Selection of the isozyme systems
Nine out of 80 combinations of electrophoretic buffers/isozyme systems, which showed better band color intensity and separation, were selected to perform this study. Among them, eight selected systems (PGI, PGM, MDH, SKDH, IDH, 6PGDH, ACP, and G6PDH) showed the best resolution in the morpholine-citrate buffer. Although the color intensity for PRX was also obtained with tris citrate/histidine buffer, this system was equally run under morpholine-citrate buffer because a higher number of activity regions of activity (four against three loci) were detected, without losing any evaluation quality. Overall, 15 putative loci were revealed by these nine systems (Auler, 2000).
Genetic diversity
The average number of alleles per locus, taking into account the entire set of the 15 putative loci, was 1.6 and ranged from 1.3 (FRA) to 1.9 (FGG). Among loci, The number of alleles detected among the loci ranged from one (loci IDH-1, G6PDH-1 and SKDH-1) to four (loci PGI-2 and 6PG-1). A total of 33 alleles were found in these nine populations, an average of 23.7 per population (Table II)
Allelic frequencies from different loci ranged from zero to 1.0, depending on the locus and the population (Table II). For all loci with two detected alleles in all populations, one of them was usually at a frequency which was 4 to 30 times higher than the frequency of the second allele. Within a population, the percentage of polymorphic loci ranged from 13.3% (FRA, FAF, and AVM) to 46.7% (FGG), and the average was 26.6% (Table III). In fact, populations FGG and ECA exhibited at least two alleles in 10 out of 15 loci. However, at species level this value reached 73.3%. When only the polymorphic loci were analyzed, the average value for the percentage of polymorphic loci was 33.3%.
Among populations and across loci, the mean expected heterozygosity (He) ranged from 0.060 (AVM) to 0.116 (FGG), average 0.084, and the mean observed heterozygosity (Ho) ranged from 0.044 (AVM) to 0.104 (URU), average 0,073 (Table III). The He average value was 0.105 when only the polymorphic loci were analyzed.
Among the nine populations, ECA and URU fitted the panmitic equilibrium (Table IV). However, by grouping the classes with the least frequent alleles, adherence to the HW equilibrium was reached by all nine populations. The adherence to the inbreeding equilibrium was performed for all loci when sufficient genotypic classes (or degree of freedom) were available. The results indicated that all populations were in inbreeding equilibrium (Table IV).
Population genetic structure
The mean total heterozygosity (HT) for all nine populations, estimated according to Nei, was 0.089, and for sub-populations (HS) it was 0.084.The GST, which indicates how much of the genetic diversity is among populations, reached the value of 0.056. Similar analysis through Wright F-statistics of Wright revealed different values of FIS, FIT and FSTvalues for different loci (Table V). While a value of 1.000 was found for FIS in the loci PGI-1 and MDH-1 loci, a value of -0.088 was obtained with locus PRX-4. For FIT, similar results were found (Table V). The greatest genetic diversity among populations, FST = 0.075, was revealed by the locus PGI-1. Although the FST values were variable from locus to locus, the mean value of 0.044 was similar to thevalue of GST (0.056) value. In addition, these statistics revealed the existence of a certain degree of inbreeding, not only within population populations but also at species level.
The FST values for selected associations of populations are included in Table VI. The lowest value of FST value (0.005) was revealed when RAC and AVM were compared, both located in areas with ecological tension. The value of this parameter jumped to 0.028 when very distant (±200 km) populations were compared (FTB and URU) and to 0.067 when populations PML, FAF and FRA were put together. Although the values of the F-statistics are low, they are statistically different from zero, as indicated by the heterogeneity (contingency) tests.
Genetic similarity among populations
The similarity among populations ranged from 0.992 to 0.999 (data not shown). In three cases, the genetic distance between two populations was near zero: RAC and AVM, FRA and PML, and FAF and URU.
Two main groups were formed by UPGMA with the Nei unbiased genetic distance of Nei (Figure 2). The first of these includes populations FAF and URU. Two sub-groups form the second: a) populations FTB, ECA, RAC and AVM and b) populations FRA, PML and FGG. While populations of the first sub-group are located in the north of Santa Catarina, the others from the second sub-group are located in the southern region of the state. Although they do not overlap geographically, the populations of each group are included in areas with similar climatic conditions and soil.
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