MATERIALS AND METHODS
Leaf samples were randomly collected from 328 adult individuals of nine naturally-occurring populations in different regions of Santa Catarina: FLONA of Três Barras (FTB), Fazenda Rancho Alegre (FRA), Fazenda Antonio Carlos (RAC), Fazenda Amola Faca (FAF), Fazenda Guamirim Gateados (FGG), Parque Municipal de Lajes (PML), Estação de Caçador (ECA), Fazenda Urupema (URU), and ARIE of Victor Meirelles (AVM) (Figure 1). The geographic location, altitude, area size, population size, forest type, and a partial history of the use of the nine populations are included in Table I. The leaf samples (needles) were taken from adult plants that had a diameter at breast height (DBH) greater than 35 cm, after which they were transported in insulated boxes to the laboratory and stored in a refrigerator (5 to 10 °C) until use.
Isozyme electrophoresis was performed with starch gels 13% (w/v) composed of corn Penetrose 30 (Cornproducts, Mogi Guaçu-SP). Four gel/electrode buffer systems were tested: 1) morpholine-citrate (citric acid 0,04 M and N-[3-amino-propyl] morpholine, pH 6.1; Cheliac and Pittel,1984), 2) tris-citrate (Tris - Citric acid, pH 6.6) / histidine [L-Histidine 0,05 M pH 6.0]; Paiva, 1992), 3) Lithium (tris-citrate [Tris - Citric acid 0,05 M pH 8.5] / Lithium hydroxide 0.06 M pH 8.1; Alfenas et al., 1991) and 4) Histidine (L-Histidine-HCl 0,05 M pH 7,0 / Tris 0.125 M pH 7.0; Alfenas et al., 1991). Electrophoretic buffer systems were used to assay 20 enzymatic systems (Auler, 2000). Nine enzymatic systems were selected to perform the population analyses: PGM, PGI, MDH, PRX, SKDH, 6PGDH, ACP, IDH, and G6PDH.
The allozymic data were utilized to estimate the genetic indexes: 1) allele number per locus (total number of alleles/number of loci); 2) percentage of polymorphic loci (total number of polymorphic loci x 100/number of loci); 3) allelic frequencies (Weir, 1990); 4) expected heterozygosity (He = 1-Spi2, pi being the mean frequency of the i allele in a locus; Nei, 1973); 5) observed heterozygosity (direct counting); and 6) Wright's fixation index. To address the population's genetic structure it was estimated the genetic diversity among and within populations (Nei, 1973, 1977, 1978) and the statistics F of Wright (Wright, 1951, 1965) were estimated. The chi-squares (c2) for the Hardy-Weinberg equilibrium deviation, based on observed and expected frequencies, grouped or not, and for the inbreeding equilibrium, were estimated as suggested by Li and Horvitz (1953). A locus was considered polymorphic when the frequency of the most common allele did not reach a frequency value higher than 0.95. The BIOSYS-1 computer program (Swofford and Selander, 1989) was used to estimate the allelic frequencies and the mentioned genetic indexes.
According to Nei (1973), the variation in gene frequency among subpopulations may be analyzed by the F-statistics of Wright: [1 - FIT = (1 - FIS)(1 - FST)], where FIT and FIS are correlations between two uniting gametes to produce the individuals relative to the total population and relative to the subpopulations, respectively, and FST is the correlation between two gametes drawn at random from each subpopulation. These three indices can then be taken as a deviation from Hardy-Weinberg equilibrium and FST can be interpreted as a genetic index of divergence among subpopulations. Using Wright's statistics, the author found that the gene diversity in the total population or total heterozygosity (HT) can be distributed within (HS) and among subpopulations (DST), and the gene differentiation among subpopulations (GST) is estimated by the proportion DST/HT.
Genetic similarities and genetic distances among populations were estimated according to Nei (1972, 1973, 1978). The obtained values were utilized to construct a dendrogram based on the unweighted paired group method (UPGMA method; Sneath and Sokal, 1973).