Journal of Heredity 2003:94(3)
© 2003 The American Genetic Association 94:218-226
Isozyme Polymorphisms Provide Evidence of Clinal Variation With Elevation in Nothofagus pumilio
From the Laboratorio Ecotono, Centro Regional Universitario Bariloche, Universidad Nacional del Comahue, Quintral 1250, 8400 Bariloche, Argentina.
Address correspondence to Andrea C. Premoli at the address above, or e-mail: apremoli{at}crub.uncoma.edu.ar.
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Abstract |
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Variable physical conditions along elevational gradients strongly influence patterns of genetic differentiation in tree species. Here, the hypothesis is tested that different growth forms of Nothofagus pumilio, which characterizes the subalpine forests in the southern Andes, will display continuous genetic variation with elevation. At each of four elevational strips in three different mountain ranges, fresh leaf tissue was sampled from 30 randomly selected individuals to be analyzed by protein electrophoresis. Allelic frequencies were used to test for heterogeneity across populations and to classify populations into different elevational strips by discriminant analysis. The degree of population divergence was estimated by FST. Clinal variation on within-population genetic characteristics was analyzed by linear regressions against elevation. Seven enzyme systems coded for 14 putative isozyme loci, 57% of which were polymorphic in at least one population. Allele frequencies significantly varied with elevation and discriminant analysis separated populations at different elevational strips. Among-population divergence within any mountain range was small, but greater than among different mountain ranges. Overall, low-elevation populations were more variable than high-elevation populations, and regression analyses suggested continuous variation in populations of N. pumilio 100 m apart. Marked stepwise phenological differences on mountain slopes are most probably responsible for the isolation of nearby populations.
Elevational gradients impose heterogeneous environmental conditions that affect plant performance, which can markedly vary within short distances. Changing physical conditions such as low temperatures accompanied by frost, frozen ground, or accumulation and permanency of the snow cover, as well as strong winds and high irradiance, will differentially affect plant populations with elevation (Larcher 1995). Because plants are sedentary, patterns of population divergence will tend to follow the underlying patterns of the environment.
Genetic differentiation among plant populations has been documented for important features of plant structure and function and also genetic markers, some of which yielded significant character variation over small scales [Linhart and Grant (1996) and references therein]. These variable patterns can result from adaptive responses to ecological gradients and/or restrictions for gene flow. In particular, if genes are subjected to natural selection, high gene flow can be overcome by selection among heterogeneous environments, producing clines associated to physical gradients (Mitton 1995). [The term cline was introduced by Huxley (1938) to designate a gradation in measurable characters, which might be continuous or discontinuous, not necessarily implying genetically based variation.]
Gene flow seems especially great in forest trees, particularly in species with pollen and seed dispersed by wind (Hamrick et al. 1992). However, even airborne pollen may not be as effectively dispersed if the timing of pollination is affected by phenological differences such as those occurring along elevational gradients. Likewise, wind-dispersed seeds of some woody species have localized seed dispersal mechanisms which may cluster genetically similar individuals into family groups (Linhart et al. 1981) as well as reproductively isolate nearby populations.
Elevational gradients impose severe constraints for reproduction and establishment, particularly in high-elevation populations, due to a shorter growing season, lower temperatures, and longer persistence of snow cover. This means that in addition to intense selection regimes, populations at the tree line are probably affected by genetic drift and increased inbreeding acting in relatively small and isolated populations. As a result, high-elevation populations will tend to be genetically depauperate.
Nothofagus pumilio (Poepp. et Endl.) Krasser, common name lenga, characterizes subalpine forests in the southern Andes. It presents marked variation with elevation in the growth habit of individuals, from erect trees through stunted forest to prostrate shrub forms (Veblen et al. 1977) (Figure 1). Morphological variation in N. pumilio has been hypothesized as a plastic response to variable environmental conditions along elevational gradients (Donoso 1987). However, remarkable differences in various life history traits of N. pumilio with elevation (see below) suggest that those character variations may have a genetic basis. This study will analyze the hypothesis that different growth forms of N. pumilio along elevational gradients in Patagonia show continuous genetic variation by means of isozymes.
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The Species
Nothofagus pumilio is a broad-leafed deciduous tree that is distributed from 35°35'S in Chile and 36°50'S in Argentina to the southernmost limit of temperate forests in austral South America at 55°S in the island of Tierra del Fuego. Although it has a wide latitudinal range, it is mostly habitat restricted and dominates subalpine communities in which prostrate individuals characterize the upper tree line (Figure 1). N. pumilio is monoecious with wind-dispersed pollen and restricted seed dispersal (Rusch 1993). Continuous variation in several traits has been measured in N. pumilio along elevational gradients. These include a significant delay in leaf budding initiation and a decrease in leaf size with elevation (Rusch 1993), a reduction in total above-ground tree biomass and radial growth, together with an increase in leaf biomass and a decrease in leaf decomposition with altitude (Barrera et al. 2000). In addition, seed characteristics vary with elevation in N. pumilio. The proportion of seed-bearing fruits decreases from 71% to 21% at low and high elevation, respectively (Cuevas 2000). Moreover, while seed weight significantly decreases with elevation, a greater proportion of insect-attacked seeds were measured in low-elevation populations (Premoli, in press). Germination capacity also decreased with elevation from 26% to less than 5% (Premoli, in press) or null (Martínez Pastur et al. 1997) in high-elevation populations. Although the genetic basis for these character variations has not yet been determined, they strongly suggest that different forces are shaping microscale differentiation in N. pumilio. Nonetheless, comparison of physiological responses of N. pumilio plants from different elevations under field and common garden conditions suggests a genetic control of net assimilation as an adaptation to nitrogen economy (Premoli, in press).
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Materials and Methods |
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Populations of N. pumilio were sampled within four elevational strips about 100 m from each other in three different mountain ranges within Nahuel Huapi National Park in northwestern Patagonia. These study sites are Challhuaco (41°15'S, 71°18'W), Cerro López (41°06'S, 71°33'W), and Cerro Otto (41°08'S, 71°21'W). Populations were chosen according to their different growth habits (Figure 1) which corresponded to different elevational strips as highest, intermediate-high, intermediate-low, and lowest elevation for each mountain range. The total altitudinal range extended from 1,100 to 1,600 m above sea level; for the elevations of sampled populations, see Table 1. In addition, these populations were compared to two other populations from the southernmost distributional limit of N. pumilio at 385 and 670 m above sea level in Volcán Martial, Tierra del Fuego (54°48'S, 68°22'W). Note that at this location the tree line occurs at a lower elevation than further north, and thus this study may contribute to understanding the effects of elevation at contrasting latitudes on the genetic characteristics of such a widespread species.
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At each location, fresh leaf tissue was collected from 30 randomly selected individuals, which was kept refrigerated until enzyme extraction in the laboratory. Protein electrophoresis was performed following the protocols described elsewhere for congener species within the same subgenus Nothofagus (Premoli 1994, 1997, 1998). Homogenates were prepared by crushing approximately 1 g of material with liquid nitrogen and adding 2 ml of the extraction buffer by Mitton et al. (1979). Samples were centrifuged and then stored at -80°C until they were analyzed by horizontal electrophoresis on 12% w/v starch gels.
Seven enzyme systems coding for 14 putative isozyme loci were resolved as follows: alcohol dehydrogenase (Adh-1, Adh-2), aldolase (Ald), isocitric dehydrogenase (Idh-1, Idh-2), malate dehydrogenase (Mdh-1, Mdh-2, Mdh-3), and 6-phosphogluconate dehydrogenase (6Pgd-1, 6Pgd-2, 6Pgd-3) in the morpholine-citrate buffer by Ranker et al. (1989), whereas glutamate dehydrogenase (Gdh) and phosphoglucoisomerase (Pgi-1, Pgi-2) in the modified system B by Conkle et al. (1982). The loci are considered putative because no formal genetic analyses were performed for N. pumilio. However, the obtained banding patterns were similar to those found in other species for which genetic control of the isozyme markers was determined (Murphy et al. 1996; Soltis and Soltis 1989).
Isozyme variation indices were calculated for each population by the mean number of alleles/locus (A), effective number of alleles (AE), total number of alleles (AT), percent of polymorphic loci under 95% and sensu stricto (P<95 and P, respectively), and observed and expected heterozygosity (Ho and He, respectively). Average within-population genetic variability parameters from 12 populations sampled along three elevational gradients at the northern range were compared to those from southernmost populations. Departures from Hardy–Weinberg expectations were evaluated for each polymorphic locus in each population by calculating Wright's (1931) fixation index (F = 1 - [Ho/He]). Significant deviations of F from zero were tested using a chi-square test formulated in terms of the fixation index F (Li and Horvitz 1953).
Population genetic structure was measured by F statistics: FIT and FIS are the total and within-population inbreeding coefficients, respectively, and FST indicates the degree of among-population divergence, which is also an indirect estimator of gene flow (Wright 1965). Means and 95% confidence intervals were computed on polymorphic loci by resampling schemes following Weir and Cockerham (1984) using FSTAT version 2.9.1. (Goudet 2000). These indices were calculated for different hierarchical levels: within and among populations at different elevations for each mountain range and among different mountain ranges. In addition, gene diversity analyses were performed for different levels of the hierarchy, that is, for populations within each mountain range and among different mountain ranges using polymorphic loci. These were performed according to the total genetic diversity (HT), which was partitioned into the within-population component (HS) and the proportion of the total diversity found among populations (GST) according to Nei (1973).
To test for possible genetic divergence among populations with elevation, heterogeneity in allelic frequencies across populations within a given mountain range was evaluated by chi-square tests following Workman and Niswander (1970). Moreover, discriminant analysis of arc sine-transformed allele frequencies at the most variable alleles was used to classify populations into different a priori alternative groups of populations, which in this study correspond to different elevational strips as highest, intermediate-high, intermediate-low, and lowest elevation at each mountain range. This multivariate technique classifies populations on the basis of linear combinations of variables, that is, in our case allelic frequencies, that represent the maximum possible separation among the groups while minimizing the variance within each group (Afifi and Clark 1990). Wilk's 
test was used to illustrate if the groups present significant differences in the position of their centroids; = 0 indicates maximum dispersion of the centroids or perfect discrimination, whereas = 1 indicates no dispersion between groups or no discrimination. Squared Mahalanobis distances between the group centroids (population averages) were calculated to study the discriminatory power between any two groups (Legendre and Legendre 1983).
Clinal variation on genetic characteristics of N. pumilio was analyzed by linear regressions of different parameters of within-population isozyme variation against elevation as an independent variable. These parameters were population averages for A, AE, AT, P<95, P, Ho, and Ho. Regressions were run for all populations as well as for populations within each mountain range separately.
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Results |
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Eight of the 14 analyzed loci (57%) were polymorphic in at least one population: Adh-1, Adh-2, Ald, Idh-2, Mdh-1, Mdh-2, Mdh-3, and Pgi-2. These results add a new polymorphic locus (Mdh-1) to the preliminary study on latitudinal variation in N. pumilio (Premoli 1998). Highest-elevation populations had a greater number of polymorphic loci with fixed alleles (7, 6, 8, and 4) than lowest elevation populations (6, 4, 3, and 3 for Challhuaco, López, Otto, and Martial respectively; see Appendix).
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Reduced within-population genetic variation was recorded for the analyzed populations along the elevational gradient. Polymorphism at the population level reached at most 36%, and in all cases heterozygosity was less than 6%, with some populations having no polymorphism or heterozygosity (Table 1). However, populations from the species' southernmost range at Volcán Martial seem to be more genetically variable than those from the northern range (Challhuaco, López, and Otto). Tests for Hardy–Weinberg equilibrium showed that out of 44 possible tests, 36% yielded significant deviations from equilibrium conditions and also homozygous excess, 4% indicated heterozygous excess, whereas 60% suggested equilibrium conditions for the analyzed loci (data not shown).
Analyzed populations were highly heterogeneous in their allelic frequencies. Within a given mountain range, allelic frequencies of populations at the four different elevational strips differed significantly (P <.05) for two of five possible tests in Challhuaco and Martial and for all five loci in López and Otto (see Appendix). Discriminant analysis on the arc sine transformed allelic frequencies significantly separated the four elevational strips which were used as a priori grouping variables (F(6, 14) = 2.9, P <.05). The first discriminant function explained 99% of the total variance and significantly separated different elevational strips (chi-square test for canonical roots: 
2 = 13.0, P <.05), particularly the two high-elevation strips from the low-elevation ones. Wilk's equaled 0.19, which is close to zero, meaning marked dispersion of centroids (i.e., among elevational strips' averages). Populations belonging to the high-elevation strip were all classified as belonging to their respective elevational strip, whereas one-third of the populations from other elevations were correctly classified. This yielded an average of 50% correct classification. Squared Mahalanobis distances were not significant between any extreme elevation and its nearby strip, that is, between high and intermediate-high as well as between low and intermediate-low elevational strips, respectively (Table 2). In contrast, all the other pairwise between-strip possible comparisons where statistically significant.
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Overall total genetic diversity measured by Nei's statistics was low (mean HT = 0.047), most of which was distributed within populations (HS = 0.045) (Table 3). Inbreeding coefficients had positive values on average and hierarchical analysis of population structure by F statistics yielded elevated total and within-population inbreeding. Reduced among-population genetic divergence was measured by FST. Average FST values varied for different mountain ranges: from 0.028 in Otto, to 0.057 and 0.131 in López and Challhuaco. However, for each mountain range these FST values were within the range or greater than the divergence calculated among different mountain ranges, FST = 0.019. These results suggest that relatively lower divergence and thus greater gene flow rates are maintained among different mountain ranges compared to the among-population divergence measured along elevation within any mountain range (Table 3).
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Regression analyses of within-population isozyme variation parameters along elevational gradients indicated a continuous reduction of genetic variation in N. pumilio with increasing elevation. Overall, low-elevation populations were significantly more variable than high-elevation ones in terms of the mean number of alleles per locus, the total number of alleles, and the percent of polymorphic loci (Figure 2, Table 4). In addition, similar reductions in within-population variability indices were recorded at individual mountain ranges. That was the case for the mean number of alleles per locus, total number of alleles, and observed heterozygosity in Challhuaco as well as for the percent polymorphism in López (Table 4).
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Discussion |
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Genetic Variation Decrease With Elevation
Levels of within-population genetic variation in N. pumilio significantly decreased with elevation and high-altitude populations tended to have reduced polymorphism and heterozygosity along with a greater proportion of loci with fixed alleles. These results fit the expectations for populations at higher elevations, which would tend to be genetically depauperate probably due to the combined effects of genetic drift and/or greater inbreeding. Data on seed characteristics of N. pumilio populations at Challhuaco and Cerro Otto indicated that high-elevation individuals tend to produce lighter seeds, which in turn had lower germination capacities than low-elevation ones (Premoli, in press). In addition, limited seed production and seedling emergence measured in the southernmost populations of N. pumilio in Tierra del Fuego suggested an overall reduced probability for adult establishment at the alpine timberline (Cuevas 2000). Therefore empirical data on demographic traits of N. pumilio indicate severe bottlenecks for tree recruitment at higher elevations, which increases the potential for genetic drift affecting those populations. In contrast, relatively more abundant regeneration by seedlings at lower elevations results in a greater potential for gene flow and thus elevated isozyme diversity. A similar pattern of greater isozyme diversity in low-elevation populations was obtained for populations of Cryptomeria japonica, which was related to abundant seedling establishment (Taira et al. 1997). On the contrary, small differences in the average heterozygosity and effective number of alleles were measured among populations of Norway spruce (Picea abies) with elevation (Lundkvist 1979). In this case, selection for distinct microhabitats in different localities may counteract intense directional selection or genetic drift that may reduce variability at species margins, such as the tree line.
Nevertheless, a population of N. pumilio exists at the highest elevational limit in Cerro López with considerable polymorphism and heterozygosity (Table 1). At this location, evidence of hybridization between N. pumilio and Nothofagus antarctica has been recently described in terms of leaf morphology and isozymes (Quiroga P, personal communication) with greater polymorphism than in pure populations. Thus isozyme characteristics of N. pumilio may be the result of complex processes taking place along elevational gradients in the southern Andes.
Positive FIT and FIS values obtained in this study suggest that significant total and within-population inbreeding occur in N. pumilio along elevational gradients. It is not yet clear whether this results from biparental inbreeding or vegetative spread. It has been documented that N. pumilio is able to resprout after damage (Rebertus et al. 1997; Veblen et al. 1996) and that vegetative spread increases with elevation in Tierra del Fuego (Martínez Pastur et al. 1997). The genetic consequences of these variable reproductive strategies, particularly at small spatial scales, deserves further analysis.
On average, within-population measures of isozyme variation (Table 1) were within the range of those reported previously for N. pumilio populations located toward the northern distributional range of the species (Premoli 1998). However, N. pumilio seems to maintain reduced isozyme polymorphisms in comparison to other relatively more latitudinally restricted South American Nothofagus with polymorphisms and observed heterozygosities of 25% and 0.08 in Nothofagus dombeyi (Premoli 1997) and 75% and 0.17 in Nothofagus alpina (Marchelli and Gallo 2001), respectively. It was suggested that widespread species may maintain greater polymorphisms, given that most probably they consist of historically larger and more continuously distributed populations (Hamrick et al. 1992). Although latitudinally widespread, N. pumilio is restricted throughout most of its range to high-elevation forests, which may suffer restrictions in gene flow as well as the effects of genetic drift, resulting in reduced isozyme variation. Hence the comparison of N. pumilio with its range-restricted congeners supports the hypothesis that levels of genetic variation in tree populations maybe predicted not only in relation to their overall geographic distribution, but in concert with the continuity of their ranges (Premoli et al. 2001).
In addition, total genetic diversity reported here (HT = 0.047) is lower than that previously measured (HT = 0.112) (Premoli 1998). This higher diversity may be a result of the sampling schedule in this early study that consisted of populations located over a wide geographic area and therefore genetic characteristics reflect the effects of drift on populations relatively isolated from each other. Similarly, elevated isozyme polymorphisms for southernmost populations (Table 1) may be a reflection of an overall highly diverse gene pool previously reported for Tierra del Fuego as a result of diverse environments occupied by N. pumilio (it is the only location throughout its entire distribution where it can be found from sea level on the dry Atlantic coast to tree line on more humid western slopes) together with a complex evolutionary history (Premoli 1998).
Relationship Between Genetic and Character Variation
The evidence presented here suggests that populations of N. pumilio along elevational gradients are in the process of genetic differentiation. Although the extent of among-population divergence can be considered marginal, for example, FST ranged from 3% to 13% within any studied mountain range, they were relatively greater than that calculated among different mountain ranges (FST = 2%). However, marked differences were found for other traits studied in N. pumilio populations from Challhuaco. Differences in maximum net assimilation measured on plants from contrasting elevations under field and common garden conditions yielded 40% and 18% significant differences, respectively, which suggested a genetic control of photosynthesis probably in relation to differences in nutrient use efficiency with elevation (Premoli et al., in review). This is in addition to preliminary data on seedlings from distinct elevations that were grown for 2 years under homogeneous environments that showed phenological differences such as early leaf burst and larger plant size in low-elevation plants (Premoli AC, personal communication). These patterns of divergence apparently have not occurred at the same rate as that of isozyme loci in N. pumilio.
Other examples in trees such as in the Hawaiian Metrosideros suggest that variation in morphology with elevation has not been paralleled by genetic differences, which has been interpreted as evidence of incipient speciation in insular environments (Aradhya et al. 1991). Shrub and tall forests of pitch pine (Pinus rigida) were essentially identical in genetic constitution (Guries and Ledig 1982), in contrast to phenotypic differences at morphological characteristics (Ledig and Fryer 1974), some of which had a genetic basis as revealed in common garden experiments (Ledig et al. 1976). Therefore the limited isozyme divergence measured in the present study may suggest that populations of N. pumilio along elevational gradients are in the early stages of genetic differentiation which may develop into distinct ecotypes.
Divergence Along Elevational Gradients
A significant heterogeneity in allelic frequencies along elevational transects together with a greater similarity of populations at a given elevational strip from different mountain ranges yielded by discriminant analysis suggest that barriers for gene flow may exist between nearby populations of N. pumilio. The extent of among-population differentiation depends on the amount of gene flow via pollen and seed movement, which will be affected by different isolating factors that act to disrupt gene exchange, such as the spatial distance between populations. Over short distances, gene flow in forest trees with airborne pollen is expected to be great, and therefore differentiation seems unlikely. However, other factors may produce divergence between populations in close proximity, such as the time of pollen release and female receptivity (phenology of flowering), which is vital in the potential effectiveness of pollination (Grant and Antonovics 1978). This is particularly relevant along elevational gradients, given that trees within a given elevational strip are more likely to be synchronized with one another than with trees progressively farther away, resulting in a temporally and spatially concentrated bloom (Rathcke and Lacey 1985). For N. pumilio, a 10-day delay in flowering initiation was measured in populations located at 1,225 and 1,390 m in northern Patagonia (i.e., approximately 6 days every 100 m) and the onset of flowering was synchronous at each elevation (Rusch 1993). In addition, female flowers are receptive for about a week (Kitzberger T, personal communication). This means that significant reproductive barriers may exist even between populations only 100 m apart due to the fact that female flowers at a given elevation will tend to fall before pollen from a different elevational strip can reach them. Similar results were obtained for limber pine (Pinus flexilis), where nonoverlapping pollination periods along elevational transects resulted in limited gene exchange occurring in any single generation (Schuster et al. 1989).
Moreover, N. pumilio seeds are mainly dispersed underneath the crown of seed-producing trees (Rusch 1987), resulting in limited gene flow via seed movement. Restrictions for seed dispersal may result in the formation of family groups, as shown in the elevated inbreeding coefficients detected by F statistics. In addition, heterozygote deficiencies may reflect a Wahlund effect probably associated with the sampling schedule which sought to maximize the detection of variability from unrelated genotypes.
Continuous Genetic Variation With Elevation
Nothofagus pumilio presents clinal variation as suggested by significant correlations of different indices of within-population isozyme variation with elevation. Similar patterns have been described for other tree species. In Engelmann spruce (Picea engelmannii) and subalpine fir (Abies lasiocarpa), a significant differentiation with elevation in the peroxidase locus was correlated with spire, flag, and krummholz morphs along elevation (Grant and Mitton 1977). In Norway spruce (Picea abies), in 4 of 11 loci, gene frequencies significantly varied with altitude (Lundkvist 1979).
The data presented here suggest that changing environmental conditions along elevational gradients in the southern Andes exert significant barriers for gene flow which, in addition to different selection regimes, determine genetically based clinal variation patterns in populations of N. pumilio as close as 100 m apart. Other traits measured on N. pumilio have yielded differences with elevation such as leaf (Rusch 1993) and seed characteristics (Cuevas 2000), structural and functional traits (Barrera et al. 2000), and ecophysiological variables, some of which show hereditary effects (Premoli AC, in review). Marked stepwise phenological differences on mountain slopes are most probably responsible for the isolation of nearby populations, which in combination with elevated within-population inbreeding result in divergent gene pools. Although differentiation seems recent, populations located at the species' extreme elevations represent a natural experiment which deserves further study to analyze processes related to intraspecific divergence in relation to changing environmental conditions.
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Acknowledgments |
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I thank M. Caldiz, F. Kitzberger, G. Lovrich, F. Premoli, P. Soares, and N. Tercero for field assistance, and the Delegación Regional Patagonia, Administración de Parques Nacionales, for providing access to protected areas. Y. B. Linhart and three anonymous reviewers made insightful comments on the manuscript, and I am also grateful to M. Barrera for providing the original version of Figure 1. This research was funded by the International Foundation for Science (D/2544-1). The author is a researcher of CONICET.
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Footnotes |
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Corresponding Editor: Jonathan F. Wendel
Received July 24, 2002
Accepted February 20, 2003
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References |
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