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0 Ecology Research Center Kiel, Schauenburger Str. 122, D-24118 Kiel, Germany
Received for publication March 5, 1999. Accepted for publication August 31, 1999.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: AFLP • cultural landscape • fen meadows • genetic variation • habitat fragmentation • Pedicularis palustris • population history • population structure • rare plant
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Fojt and Harding, 1995
) led to the destruction of large areas of species-rich fen meadows. The remaining fen-meadow patches are often isolated from each other and contain only small populations of typical species. As a consequence, many formerly common species of these habitats are rare today.
There is a paradigm in conservation biology that genetic variation and population viability increase with population size, but decrease with population isolation (Gilpin and Soulé, 1986
; Ellstrand and Elam, 1993
; Young, Boyle, and Brown, 1996
; Menges and Dolan, 1998
). Many recent investigations support the hypothesis that low population size leads to reduced per capitata reproductive rates (Allee effect; Menges, 1991
; Lamont, Klinkhammer and Witkowski, 1993
; Ågren, 1996
; Groom, 1998
, Fischer and Matthies, 1998a
; Oostermeijer et al., 1998
) and thus population size itself is the best predictor of population viability. Random variation in environmental conditions ("environmental uncertainty"), demographic parameters ("demographic uncertainty"; Shaffer, 1987
), the interruption of biotic interactions (e.g., plant-pollinator interactions; Jennersten, 1988
; Olesen and Jain, 1994
) and genetic processes (genetic drift, inbreeding, and low levels or absence of gene flow; Charlesworth and Charlesworth, 1987
; Ellstrand and Elam, 1993
) all lead to increased extinction probabilities in small and isolated populations. The importance of random genetic processes for the survival of rare species is still subject to question. Genetic processes could result in (1) decreasing genetic diversity within populations; (2) increasing genetic differentiation among populations; and (3) the reduction of plant fitness due to inbreeding depression or due to a higher susceptibility to pathogens (Schmid, 1994
). Furthermore, populations with low levels of genetic diversity may also have a reduced potential to adapt to changing environments (Ellstrand and Elam, 1993
).
It is supposed that "naturally rare" species (Huenneke, 1991
) are adapted to conditions and processes connected to small population size (e.g., adaptation to inbreeding or to low pollinator availability). In contrast, formerly common species, which occurred in large populations and have become rare during the last few decades, may be especially vulnerable to the abovementioned processes. It is probable that genetic considerations and pollen limitation are most important for short-lived, animal-pollinated, mainly outcrossing species, as persistence of populations of these species depends on reproduction by seed (Charlesworth and Charlesworth, 1987
; Fischer and Matthies, 1997
).
A positive correlation between genetic diversity and population size and between genetic diversity and plant fitness components has been reported for some primarily outcrossing, insect-pollinated plants (Van Treuren et al., 1991
; Raijmann et al., 1994
; Fischer and Matthies, 1998b
). Furthermore, genetic differentiation among populations has been found for several rare species, most likely a result of low levels of gene flow among isolated populations (Raijmann et al., 1994
; Travis, Maschinski, and Keim, 1996
; Fischer and Matthies, 1998b
; Allphin, Windham, and Harper, 1998
). Further investigations are necessary to improve theoretical postulations about the effects of random genetic processes on genetic structure of populations and on the evolutionary potential and survival of small and isolated populations in nature (Oostermeijer, Berholz, and Poschlod, 1996
).
The short-lived, monocarpic, primarily outcrossing (Macior, 1993
) hemiparasite Pedicularis palustris (called P. palustris in this paper) was formerly common in natural fens and fen meadows in different regions of Europe. It has decreased in abundance due to changes in land-use practices of fen meadows during the last few decades (Rosenthal and Fink, 1996
; Schrautzer and Jensen, 1998
). In Schleswig-Holstein (northwestern Germany) more than 175 populations of P. palustris were recorded in the 1950s (Raabe, 1987
), while in 1997 only 12 populations still remain. These 12 populations and one population from southern Norway were included in this investigation. Today, P. palustris is considered endangered in Germany and elsewhere in Europe (Germany—Korneck, Schnittler, and Vollmer, 1996; Tchechia—Hendrych and Hendrychová, 1989; The Netherlands-Weeda, van der Meijden, and Bakker, 1990), although it is still common in areas of Europe with large undisturbed fens (e.g., Scandinavia—Lid, 1987
).
Demography, germination ecology, pollination biology (Karrenberg and Jensen, in press
), and population genetics of P. palustris were studied within the context of a study on patterns and mechanisms of abandoned fen meadows succession (Jensen, 1998
; Schrautzer and Jensen, 1998
; Jensen and Schrautzer, 1999
). In this study, genetic structure both within and among P. palustris populations at the molecular level was analyzed. The dominant DNA marker system AFLP (amplified fragment length polymorphism), which is based on two selective polymerase chain reactions, was used. AFLP has a high potential to discover genetic variation in any part of the genome and is therefore a suitable tool for population genetic studies (Travis, Maschinski, and Keim, 1996
). The aim of this study was to investigate the genetic effects of habitat fragmentation and reduction in population size of P. palustris populations by asking the following questions: (1) How is genetic variation distributed among and within populations? (2) Is there a relation between population size and genetic variation? (3) Are population size and/or genetic variation related with reproductive components of P. palustris? (4) Which conclusions can be drawn for the conservation of P. palustris in a cultural landscape?
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Pedicularis palustris is a monocarpic hemicryptophyte with a primilary biennial life cycle, which reproduces exclusively by seed. Seeds are buoyant and can be dispersed by water (Kleinschmidt and Rosenthal, 1995
). Only a minor fraction of seeds germinate immediately after seed-shedding in summer. Most seeds exhibit a primary dormancy, delaying germination until the following spring (Jensen, unpublished data). During the first growing season, a rosette develops, which forms a winterbud in autumn. Flowering takes place during June and July of the second growing season. Flowers are pollinated by bumble bees (in Europe mainly by Bombus hortorum, B. lucorum, B. lapidarius, and B. muscorum; Kwak, 1979
; Karrenberg et al., in press
). Seed set depends on visitation of pollinators (i.e., low autofertility). Even when self-pollination led to a high seed set (high self-compatibility), significant differences in seed number per capsule between experimentally selfed and outcrossed individuals were found in the smaller of two studied populations (Karrenberg and Jensen, in press
), perhaps due to inbreeding. In addition, the number of seeds per capsule (but not the number of seeds per plant) of open-pollinated flowers were reduced in the smaller population in comparison to those pollinated by hand ("pollen limitation"; Karrenberg and Jensen, in press
).
Study site, population size, and sample collection
In summer 1997 the remaining 12 populations of P. palustris in Schleswig-Holstein (northwestern Germany) were investigated. These all occur on secondary habitats such as fen meadows and pastures. To compare site conditions of these secondary habitats with those of a primary habitat a population from a fen in south Norway (located ~800–900 km north from the study sites in Germany) was analyzed. This also provided an outgroup in cluster analysis of genetic similarity. All 13 populations were sampled for ecological and genetic analyses.
The same individually marked plants were used for both measurements of reproductive components and for genetic studies. The number of sampled individuals was determined by population size, estimated as the number of flowering individuals in summer 1997. In small populations with less than ten reproductive individuals, all plants were investigated. In populations with more than ten reproductive individuals, 8–15 plants were selected along a diagonal crossing through the population area. In populations of large areal extent samples were taken at both ends of the transect, representing subpopulations of similar area as small populations. Altogether, a total of 129 P. palustris individuals were subjected to the examination. Because of the very low population size and thus the unbalanced sampling scheme, the two smallest populations (4 and 12; see Table 1) were only included in the cluster analysis, but excluded from all other analysis (see below).
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). Biomass of standing crop (including P. palustris) was also estimated. Samples from six randomly chosen subplots (0.25 m²) were harvested and returned to the laboratory, dried at 65°C for at least 48 h and weighed.
To characterize environmental conditions of the sites, soil pH and the C:N ratio of the rooting zone (0–30 cm depth) were measured. The C:N ratio indicates the nutrient status of the habitat. Soil profiles were drilled to a depth of 1 m and classified into soil types (AG Bodenkunde, 1982
).
Reproductive components of all marked individuals were determined in summer 1997 by counting the total number of capsules per plant and the number of developed seeds of ten selected capsules, situated at the bottom of the primary shoot. The average number of seeds per individual was calculated as the number of seeds per capsule times the total number of capsules per plant. In summer 1998 the number of seedlings on subplots (0.5 x 0.5 m) was also counted. As a fitness parameter the relation between number of seedlings per population (mean number of seedlings per square metre in 1998 times population area) and flowering individuals per population (in 1997) was calculated.
DNA extraction, AFLP analysis, and scoring procedure
In June 1997 tissue samples (young leaves; 0.5–1 g) from each plant were collected. Leaf samples were freeze-dried in a vacuum for 48 h, stored at -80°C, and then homogenized to a fine powder. DNA was extracted using the GenomeCleanTM System (Angewandte Gentechnologie Systeme GmbH, Heidelberg) according to the manufacturer's protocol. DNA was quantified by means of a fluorescence photometer (DyNA Quant 200, Hoefer Corporation, Minnesota, San Francisco, USA) and adjusted to an average of 12 µL per extraction. AFLP analysis was carried out following the method of Vos et al. (1995) with some minor modifications. (1) We digested only 100 ng instead of 500 ng genomic DNA and (2) omitted the selection of biotinylated fragments after ligation. Amplification was performed with four primer combinations, with each primer having three selective nucleotides. Preamplification, labeling reactions, amplification, gel electrophoresis, and gel exposure followed the method of Schondelmaier, Steinrücken, and Jung (1996). Only AFLP fragments that could be scored unambiguously were included in the analysis. All monomorphic and polymorphic AFLP bands visible in at least 95% of the individuals by eye were scored.
Statistical analyses
The presence/absence data from the AFLP samples were used to calculate genetic similarities for all possible pairwise comparisons of individuals within and among populations. Genetic similarity was calculated as GSxy = 2a/[2a + b + c], where a is the number of bands common for samples x and y, b is the number of bands present only in sample x, and c is the number of bands present only in sample y (Dice, 1945
). Based on GS values, associations among individuals were revealed by a cluster analysis with the unweighted pair-group method using arithmetic averages (UPGMA; NTSYS-pc-p package; Rohlf, 1993
), in which we used the Norwegian population as an outgroup.
Both the genetic structure of the populations from nortwestern Germany (except the small populations 4 and 12, see above) and the levels of variation in AFLP patterns were analyzed by analysis of molecular variance (program AMOVA, version 1.55; see Excoffier, Smouse, and Quattro, 1992
). AMOVA analyses were based on the pairwise squared Euclidean distances among molecular phenotypes (Stewart and Excoffier, 1996
). By using AMOVA it was possible to calculate variance components and their significance levels (based on permutation procedures) for the following hierarchical levels: among populations, among subpopulations within populations, and among individuals within subpopulations. Differences in molecular variance among populations were tested by Bartlett's statistics. Genetic differentiation (
ST) among pairs of the P. palustris populations from northwestern Germany and their levels of significance were also calculated. The ST values were also obtained from the AMOVA package and are analogous to traditional F statistics. A Mantel test was used to estimate the association between the matrix of geographic distances with the matrix of genetic distances (1000 permutations; NTSYS-pc-package; Rohlf, 1993
). Furthermore, gene flow between the pairs of populations (Ne m = 0.25 x (1/FST - 1) was calculated from ST values (Wright, 1951
).
Correlations between population size and molecular variance within populations (AMOVA sum of squares divided by n - 1; see Fischer and Matthies, 1998b
) were calculated. Correlations between population size and reproductive components and between molecular variance and reproductive components were also calculated. To determine whether molecular variance and population size had an influence on reproductive components when site conditions, vegetation composition, and standing crop were taken into account, stepwise forward multiple regressions were carried out. Principal Components Analysis (PCA) with Varimax rotation was used to analyze gradients in vegetation composition of the P. palustris sites. Site scores of the first two axes of the PCA as well as molecular variance, population size, standing crop, soil pH, and C:N ratio were included in multiple regressions on reproductive components (Statsoft, 1997
).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The habitat of population 9 was formed by lowering the water table of lake Westensee in combination with a human deposition of sandy material ~50 yr ago (Table 1).
AFLP patterns and genetic similarity
With four primer combinations the AFLP analysis yielded a total of 262 AFLP loci among the 129 individuals. Of these loci 167 (=64%) were polymorphic and 95 monomorphic (=36%; Table 2). A sample autoradiograph depicting the AFLPs generated from the four primer combinations is shown in Fig. 1.
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Associations among GS of all 129 P. palustris individuals revealed by cluster analysis (Fig. 2) that (1) most individuals of the single populations were arranged in population-specific clusters, and only the individuals of the populations of the floodplains in the Eider-Treene-Sorge area form a single central cluster. Individuals of these populations are not distributed at random within the central group, but subclusters often consist of individuals of more than one population. Moreover, (2) the order of exclusions of clusters occurred with decreasing geographical distance of the populations. The cluster of the Norwegian population is excluded first and showed only 59.8% GS to the populations in northwestern Germany. In the next steps clusters of the most isolated populations (numbers 1, 11, and 8) in the western, eastern, and northern parts of this region were separated from the other populations. (3) Values of GS between individuals within population-specific clusters and the cluster of the Eider-Treene-Sorge area vary enormously. In particular, populations 6 (94.8% GS), 7 (88.6% GS), and 9 (92.3% GS) stand out because of their high genetic similarities within populations.
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st) between most pairs of populations was high. Most values varied between 0.27 and 0.89, only genetic differentiation between pairs of populations 2, 3, and 5 was considerably lower (0.05–0.15; Table 3). The matrix of the pairwise genetic distances was not correlated with the corresponding matrix of pairwise geographic distances (Mantel test: r = 0.077; P > 0.409).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Furthermore, the technique needs only small amounts of tissue (Vos et al., 1995
) and is therefore very suitable for studies on rare species. However, there are some disadvantages of AFLP markers due to its dominant character. Common measures of genetic diversity in studies using allozymes or codominant markers (e.g., RFLP) are (1) heterozygosity and (2) the number of polymorphic loci, which are supposed to be lowered in small populations due to inbreeding and genetic drift, respectively (Oostermeijer, Berholz, and Poschlod, 1996
). Homozygotes are not directly distinguishable from heterozygotes by using dominant markers and thus heterozygosity has to be calculated indirectly by assuming that gene frequencies in the studied populations are in Hardy-Weinberg-Equilibrium (Lynch and Milligan, 1994
; Travis, Maschinski, and Keim, 1996
). However, this is often not the situation in nature, specifically in small plant populations, which are subjected to nonrandom mating processes (e.g., Raijmann et al., 1994
; Stewart and Excoffier, 1996
; Allphin, Windham, and Harper, 1998
). Hence, we preferred to use a phenetic approach (AMOVA) for testing genetic population structure and genetic variability within populations (Stewart and Excoffier, 1996
).
Population genetic structure
The degree of geographic isolation of the remaining 12 P. palustris populations in Schleswig-Holstein is very high. Median distance between pairs of populations was 40.75 km. In accordance with this spatial pattern, genetic differentiation among the studied populations was also pronounced. The amount of variance among populations (44%) was higher than in a comparable study on Gentianella germanica (37%; Fischer and Matthies, 1998b
), but considerably lower than in Astragalus cremnophylax var. cremnophylax (63%; Travis, Maschinski, and Keim, 1996
). Corresponding to the high degree of spatial isolation and genetic differentiation between pairs of populations, we also found an extremely low approximated average number of individuals exchanged between populations per generation (0.298).
Even though the amount of genetic variance among subpopulations (4.63%) of P. palustris was comparatively low, it was significantly higher than zero (P < 0.001) and similar to values reported by Travis, Maschinski, and Keim (1996
; 5%) and Fischer and Matthies (1998b; 13%). This indicates that P. palustris subpopulations are differentiated from each other and that gene flow between them is also restricted.
Population size, molecular variance, and reproductive components
In contrast to Van Treuren et al. (1991)
, Raijmann et al. (1994)
, Travis, Maschinski, and Keim (1996)
, and Fischer and Matthies (1998b)
we found no significant correlation between population size and the amount of genetic diversity within the investigated populations (but in accordance to Oostermeijer, van Eijck, and den Nijs; 1994
). This can be due to other factors including historical development of population sizes (Ouborg and van Treuren, 1995
) and the degree of isolation from other populations. Historical population processes can be illustrated by our results in combination with information gathered from both local botanists and old vegetation maps (Dahm, 1964
; K. Voß, personal communication, Kiel, Germany). Large molecular variation in all (small and large) populations in the Eider-Treene-Sorge area (Table 1) was found. As recently as in the 1960s P. palustris was a common plant in this region (Dahm, 1964
). Today, the area is drained and species-poor vegetation types occur. Currently, the remaining individual P. palustris populations seem to be isolated (mean distance: 6.7 km) with no actual gene flow occurring. The estimated high gene-flow values (Fig. 3) may reflect historical genetic exchange processes rather than the actual amount of gene flow among populations (see also, Templeton, 1998
). Molecular variance of the populations is still high, indicating that processes of genetic erosion (genetic drift and inbreeding) have not lowered genetic diversity during the last 15 to 20 generation cycles of P. palustris.
The smallest amount of molecular variance was found in population nine, despite its large population size (1900 flowering plants in 1997). This population is of recent origin, and its low amount of genetic diversity may be caused by a founder effect. Thus, it can be concluded that both actual population size and historical population processes have to be taken into account when looking for correlations between population size and genetic diversity.
Correlations between molecular variance and reproductive traits were found. The higher the degree of genetic variation, the higher the number of capsules and seeds per plant. A loss of genetic variation can have both consequences in an evolutionary and ecological context. As selection operates on variance among individuals within a population, low levels of genetic variability may lead to a reduced potential to adapt to changing environments (Barrett and Kohn, 1991
). Furthermore, a loss of genetic variability may lead to a higher susceptibility to pathogens (Schmid, 1994
) and in combination with small population size increase the level of inbreeding. Potentially, this is followed by inbreeding depression and results in reduced biomass, growth, number of flowers, seed number and mass, germination rate, or seedling establishment (Menges, 1991
; Oostermeijer, van Eijck, and den Nijs, 1994
; Ouborg and Van Treuren, 1995
; Fischer and Matthies, 1998b
). Whether the reduced fitness of P. palustris in populations with low levels of genetic variability is caused by inbreeding depression cannot concluded from our results. Inbreeding leads to reduced heterozygosity (Oostermeijer, Berholz, and Poschlod, 1996
), which cannot be calculated directly from AFLP variation. Nevertheless, results of a study on pollination biology of P. palustris (lower seed number per capsule in selfed than in outcrossed individuals; Karrenberg and Jensen, in press
) indicate some evidence for inbreeding depression. However, effects of inbreeding depression are most pronounced during late stages of the life cycle of the offspring (Dudash, 1990
; Oostermeijer, van Eijck, and den Nijs, 1994
; Husband and Schemske, 1996
; Fischer and Matthies, 1998b
), which we have not yet analyzed.
Disregarding the possibility of inbreeding depression, other factors including habitat quality, host availability and quality (Matthies, 1996
; Marvier and Smith, 1998), and pollen limitation may be responsible for the variation in reproductive components in the studied populations. Our results of multiple regression analysis indicate that beside molecular variance and population size other factors (vegetation composition, standing crop, C:N ratio, and the soil pH) do have an impact on the variation on reproductive components.
Karrenberg and Jensen (in press) found that the number of seeds per capsule of open-pollinated flowers were reduced in comparison to hand-pollinated ones in a small population with low density of P. palustris individuals (number 5). This indicates that pollen limitation can lead to a reduced number of seeds per capsule, thus explaining why none of the tested independent variables explained a significant amount of the variation in number of seeds per capsule. In fact, we found a reduced number of seeds per capsule in three small populations (numbers 1, 4, and 7; population <10 flowering individuals), in which vegetation is dominated by graminoids and dicots are underrepresented. In contrast, seed number per capsule was not reduced in population 12, which also consisted of <10 flowering individuals, but was characterized by a high number of dicots in vegetation and thus may be more attractive for pollinators of P. palustris. Even though we do not have any quantitative data on visitation rates of the study populations, we can postulate that species-rich vegetation attracts more pollinators and thus facilitates pollination and seed set in P. palustris (see also Oostermeijer et al., 1998
).
Implications for nature conservation
One primary objective of nature conservation is the maintenance of genetic diversity. The high amount of genetic differentiation among the studied populations of P. palustris indicates that a considerable amount of the overall genetic variation of the species in the study area would be lost if management of the remaining populations concentrated only on large populations and if the small populations were to decline further or even become extinct.
Although we found that molecular variance was positively correlated with some reproductive components, the importance of genetic processes in the population decline of P. palustris in many parts of Europe can be questioned. After a period of isolation of ~30 yr the small populations of the Eider-Treene-Sorge area still have a high molecular variance and are probably in an early phase of the genetic erosion process (see also Ouborg and van Treuren, 1995
). However, P. palustris is very vulnerable to rapid changes in site conditions (Schrautzer and Jensen, 1998
) due to its short life span and the absence of a long-term persistent seed bank (Thompson, Bakker, and Bekker, 1997
; Jensen, unpublished data). We can conclude that the fast decline of populations in Schleswig-Holstein during the last 50 yr is a consequence of human-related habitat destruction and land use changes rather than slowly operating genetic processes. However, genetic processes have to be considered when planning future conservation efforts for the species.
Conservation of P. palustris is a question of managing succession (see also Menges, 1990
; Silvertown, Franco, and Menges, 1996
). Management plans have to include either moderate grazing or mowing and haymaking, which reduce the amount of light competition and lead to suitable conditions for germination and establishment (see also Oostermeijer, van't Veer, and den Nijs, 1994
; Schrautzer, Asshoff, and Müller, 1996
). In addition, artificial gene flow (human-related pollen transport or seed dispersal) between populations may increase molecular variance and thus fitness of individuals. Even if P. palustris is not considered as endangered within its whole distribution area, it is an instructive example for the decline of many species in seminatural habitats in Europe. Managing populations of P. palustris through moderate grazing or cutting regimes also leads to the conservation of a large number of other typical fen-meadow species. Thus, the occurrence of a large P. palustris population can be considered as an indicator for fen-meadow habitat quality.
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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