ESTERASE POLYMORPHISM IN REMANANT POPULATIONS OF Aspidosperma polyneuron Müll . Arg . ( APOCYNACEAE ) 1

R. Árvore, Viçosa-MG, v.28, n.5, p.625-631, 2004 ABSTRACT – The population genetic structure of the endangered tree species Aspidosperma polyneuron Mull.Arg. (Apocynaceae) was reported based on analysis of esterase polymorphism in two remanant populations. Allelic variation was detected at three isoesterase loci (Est-3, Est-9, and Est-10). The proportion of polymorphic loci for both populations was 30% and deviation from Hardy-Weinberg equilibrium was observed for the Est3 locus observed in the northern population. Segregation distortion and the lower level of observed and expected heterozygosity in this population were attributed to founder genotype. The high genetic identity values for northern and northwestern populations are in accordance with the low levels of interpopulation genetic divergence demonstrated by the F(ST) (0.03) value. The F(IS) value (0.23) indicated moderate levels of inbreeding. A. polyneuron can be indicated as an example of endangered species suggesting high genetic variation in contrast to the low genetic variation reported for endangered species. The esterase isozymes may be a good genetic marker for studies of natural A. polyneuron populations.

In the State of Parana, the remanant populations of A. polyneuron are limited to small isolated sites and the study of these populations has mainly focused on morphological characteristics of flowers and fruits (CONSTANTINO, 1990), and experimental culture of seedlings obtained by seed germination (SOUZA and MOSCHETA, 1992).Studies of genetic diversity in A. polyneuron are important for the conservation of this species and for breeding work.However, no information about biochemical markers and the genetic variability of this species is available in the specialized literature.Estimates of plant genetic diversity are necessary if we are to understand the forces that affect the genetic organization of natural plant populations (GITZENDANNER and SOLTIS, 2000).Maintaining the existing genetic diversity in populations is one of the most important measures for species conservation.
The use of allozyme variation has a long tradition in population genetics, and more recent application in conservation biology (GITZENDANNER and SOLTIS, 2000).Starch gel electrophoresis for isozyme analysis is of significant value as a method for determining genetic diversity in plants of economic importance because selective neutrality at isozyme loci can be assumed in most cases, and therefore the product of these loci provide a measure of relatedness among the plants that have been severely altered by natural or experimental selection processes (KARHU et al., 1996;STORFER, 1996).Nonspecific esterases are usual markers in genetic studies of animals, plants and microorganisms because they are easy to detect and appear to be highly polymorphic (DAVIS, 1964;MANGOLIN et al., 1997;RESENDE et al., 2000;MACHADO and CASTRO-PRADO, 2001).For most esterases a rather general substrate specificity is observed, indicating that they may have a broad biological function.
In the present study we reported esterase polymorphism as a biochemical marker in order to obtain preliminary data on the genetic variability present within and between two remanant populations of the A. polyneuron species.

MATERIAL AND METHODS
Samples of young leaves (8-10 mm) of A. polyneuron were collected from two different populations in the northern and northwestern regions of the State of Paraná (PR), Brazil (BR).The northern population consisted of 15 plants of medium height (4-5 m) established on the campus of the state University of Londrina (Londrina, PR) which were obtained from seedlings grown from germinated seeds in green house.The seeds were collected from plants previously native to this region but that were cut for different reasons (dying from diseases or urbanization processes).
The northwestern population consisted of 48 native plants naturally conserved for 54 years in the "Horto Florestal Dr. Luiz Teixeira Mendes" situated in an urban area (approximately 37 hectares) of Maringa (Maringa, PR).The "Horto Florestal" is an area protected from destruction established to conserve native and specific rare taxa, which often occur in small isolated populations or fragmented populations that have been reduced in size.The distance between Londrina and Maringa is 100 km.The plant height of the Maringa population ranged from 3 to about 10 m.
The electrophoretic evaluations were carried out on samples consisting of young leaves collected from each 'peroba-rosa' tree.The leaves were individually homogenized with a glass rod in an Eppendorf microcentrifuge tube using 60 ml of 1.0 M phosphate buffer, pH 7.0, containing 5% PVP-40, 0.01 M DTT (dithiothreitol), 10 mM sodium metabisulfite, 50 mM ascorbic acid, 1.0 mM EDTA, and 0.5% β-mercaptoethanol solution (RESENDE et al., 2000).After homogenization, the samples were centrifuged at 25,000 rpm for 30 minutes at 4 °C in a Sorval 3K-30 centrifuge.
In the electrode chambers we used 0.1 M Tris and 0.028 M citric acid, pH 7.5.Electrophoresis was carried out at 4 °C for approximately 5-6 hours at 35 mA (8.5 V/ cm of gel).
4-Methylumbelliferyl esters (acetate and propionate) and α-naphthyl acetate and ß-naphthyl acetate were used as substrates (TASHIAN, 1969, modified by RESENDE et al., 2000) for isoesterase detection and comparative analysis of the plants from each population.4-Methylumbelliferyl acetate (4 mg) or propionate was dissolved in 500 ml acetone and the volume was completed to 10 ml using twice-distilled water.After staining with 4-methylumbelliferyl esters the gels were washed with tap water and separately incubated for 30-60 min in a solution containing 50 ml 0.05 M sodium acetate, pH 6.5, 40 mg fast blue RR salt and 4 mL 1% a-naphthyl acetate or 4 mL 1% ß-naphthyl aceatae (RESENDE et al., 2000).
Genetic variability in the two A. polyneuron populations was analyzed using the BIOSYS-1/A Computer Program for the Analysis of Allelic Variation in Genetics (SWOFFORD and SELANDER, 1989).Allele frequencies, mean heterozygosity and mean number of alleles per locus, percentage of polymorphic loci, and genetic similarity were calculated for the two A. polyneuron populations.
Two alleles were observed in each of the Est-3 (Est-3 1 and Est-3 2 ), Est-9 (Est-9 1 and Est-9 2 ), and Est-10 (Est-10 1 and Est-10 2 ) loci for the NO and NW populations (Table 1).The allele 2 variants were found to have a faster electrophoretic mobility than the allele 1.The proportion of polymorphic isoesterase loci for both A. polyneuron populations was 30%.Allele frequencies per locus are listed in Table 1.
Chi-square analysis showed no differences between observed genotypic frequencies and expected Hardy-Weinberg frequencies for the Est-3, Est-9, and Est-10 loci of the NW population, but departure from expected equilibrium was observed in the NO population for the Est-3 locus (Table 2).Deviation from Hardy-Weinberg expectation represented by the fixation index (F) was greater at the Est-3 locus of the NO population (F = 0.70).Genetic diversity calculated by NEI' s (1972) genetic identity (I) or distance coefficient (D) showed high I and low D values for the two A. polyneuron populations (Table 3).The high genetic identity values for the NO and NW populations are in accordance with the low levels of interpopulation genetic divergence demonstrated by F (ST) (Table 3).F-statistic values calculated for the two populations indicated no differences in allele fixation between the two populations.

DISCUSSION
Despite the history of fragmentation of the A. polyneuron species, the isozyme esterase pattern showed a large amount of genetic variation in this species.Genetic diversity values (% polymorphic loci, observed and expected heterozygosity) were higher than mean values for dicotyledonous species reported by Hamrick and Godt (1989).Based on the high diversity and low level of population differentiation (F ST = 0.033) we    conclude that the A. polyneuron plants found in the remaining fragment at "Horto Florestal" (NW population) is likely to reproduce primarily sexually by outcrossed matings.The lack of major differentiation between the NO and NW populations suggests either substantial gene flow among populations or that fragmented populations have not yet been isolated long enough for genetic drift (or selection) to have caused population differentiation.
On the other hand, because most populations of A. polyneuron have been interconnected at least until the recent past, severely reducing population sizes could alter the mating structure within populations by increasing mating among relatives and thus increase total inbreeding (F IT = 0.263).Fragmentation isolating previously connected populations could have deleterious effects by disrupting gene flow among populations and could exacerbate loss of diversity through drift and further increase inbreeding in remaining fragments (TEMPLETON et al., 1990;YOUNG et al., 1996;HUSBAND and SCHEMSKE, 1996).
The restoration of degraded sites to increase population sizes and to connect fragmented populations could be useful to prevent the deleterious effect of the fragmentation process.Reintroduction of plants is recommended in the restoration plan of a taxon, but the analysis of isoesterases in the NO population of A. polyneuron indicated that it is important to select the founder genotype to prevent a loss of genetic diversity.Isoesterase study in A. polyneuron showed a lower level of observed and expected heterozygosity in the NO than in the NW population.Heterozygosity and inbreeding are specific measures of diversity that should be targeted because of the direct influence of levels of diversity on fitness (HUENNEKE, 1991;LINHART and GRANT, 1996;LATTA and MITTON, 1997).
The lower level of observed and expected mean heterozygosity in the NO population than the level of observed and expected mean heterozygosity in NW population may be attributed to the effect of founder genotype or may be due to a severe reduction in size of this population since the NO population has recently recolonized its current distribution area.Foundation events usually reduce within-population diversity and could increase differentiation among populations (AUSTERLITZ et al., 2000).
Segregation distortion observed for the Est-3 locus in the NO population of A. polyneuron may be also attributed to the method used to establish this population: the seedlings grown from germinated seeds in house gardens were used to establish the actual NO population.Thus, the founder genotype can produce nonrandom phenotypic distribution and an absence of Hardy-Weinberg equilibrium.
The lower number of plants sampled in the NO population seems not to be a factor leading to the absence of Hardy-Weinberg equilibrium since no evidence of random phenotypic distribution was detected by isoesterase analysis of 13 plants reaching 4-5 m in height found in the "Horto Florestal" fragment (data not shown).We examined the diversity among plants of the three age classes (11 plants higher than 8 m, 13 plants reaching 4-5 m in height, and 20 plants reaching 6-8 m in height) within the NW population and the homogeneity of genetic structure and genetic diversity among the three age classes seems to reflect the occurrence of similar reproductive events year after year (unpublished results).
Esterase polymorphism in remanant population of A. polyneuron suggest this species as an example of an endangered species showing high genetic variation in contrast to the low genetic variation commonly reported for others endangered species (FRANKHAN, 1995).However, we suggest that caution should be taken in performing a balanced recolonization of NO population of A. polyneuron species since genetic consequences of founding events may strongly affect the genetic structure of this population.Under conditions causing loss of genetic diversity and decreased gene flow, decreased heterozygosity and increased differentiation among populations are expected.Such patterns are thought to be detrimental and to increase extinction risks.Esterase polymorphism in A. polyneuron species can be useful as a genetic biochemical marker for choosing the founder genotypes or in order to supply the restoration program.