the chemical parameters (quali- and quantitative variations) is what can establish the real value of a component as a useful taxonomical marker at the species level, and the statistical analyses of the chemical data can give us indications to their adequacy (Maffei and Scannerini, 1993). One aspect that has not received much attention in this context is the variation of intra-specific phenotypic expression, which depends on environmental factors, as well as the age and general situation of each individual (Vieira et al., 2001).

In the present study, the hydrocarbon fractions of two native groups, Butia and Syagrus were analyzed. The samples were collected in the regions of the Serra Gaucha, Atlantic Littoral and the Planalto (Missoes) of Rio Grande do Sul (Brazil), and Rocha (Uruguay), with the aim of establishing their value as taxonomic markers and to correlate their compositions with proposed species and geographical origins.

[FIGURE 2 OMITTED]

2. Material and Methods

2.1. Sample collection and preparation

24 Butia leaf samples and 20 Syagrus leaf samples were collected in the Serra Gaucha, Atlantic Littoral and Planalto (Missoes) of the State of Rio Grande do Sul (Brazil/ and Rocha (Uruguay) (Figures 2 and 3). Voucher samples were deposited at the Herbario (HUCS) and Banco Ativo de Germoplasma (BAG) of the Universidade de Caxias do Sul (Figures 2 and 3). The leaves were separated from the stems and dried at 35 [degrees]C until constant weight.

[FIGURE 3 OMITTED]

2.2. Wax extraction and fractionation

100 g of dry leaves were washed using chloroform (40 seconds). The solvent was removed under vacuum in a rotary evaporator.

The residues were fractionated by Preparative Thin Layer Chromatography using Silica Gel plates, with hexane as development solvent. The hydrocarbon fractions were separated on the basis of the Rf of an authentic sample of icosane (Sigma-Aldrich Co.). For spot development a mixture of CuS[O.sub.4] 5% (aq.) and [H.sub.3]P[O.sub.4] 10% (aq.) (1:1) was sprayed, with heating in an oven to complete the process.

2.3. Chromatographic analysis

The isolated hydrocarbon fractions were analyzed using a Gas Chromatograph coupled to a Mass Spectrometer (Shimadzu CG 17A, CG/EM, QP 5050A), in sweep mode, using a DB-5 capillary column (30 m x 0.25 mm x 0.25 [micro]m). The column temperature was programmed at 100 [degrees]C (2 minutes); 250 to 300 [degrees]C at a rate of 2 [degrees]C/min.; 10 minutes at 300 [degrees]C. Helium gas was used as carrier. Injector temperature was 290 [degrees]C and detector temperature was 310 [degrees]C. The injection was done in the split mode (ratio 1:50), with electronic impact at 70 eV. Biphenyl (Sigma-Aldrich) at 100 mg.[L.sup.-1] concentration was used as internal standard. Peaks were integrated manually, and quantitation was achieved by comparison with the internal standard. Constituents of the sample were identified comparing their retention times to those of authentic compounds (n-pentacosane, n-heptacosane, n-nonacosane, n-hentriacontane and n-tritriacontane--Sigma-Aldrich) and their mass spectra with those of the Wiley Library.

2.4. Scanning electronic microscopy

The dried leaves were metalized by gold sputtering and their SEM microphotographs recorded with a Jeol Scanning Electron Microscope JSM-5900LV, at the SEM facilities of the Nucleo de Servicios de Alta Tecnologia, Facultad de Ciencias, UDELAR, Montevideo, Uruguay.

2.5. Statistical analysis

Samples were injected three times and the statistical analyses for means comparisons (Tukey and t-Student, p < 0.05) and of groupings (Euclidian distance with simple links) were made with the Statistica 5.0 program.

3. Results and Discussion

The average yields of crude epicuticular waxes on dry leaves were 0.31% for Butia leaves and 0.28% for Syagrus leaves. Saturated linear hydrocarbons are, on average, 15.3% of Butia wax extract and 13.7% of Syagrus waxes. SEM microphotographs were recorded to observe the distribution of the waxes on the surface. Figure 1 shows typical distributions.

The GC-MS analysis permitted the identification and quantification of 19 hydrocarbons ([C.sub.13] to [C.sub.33]) in the Butia and Syagrus samples. The hydrocarbons of higher molecular weights are present in all samples (Tables 1 and 2), while the lower weight hydrocarbons were present as traces in most samples (less than 0.001 g.[g.sup.-1] of the fraction). Hentriacontane ([C.sub.31]) and tritriacontane ([C.sub.33]) are responsible for 81.7% of the total mass of the Butia hydrocarbons and 72.3% in Syagrus samples. Higher concentrations of high molecular weight hydrocarbons, which should result in higher melting ranges and less water permeability, seem to be an important evolutionary strategy for the Family, as they would also imply higher resistance to insect attack and climatic variations.

[FIGURE 4 OMITTED]

Significant differences (Tukey p < 0.05) in the average masses of the main hydrocarbons happened both in Butia as in Syagrus. The means comparison analysis following the t-Student Test for independent samples (p < 0.05), showed significant differences in the concentration of the main hydrocarbons both in Butia and Syagrus, with the exception of docosane ([C.sub.22]).

The grouping of populations made through Euclidian distances through simple links of the quantitative differences of the hydrocarbons analyzed, allowed the separation of all the samples.

For Butia samples the statistical analysis showed the formation of two main groups, with 3 samples outside of these populations. The relation of these groups with their geographical distribution and traditional species descriptions is shown in Figures 2 and 4. Group 1, with high similarity of compositions, included all samples collected in the Littoral of Rio Grande do Sul and in the Uruguayan coast, as well as samples from Jaquirana, Erechim e Ijui. Some authors (Cerqueira and von Behr, 2004) describe this as the habitat Butia capitata var. odorata (Barb. Rodr.) Becc. The outlying samples from the Serra Gaucha, Planalto and Missoes are probably results of anthropic activities.

Group 2, with greater variability, was also geographically coherent, with most samples coming from the regions of Serra Gaucha and Planalto, which corresponds to the habitat of B. eriospatha Becc (Cerqueira and von Behr, 2004). The samples collected in Sao Miguel das Missoes, Entre Ijuis and Tupancireta, all in the Missoes region, to the Northwest of Rio Grande do Sul, could not be related to Groups 1 or 2, did not establish a coherent group themselves, and can be related to other Butia species, particularly B. paraguayensis (Barb. Rodr.) L. H. Bailey and B. yatay (Mart.) Becc (Cerqueira and von Behr, 2004). Thus, the different groupings made by average masses of the main hydrocarbons in the waxes could reflect interspecific differences, something that would make them very useful taxonomic markers.

In the case of S. romanzoffiana (Cham.) Glassman, statistical analysis shows the formation of only one group, with the exception of the sample from Viadutos (Figure 5). Groups can be established, but they are statistically much less consistent than those for Butia (Figure 5), and which show no relevant connection with their geographical origin (Figure 3). Although sub-species with a less defined sub-regional distribution could be the case for Syagrus, other evaluations also seem to point to the presence of one species in all its distribution range (Iriarte et al., 2005). In the case of these populations, factors connected with the microclimate, age of the plant or leaves, sunlight, soil, nutritional level, etc., which were not considered in the present study but have been discussed by Vieira et al. (2001) should be considered. Although some authors (Cerqueira and von Behr, 2004) mention 32 Syagrus species in Brazil, S. romanzoffiana (Cham.) Glassman seems to be the only abundant species in the Southern region.

[FIGURE 5 OMITTED]

4. Conclusions

The epicuticular waxes of Butia and Syagrus have high contents of linear hydrocarbons, with hentriacontane ([C.sub.31]) and tritriacontane ([C.sub.33]) being the main constituents. Differences in the individual hydrocarbon contents were observed in the compositions of both Genera. The statistical analysis of the hydrocarbon fractions in Butia established two distinct groups and a few outliers, which could be connected to their geographical origin and to the species traditionally described for each region. In the case of Syagrus waxes no groupings could be established. The small differences could be associated to environmental responses or to the existence of subspecies. The study of the wax linear hydrocarbon compositions seems to be a useful tool in the taxonomical study of the Palmae.

Acknowledgements--The authors are indebted to Drs. L. Noblick and M.Rivas for useful comments and insights. The authors (NP, RLC, MR and GFP) are grateful for the support received from URI-Campus de Erechim and UCS (Brazil) that made their work possible, and to the Science and Technology Secretariat and FAPERGS (RS, Brazil) for financing purchases of equipment. PM and HH thank the financial support of CSIC (UDELAR, Uruguay), and PDT (DINACYT-Uruguay).

Received June 1, 2007--Accepted August 20, 2007--Distributed May 31, 2009 (With 5 figures)

References

BALICK, MJ., 1988. The palm-tree of life: biology, utilization and conservation. Advances in Economic Botany, vol. 6.

CERQUEIRA, LSC. and Von BEHR, N., 2004. Palmeiras no Brasil. Sao Paulo: Plantarum.

CORNER, EJH., 1966. The natural history of palms. London: Weidenfeld and Nicholson.

Del CANIZO, JA., 1991. Palmeras. Bilbao: Grafo.

ESCALANTE-EROSA, F., GAMBOA-LEON, MR., LECHER, JG., ARROYO-SERRALTA, GA., ZIZUMBO-VILLAREAL, D., OROPEZA-SALIN, C. and PENA-RODRIGUEZ, LM., 2002. Major Components from the Epicuticular Wax of Cocos nucifera. Revista de la Sociedad Quimica de Mexico, vol. 46, no. 3, p. 247-250.

GARCIA, S., HEINZEN, H., HUBBUCH, C., MARTINEZ, R., DEVRIES, JX. and MOYNA, P., 1995. Triterpene methyl ethers from Palmae epicuticular waxes. Phytochemistry, vol. 39, no. 6, p. 1381-1382.

GLASSMAN, SF., 1972. Dahlgren's index of American palms. Stuttgart: Straus and Cramer. Phanerogamarum Monographie. Tomus VI. Revision of B.E.

HENDERSON, A., 1988. Pollination biology of economically important palms. In BALICK, MJ. (Ed.). The palm-tree of life: biology, utilization and conservation. Advances in Economic Botany, vol. 6. p. 36-41.

HENDERSON, A., GALEANO, G. and BERNAL, G., 1995. Field guide to the palms of the Americas. Princeton: Princeton Univ. Press.

IRIARTE, J., HOLST, I., MAROZZI, O., LISTOPAD, C., ALONSO, E., RINDERKNECHT, A. and MONTANA, J., 2005. Evidence for cultivar adoption and emerging complexity during the mid-Holocene in the La Plata basin. Nature, vol. 432, p. 614-617.

JONES, DL., 1994. Palms throughout the world. Washington: Smithsonian Press.

KOLATTUKUDY, PE. (Ed.)., 1976. Chemistry and biochemistry of natural waxes. Amsterdam: Elsevier.

LOMBARDO, A., 1964. Flora arborea y arborescente del Uruguay. 2 ed. Montevideo: Concejo Dept. Montevideo.

LOTSCHERT, W., 1985. Palmen. Stuttgart: Ulmer.

MAFFEI, M. and SCANNERINI, S., 1993. Fatty acid variability from non-polar lipids in some Lamiaceae. Biochemical Systematic and Ecology, vol. 21, no. 4, p. 475-486.

MOORE, HE. and UHL, NW., 1982. Major trends of evolution in palms. The Botanical Review, vol. 48, no. 1, p. 1-69.

REITZ, R., 1974. Palmeiras. In REITZ, PR. (Ed.). Flora Ilustrada Catarinense. Itajai: Herbario Barbosa Rodrigues.

TOMLINSON, PB., 1961. Palmae. In METCALFE, CR. (Ed.). Anatomy of the Monocotyledons II. Oxford: Clarendon.

VIEIRA, VF., GRAYER, RJ., PATON, A. and SIMON, JE., 2001. Genetic diversity of Ocimum gratissimum L. based on volatile oil constituents, flavonoids and RAPD markers. Biochemical Systematic and Ecology, vol. 29, no. 3, p. 287-304.

XU, R., FAZIO, GC. and MATSUDA, SPT., 2004. On the origins of triterpenoid skeletal diversity. Phytochemistry, vol. 65, no. 3, p. 261-291.

Paroul, N. (a) *, Cansian, RL. (a), Rossato, M. (b), Pauletti, GF. (b), Serafini, LA. (b), Rota, L. (b), Moyna, P. (c) and Heinzen, H. (c)

(a) Departamento de Quimica, Universidade Regional Integrada do Alto Uruguai e das Missoes--URI, Av. Sete de Setembro, 1621, CEP 99700-000, Erechim, RS, Brazil

(b) Instituto de Biotecnologia, Universidade de Caxias do Sul--UCS, Rua Getulio Vargas, 1130, CEP 95070-560, Caxias do Sul, RS, Brazil

(c) Facultad de Quimica, Universidad de la Republica--UDELAR, Av. General Flores, 2124, CP 11800, Montevideo, Uruguay

* e-mail: nparoul@uricer.edu.br
Table 1. Main Hydrocarbons Weights (g) per lg Crude Wax in Butia. l)
Jaquirana; 2) Caxias do Sul; 3) Uruguai; 4) Uruguai; 5) Erechim; 6)
Erechim; 7) Ijui; 8) Sao Miguel das Missoes; 9) Entre Ijuis; 10)
Entre Ijuis; 11) Tupanciret5; 12) Santa Cruz do Sul; 13) Bom Retiro
do Sul; 14) Morro Azul; 15) Sertao; 16) Coxilha; 17) Coxilha; 18)
Passo Fundo; 19) Santa Vitoria do Palmar; 20) Pelotas; 21) Sao
Lourenco do Sul; 22) S5o Lourenco do Sul; 23) Barra do Ribeiro; 24)
Morro Azul. a) Planalto and Serra Gaucha (Altitude over 600 m); b)
Missoes and Central Depression (Altitude from 300 to 600 m); and c)
Littoral (Altitude below 200 m).

         C15      C16      C17      C18      C19

01 - A   --       --       0.0008   0.0024   --
02 - A   --       --       --       --       --
03 - C   0.0037   0.0033   0.0017   0.0022   --
04 - C   0.0030   --       0.0030   --       0.0030
05 - A   --       --       --       --       --
06 - A   --       --       --       --       --
07 - B   --       --       0.0030   0.0030   0.0030
08 - B   0.0275   --       --       --       --
09 - B   --       --       --       --       --
10 - B   --       --       --       --       --
11 - B   0.0021   0.0026   0.0036   0.0048   -
12 - B   --       --       0.0420   --       --
13 - B   --       --       --       --       --
14 - C   --       0.0005   --       --       --
15 - A   --       --       --       --       --
16 - A   --       --       --       --       --
17 - A   --       --       --       --       --
18 - A   --       --       --       --       --
19 - C   --       --       --       --       --
20 - C   --       --       --       --       --
21 - C   --       --       --       --       --
22 - C   --       --       --       --       --
23 - C   --       --       --       --       --
24 - C   --       --       --       0.0021   0.0011

         C20      C21      C22      C23      C24

01 - A   0.0006   --       0.0394   0.0130   --
02 - A   --       --       0.0326   --       --
03 - C   --       --       0.0193   --       --
04 - C   0.0030   --       0.0095   0.0020   0.0035
05 - A   --       --       0.0093   --       --
06 - A   --       --       0.006l   0.0016   0.0013
07 - B   0.0030   0.0020   0.0140   0.0030   0.0020
08 - B   --       --       0.0075   --       --
09 - B   --       --       0.0085   0.0050   --
10 - B   --       --       0.0330   -        --
11 - B   0.0029   0.0046   0.0381   0.0186   0.0040
12 - B   --       --       0.0160   --       --
13 - B   --       --       0.0440   --       --
14 - C   --       --       0.0035   --       --
15 - A   --       --       0.0150   0.0043   --
16 - A   --       --       0.0170   --       0.0023
17 - A   --       --       0.0166   --       --
18 - A   --       --       0.0081   0.0029   --
19 - C   --       --       0.0155   --       --
20 - C   --       --       0.0474   --       --
21 - C   --       --       0.0047   --       --
22 - C   --       --       0.0035   --       --
23 - C   --       --       0.0075   0.0019   --
24 - C   --       --       0.0180   --       0.0035

         C25      C26      C27      C28      C29

01 - A   0.0022   --       0.0170   0.0164   0.0360
02 - A   --       --       0.0233   0.0306   0.0563
03 - C   0.0055   0.0073   0.0122   0.0152   0.0271
04 - C   --       0.0050   0.0080   0.0100   0.0275
05 - A   0.0022   0.0088   0.0153   0.0203   0.0365
06 - A   0.0037   0.0027   0.0098   0.0137   0.0271
07 - B   0.0040   0.0050   0.0200   0.0080   0.0330
08 - B   --       --       0.0317   0.0501   0.0709
09 - B   --       0.0073   0.0190   0.0170   0.0358
10 - B   --       --       0.0134   --       0.0336
11 - B   0.0045   0.0038   0.0150   0.0046   0.0211
12 - B   --       --       0.0116   0.0190   0.0322
13 - B   --       --       --       --       0.0028
14 - C   --       --       0.0015   0.0015   0.0095
15 - A   --       --       0.0110   0.0146   0.0380
16 - A   0.0033   0.0l66   0.0203   0.0290   0.0343
17 - A   --       0.0040   0.0140   0.0193   0.03l0
18 - A   0.0023   0.0072   0.0135   0.1150   0.0332
19 - C   0.0030   0.0050   0.0155   0.0205   0.0340
20 - C   0.0048   0.0138   0.0216   0.0298   0.0446
21 - C   --       --       --       --       0.0015
22 - C   --       --       0.0020   0.0030   0.0140
23 - C   0.0015   0.0020   0.0092   0.0074   0.0345
24 - C   --       --       0.0025   --       0.0092

         C30      C31      C32      C33

01 - A   --       0.3511   --       0.1752
02 - A   0.0063   0.5280   --       0.2063
03 - C   0.0256   0.3949   0.0286   0.2338
04 - C   0.0215   0.3740   0.0285   0.2686
05 - A   0.0318   0.4846   0.0232   0.1617
06 - A   0.029l   0.3670   0.0322   0.2603
07 - B   0.0320   0.3170   0.0270   0.2040
08 - B   0.07l7   0.3200   --       --
09 - B   0.0285   0.0503   0.0346   0.3090
10 - B   --       0.6092   --       0.2072
11 - B   0.0108   0.0648   --       0.0215
12 - B   0.0508   0.5200   0.0430   0.2842
13 - B   --       0.6478   0.0228   0.2223
14 - C   0.0060   0.3360   0.0090   0.2025
15 - A   0.0343   0.6040   --       0.2463
16 - A   0.0373   0.4783   0.0360   0.3030
17 - A   0.0350   0.4973   0.0326   0.3243
18 - A   0.0230   0.5338   0.0325   0.3192
19 - C   0.0445   0.3270   0.0315   0.2290
20 - C   0.0472   0.3250   --       0.2130
21 - C   0.0137   0.3540   --       0.2410
22 - C   0.0160   0.3170   0.0265   0.2300
23 - C   0.0315   0.3320   0.0364   0.2230
24 - C   --       0.3280   0.0130   0.2108

Table 2. Main Hydrocarbon Weights (g) per 1 g Crude Wax in Syagrus.
1) Nova Petropolis; 2) Cacapava; 3) Cacapava; 4) Caxias do Sul; 5)
Camaqud; 6) Marcelino Ramos; 7) Marcelino Ramos; 8) Gaurama; 9)
Viadutos; 10) Erechim; 11) Entre Ijuis; 12) Ijui; 13) Pejucara; 14)
Candelaria; 15) Santa Cruz do Sul; 16) Bom Retiro; 17) Sert5o; 18)
Coxilha; 19) Pelotas; 20) Sao Lourenco do Sul. a) Planalto and Serra
Gaucha (Altitude over 600 m); b) Missaes and Central Depression
(Altitude from 300 to 500 m); and c) Littoral (Altitude below 200 m).

         C15     C16     C17     C18     C19

01 - A   --      --      --      --      --
02 - C   --      --      --      --      --
03 - C   --      --      --      --      --
04 - A   --      --      --      --      --
05 - C   --      --      --      --      --
06 - A   --      --      --      --      --
07 - A   --      --      0.001   0.001   0.001
08 - A   0.003   --      --      --      --
09 - A   --      0.001   --      --      --
10 - A   --      --      --      --      0.006
11 - B   --      --      0.001   0.001   --
12 - B   --      --      --      --      --
13 - B   --      --      --      --      --
14 - B   --      --      --      --      --
15 - B   --      --      --      --      --
16 - B   --      --      --      --      --
17 - A   --      --      0.002   0.003   0.002
18 - A   --      --      --      --      --
19 - C   --      --      --      --      --
20 - C   --      --      --      --      --

         C20     C21     C22     C23     C24

01 - A   --      --      0.024   --      --
02 - C   --      --      0.015   0.007   0.002
03 - C   --      --      0.012   0.005   0.003
04 - A   --      --      0.008   0.005   0.002
05 - C   --      --      0.029   --      --
06 - A   --      --      0.023   --      --
07 - A   0.001   0.001   0.008   0.004   0.002
08 - A   --      --      0.023   0.010   0.011
09 - A   --      --      0.016   0.002   0.001
10 - A   --      --      0.023   --      -
11 - B   --      --      0.005   --      0.006
12 - B   --      --      0.017   --      --
13 - B   --      --      0.020   0.004   --
14 - B   --      --      0.051   0.012   --
15 - B   --      --      0.019   0.015   --
16 - B   0.011   --      0.007   --      0.002
17 - A   --      --      0.009   --      --
18 - A   --      --      0.021   --      0.006
19 - C   --      --      0.023   --      --
20 - C   --      --      0.025   --      --

         C25     C26     C27     C28     C29

01 - A   0.016   --      0.031   --      0.059
02 - C   0.001   0.001   0.035   0.014   0.044
03 - C   0.011   0.015   0.041   0.029   0.101
04 - A   0.009   0.011   0.050   0.014   0.075
05 - C   --      --      0.029   --      0.089
06 - A   0.015   0.018   0.029   0.034   0.076
07 - A   0.007   0.002   0.024   0.010   0.055
08 - A   0.028   0.030   0.105   0.048   0.096
09 - A   --      --      0.014   0.015   0.052
10 - A   0.013   -       0.032   0.035   0.058
11 - B   0.053   0.009   0.024   0.011   0.076
12 - B   --      --      0.030   0.034   0.059
13 - B   0.006   0.006   0.003   0.030   0.075
14 - B   0.022   0.020   0.063   0.025   0.072
15 - B   0.015   0.002   0.058   --      0.056
16 - B   0.007   0.004   0.041   0.020   0.064
17 - A   --      --      0.011   0.009   0.026
18 - A   0.022   0.013   0.072   0.042   0.101
19 - C   0.020   0.017   0.081   0.047   0.121
20 - C   0.013   0.030   0.061   0.049   0.095

         C30     C31     C32     C33

01 - A   --      0.511   --      0.169
02 - C   0.023   0.408   0.023   0.252
03 - C   0.040   0.533   --      0.193
04 - A   --      0.439   0.024   0.278
05 - C   0.038   0.462   --      0.225
06 - A   --      0.345   --      0.226
07 - A   0.020   0.409   0.027   0.291
08 - A   0.046   0.210   --      0.109
09 - A   0.044   0.429   0.033   0.033
10 - A   0.050   0.254   --      0.122
11 - B   0.020   0.284   0.021   0.204
12 - B   --      0.234   --      0.236
13 - B   0.049   0.351   0.027   0.190
14 - B   --      0.333   --      0.255
15 - B   --      0.269   --      0.317
16 - B   0.022   0.489   0.024   0.266
17 - A   0.020   0.330   --      0.142
18 - A   0.043   0.412   --      0.153
19 - C   0.063   0.417   --      0.106
20 - C   0.049   0.395   --      0.205
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Author:Paroul, N.; Cansian, R.L.; Rossato, M.; Pauletti, G.F.; Serafini, L.A.; Rota, L.; Moyna, P.; Heinzen
Publication:Brazilian Journal of Biology
Date:May 1, 2009
Words:4059
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