This study demonstrated that nine out of 80 electrophoretic buffer/isozyme system combinations could be used routinely, because they provided high resolution and unambiguous zimogram patterns. Some of the selected systems, such as tris-citrate/histidine, also showed also their potential for use with other gel/electrode buffers, depending on further adjustment in their protocols.
Since no genetic studies of allozymic loci have been carried out on this species, the inference of the existence of a locus was based on studies of other plant species. Nevertheless, several genetic indexes were estimated to describe the genetic structure of the remaining natural populations in Santa Catarina state, based on these non-adaptive markers.
As expected, with the increase in the number of analyzed individuals, the number of alleles per locus increased. At populational level, the number of alleles ranged from 19 to 28 in 15 loci, when 25 to 45 plants were assayed. However, at species level, in the same loci, 33 alleles were captured when the sample reached 328 plants.
Unique alleles were detected in FGG and PML, the most preserved and degraded areas, respectively, both in the Lages region. Rare alleles were shared by populations the RAC and FGG, FTB and FGG, ECA and FGG, and FAF and RAC populations. It is intriguing that unique or rare alleles were found in six of the nine populations. However, populations located in the Lages region maintain most of them. Thus, it is reasonable to advocate that any plan for conservation should include this region.
From the amount of survey of different plants sampled across the state of Santa Catarina State (328), three out of the 15 loci were monomorphic. The other 12 loci were polymorphic at species level, but not all of them were polymorphic at populational level. In addition, for polymorphic loci, it is relevant to mention that the proportion between the frequencies of the most frequent allele and its companion allele, in all loci with two alleles, was very high, usually higher than 4.
The percentages of polymorphic loci (mean 43.3%, Table III) and numbers of alleles per locus (mean 1.8) were substantially higher in the populations FGG and ECA populations, both under good conservation status, in comparison with the values (mean 20.0% and 1.4, respectively) revealed by FTB, FAF, PML and AVM, the most degraded populations. This comparison is very useful, since all populations are fragments of the same forest, with different degrees of degradation and conservation. In fact, the indiscriminate exploitation of pinheiro-do-Paraná is responsible for a significant proportion of the genetic erosion of this species.
The two most conserved populations, FGG and ECA, also showed the highest values for observed (Ho = 106) and expected (He = 0.112) heterozygosity. The opposite (Ho = 0,053; He = 0,059) was verified with the populations FTB, FAF, PML and AVM populations, the most exploited among the surveyed populations. Shiraishi (1983) classified the conifers as the taxa with one of the greatest values for He values (= 0.207) in comparison with other taxa. An exception was indicated by Hamrick et al. (1989), since they found the lowest value for genetic diversity in Pinus longeava among 653 reports on 449 species from 165 genera. More recently, the mean heterozygosity values estimated for species of the genus Larix (a Pinaceae) ranged from 0.048 to 0.170 (Semerikov et al., 1999). All heterozygosity values estimated for pinheiro-do-Paraná, in the present study, are included in this interval.
For a group of tropical trees and a wind-pollinated species group, Hamrick and Godt (1989) obtained mean heterozygosities of 0.109 and 0.123, respectively. Taking into account the fact that pinheiro-do-Paraná is a wind-pollinated tree, its heterozygosity values are lower than those estimated by Hamrick and Godt (1989). Additionally, for some Atlantic Rain Forest species such as Euterpe edulis (Reis et al., 1998), Cedrela fissilis (Gandara, 1996) and Myracrodruon urundeuva (Moraes, 1992; Lacerda, 1997) the mean heterozygosity values ranged from 0.378 to 0.570; 0.222 to 0.222, and from 0.076 to 0.160, respectively.
In seven of the nine populations, The observed heterozygosity values were lower than expected in seven of the nine populations, indicating the presence of a certain level of inbreeding (F ranged from 0.061 to 0.402). In fact, the adherence of the nine populations to the inbreeding equilibrium model reinforced the existence of inbreeding in this species. This inbreeding could in part have arisen from non-random mating, since it is possible that the surveyed populations still represent the population structure's pre-fragmentation structure, and that insufficient time has passed for the reconstruction of an entire structure. Additionally, genetic drift could have played a role in this case. However, it cannot be ruled out that a small proportion of new individuals could have appeared after the forest fragmentation in some of the populations. In the remaining two populations, ECA and URU, an excess of heterozygotes was found.
Both approaches to address the populational structure features indicated similar patterns. Thus, among populations, the degree of genetic diversity was not so great, varying from 0.044 (FST) to 0.056 (GST). This low amount of genetic diversity among populations was also revealed by the estimates of genetic distances. However, this kind of data should be taken with caution because the variation under analysis is of a non-adaptive type. To further define conservation strategies, other parameters, such as adaptiveness (e.g.: fitness and development) or agronomic interest (e.g.: woody quality) should be taken into account.
Among gymnosperms, there is a strong variation in the population structure among gymnosperms. The values of the Araucaria angustifolia genetic indexes are either higher or lower than the values obtained in other species of this taxa group, depending on the comparison. Hence, Ge et al. (1998) found a very high divergence (FST = 0.441) among eight populations of Cathaya argyrophylla (a subtropical conifer from China). Kitamura and Rahman (1992) also obtained higher values with natural populations of Agathis borneensis (Araucariaceae) from southeast Asia (HT = 0.122, HS= 0.106 and GST = 0.140). In contrast, Hamrick and Smith (1987) obtained a value of GST = 0.016 for 17 populations of Pinus contorta, a temperate climate wind-pollinated conifer, which was three times smaller than the value (GST = 0.056) estimated for Araucaria angustifolia. In fact, the value of 5.6% is very close to 6.8%, a GSTaverage for wind-pollinated conifers based on isozyme markers (Hamrick and Godt, 1989).
Although polyzygotic polyembryogenesis occurs in pinheiro-do-Paraná (Guerra et al., 2000), only one embryo reached maturity within the magagamephophyte, a maternal tissue responsible for feeding the germinating embryo and the development of the new plant. If it is a filter, a stabilizing selection could be conservative in this case. But the role of this event in the amount of genetic diversity of the species, whether adaptive or non-adaptive, is still not known.
The geographically closed populations FAF, PML, and FRA populations showed discrepant levels of variation. When they were taken as a whole the FST was 0.067, higher than the average (0.044). This value is considerably higher than that exhibited by comparing two distant populations, FTB and URU (0.028). This and other comparisons are not in agreement with the hypothesis that geographically close populations located near one another show low genetic divergence due to the intense gene flow. The pinheiro-do-Paraná is a typical dioecious species, with a low frequency of monoecious plants, indicating the obligatory pollen movement. However, it not known how far the pollen travels. In addition, before European contact, the araucaria forest was a continuum forest with many natural seed dispersers. Thus, it is reasonable to invoke genetic drift as a cause of those discrepancies, as a consequence of the intense timber exploitation. This clue was given by the difference between the genetic diversity indexes for FGG (the most conserved) and the nearby populations FRA, PML, and FAF populations (most degraded ones), which could be attributed to anthropoid action.
Concerns raised about genetic erosion are pertinent under such an exploitation rate, notably in small populations. Hall et al. (1996) examined the genetic diversity and population differentiation of Pithecellobium elegans, a neotropical rain forest canopy tree from Costa Rica. Eight forest fragments and a large reserve (1,500 ha) were compared for several parameters of population genetics. Allozyme heterozygosity (0.13), polymorphism (35%) and effective number of alleles (1.24) were all similar to the values reported for other tropical tree species that also occur at densities of less than one individual per hectare. However, these measures of genetic variation were lowest in populations of the smallest size, farthest from the reserve, and more isolated from other populations. In addition, differentiation among samples collected in small forest fragments and the reserve population accounted for 10% of the total genetic variation observed. The authors found a positive relationship between the level of differentiation of populations from the reserve population and their distance from the reserve. Thus, the fragmentation of what was once a large, continuous forested area is now resulting in the genetic erosion of small, isolated populations of Pithecellobium elegans.
In fact, the results presented here are dependent on both fragmentation and degradation. The trend the lower the fragment size, the lower the genetic diversity' has been tested at various different opportunities. Prober and Brown (1994) detected a significant correlation among the population size and the genetic diversity values for Eucalyptus albens in Australia. The more far-apart populations and those with less than 500 individuals were the ones that showed the least genetic variation. A similar correlation was found by Billington (1991) in Halocarpus bidwillii, a dioecious conifer native to New Zealand. The fragmentation of Pithecellobium elegans populations of Pithecellobium elegans was the cause of the genetic erosion according to Hall et al. (1996).
In the case of pinheiro-do-Paraná, the highest values for numbers of alleles per locus, percentages of polymorphic loci, and observed heterozygosity were verified in the most conserved populations, even though they were isolated by fragmentation. The history of use of these populations revealed that they had not been exploited at all or the exploitation level was very low in comparison with that of the populations FAF, PML, AVM and FTB populations, which are fragments that were highly exploited before the second half of the 20th century, and showed a low level of genetic diversity.
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