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Reproductive Biology |
Estación Biológica de Doñana, Consejo Superior de Investigaciones Científicas, Apartado 1056, E-41080 Sevilla, Spain
Received for publication February 22, 2002. Accepted for publication April 26, 2002.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: gametophyte competition • geographical variation • Helleborus foetidus • microgametophyte populations • pollen limitation • pollen tube numbers • Ranunculaceae • winter flowering
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Schemske and Fenster, 1983
; Waser and Fugate, 1986
; Bertin, 1990
; Waser and Price, 1991
; Holm, 1994
; Plitmann and Levin, 1996
; Mitchell, 1997a
; Bosch and Waser, 1999
). The amount of pollen may also influence the quality of the progeny (Mitchell, 1997b
; Winsor, Peretz, and Stephenson, 2000
; and references therein), an effect that may be mediated by both pre-fertilization (competition among male gametophytes; Lee, 1984
; Snow, 1986
; Schlichting et al., 1987
; Winsor, Davis, and Stephenson, 1987
; Winsor, Peretz, and Stephenson, 2000
) and post-fertilization mechanisms (selective abortion exerted by the pistil-bearing individual; Willson and Burley, 1983
; Stephenson and Winsor, 1986
; Marshall and Ellstrand, 1988
; Niesenbaum and Casper, 1994
; Rigney, 1995
; Havens and Delph, 1996
; Niesenbaum, 1999
).
From a biological viewpoint, however, pollen load size is only a convenient proxy for the parameter most directly influencing seed production and progeny quality, namely the number of pollen tubes that penetrate the stigma and enter the transmitting tissue of the style (designated "microgametophytes" hereafter). It is the size of this microgametophyte population that will most directly set an upper limit to the number of fertilizable ovules and also, under some circumstances (synchrony in pollen arrival and germination; Snow, 1986
; Thomson, 1989
), will determine the possibilities of gametophyte competition. Pollen grain and pollen tube numbers will generally be correlated. This justifies, for example, inferences on gametophytic competition in natural populations based on observations of the number of deposited pollen grains alone (e.g., Spira et al., 1992
). Nevertheless, the relationship between the number of conspecific, compatible pollen grains on the stigma and the number of pollen tubes in the style is generally a very loose one (Snow, 1986
; Herrera, 1997
) due, among other factors, to environmental conditions, pollen–pistil interactions, pollen tube attrition effects, and to the marked stochasticity and strong context dependence of the germinating behavior of pollen grains (Cruzan, 1986
, 1989
; Thomson, 1989
; Stephenson et al., 1992
; Holm, 1994
). Therefore, although both seed set and levels of gametophyte competition levels will ultimately be related to pollen load size, the number of pollen tubes that penetrate the stigma is a more proximate variable in the reproductive success of flowers (Plitmann and Levin, 1996
; Quesada, Fuchs, and Lobo, 2001
).
In comparison with the multitude of manipulative experiments examining the effects of variations in the size of stigmatic pollen loads, only a handful of investigations have so far provided information on patterns and correlates of variation in microgametophyte populations for naturally pollinated plants in the wild (Levin, 1990
; Honig, Linder, and Bond, 1992
; Niesenbaum, 1994
; Plitmann and Levin, 1996
; Herrera, 1997
; Quesada, Fuchs, and Lobo, 2001
). Direct measurements of the size of natural microgametophyte populations and its relationship to ovule number are essential to assess where individual plants, populations, or species stand along the conceptual gradient running from extreme pollination deficit (microgametophyte/ovules ratio << 1) to potentially intense competition between gametophytes (microgametophyte/ovules ratio >> 1). Furthermore, knowledge of natural patterns of spatial and temporal variation in microgametophyte numbers is a prerequisite for evaluating the prevalence and biological significance in naturally pollinated plant populations of the mechanisms and effects elucidated by experimental manipulations of pollen loads, including gametophyte competition. Documenting temporal or geographical variation in microgametophyte populations for a given species, for instance, may provide insights on possible spatiotemporal mosaics in the degree of pollen limitation and intensity (or likelihood) of gametophyte competition and associated selective processes. Furthermore, information on the hierarchical apportionment of variance in per-pistil microgametophyte populations among and within individual plants is essential to evaluate the actual opportunities of selection on any influential plant trait. Of particular relevance from an evolutionary perspective are the relative magnitudes of within- vs. between-individual variance in number of microgametophytes per pistil, as the possibilities of natural selection to act on whichever trait is influential on pollen tube numbers would be directly related to the fraction of total variance accounted for by between-individual differences. In one of the few studies examining patterns of variation of microgametophyte populations among individual plants in the wild, Niesenbaum (1994)
found that, in Lindera benzoin, as little as 7% of total population-wide variance in pollen tube numbers was accounted for by differences among individual plants. This intriguing finding raises doubts on the opportunities for evolutionary change of any trait correlated with pollen tube numbers in that species and calls for confirmation in other plants.
The present study was undertaken to assess patterns of spatial and temporal variation in microgametophyte numbers in the style of naturally pollinated flowers of the winter-flowering, perennial herb Helleborus foetidus (Ranunculaceae). The main goals of this paper are (1) to provide a comprehensive picture of the magnitude and scales of spatial and temporal variation in microgametophyte populations for this species and identify the main sources of variation in microgametophyte numbers and (2) to evaluate the relative frequency of occurrence of situations of pollen deficit and gametophyte competition, information that is still hardly available for any other plant species under natural conditions. A series of hierarchically nested spatial scales spanning several orders of magnitude is considered, ranging from hundreds of kilometers (between separate regions within the Iberian Peninsula) through a few kilometers (between populations in the same region) to individuals within populations (dozens to hundred meters), flowers within plants (centimeters) and, finally, distinct styles within individual flowers (millimeters; H. foetidus flowers are apocarpous, each carpel bearing a distinct style). To investigate the temporal component of variation, all the populations in the most thoroughly studied region were sampled during two consecutive years.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). In the Iberian Peninsula, the species typically grows in the understory of deciduous and mixed forests. Flowers are hermaphroditic, protogynous, last for 2–3 wk, and the stigmas are receptive for 6–12 d (Vesprini, Nepi, and Pacini, 1999
; Vesprini and Pacini, 2000
; Herrera et al., 2001
). Flowers are apocarpous, with the number of carpels ranging between one and six (mostly 2–3) per flower. Pollination is performed by bumble bees and, to a lesser extent, anthophorid bees (Herrera et al., 2001
). Although the species is self-compatible and a small proportion of flowers set fruit in the absence of pollinators via spontaneous self-pollination, insect pollination is required for abundant seed production (Vesprini and Pacini, 2000
; Herrera et al., 2001
).
This study was conducted in 2000 and 2001 at three widely separated regions in the Iberian Peninsula (Fig. 1). The two most distant regions (Caurel and Cazorla) were 
675 km apart, while the two nearest ones (Mágina and Cazorla) were only 85 km away. The flowering period was roughly similar at the three study regions, extending mainly from January to March (in some Cazorla localities at low elevations, flowering started in December). A variable number of H. foetidus populations were selected for study in each region: four populations in Caurel (elevation range = 950–1350 m), three populations in Mágina (elevation range = 1640–1670 m), and 22 populations (elevation range = 760–1800 m) in Cazorla. At the northwestern region (Caurel), the H. foetidus populations chosen for study grew in pine (Pinus sylvestris) plantations, open successional scrublands, and Brachypodium rupestre meadows. At the two southern regions (Cazorla and Mágina) the selected populations were in pine- (Pinus nigra) or oak- (Quercus rotundifolia) dominated forests. Cazorla populations encompassed and were more or less evenly distributed over the whole elevational range of H. foetidus in the region. Caurel and Mágina populations were studied only in 2000. The 22 Cazorla populations were studied in 2000 and 2001, and the extensive data from this region provided the core information for this study. Information on the pollination ecology of H. foetidus at the three study regions is given by Herrera et al. (2001)
.
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Martin's (1959)
epifluorescence method was used to reveal pollen tubes in the collected styles. Styles were kept at 65°C for 20 min in 5 mol/L NaOH for softening, rinsed in distilled water, and stained for 20 min at 65°C in decolorized aniline blue. The number of pollen tubes that had penetrated the stigma and were in the stylar canal was then counted under a fluorescence microscope. Tubes were counted on or near the midpoint between the stigma and the bifurcation of the transmitting tissue that leads to the two marginal placentae (see Weberling [1989
, pp. 139–140] for drawings and descriptions of gynoecium structure in H. foetidus).
Assessing the implications of variation in the number of pollen tubes requires information on the size of the ovule complement of each carpel and on the effect of pollen tube numbers on seed and fruit set. Detailed information on the number of ovules per carpel was available for several populations in each of the three regions, obtained in the course of other investigations on H. foetidus. The range of variation of the number of ovules per carpel was very narrow both among regions (mean ± 1 SD = 11.1 ± 1.2 [N = 1313 carpels], 11.3 ± 1.1 [N = 438], and 10.8 ± 1.4 [N = 1025] ovules/carpel in Caurel, Cazorla, and Mágina populations, respectively) and within regions (interquartile range = 10–12 ovules/carpel at each of the three regions). An average figure of 11 ovules per carpel will thus be used throughout this paper. The relationship between number of pollen tubes and seed set was investigated in three Cazorla populations. In May 2000, samples of developing fruits (= the set of follicles originating from the same gynoecium) were collected in three populations. At each site, between six and ten fruits were collected from each of ten plants and preserved in FAA. For every follicle, the number of pollen tubes in the persistent, dry style was counted using the same epifluorescence technique described above, along with the number of enclosed undeveloped ovules and nearly ripe seeds.
Statistical analyses
All statistical analyses were carried out using the SAS statistical package. Variance components of microgametophyte numbers at the various spatial scales considered were estimated using the restricted maximum likelihood method, as implemented in the SAS procedure MIXED (SAS, 1996
). This procedure also provides approximate standard errors of variance component estimates and two-tailed tests of significance based on standard normal deviates.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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0.73–0.86 in the number of seeds produced (Table 1).
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2 = 110.9, df = 2, P < 0.0001; Kruskal-Wallis analysis of variance, data from all populations in each region pooled). In absolute terms, however, the magnitude of differences between regional means was quite small (Table 2), with the largest and smallest means (Cazorla and Mágina, respectively) differing by only two pollen tubes. Within-regional variability, as assessed by the interquartile ranges and the proportion of styles with a number of pollen tubes smaller than the number of ovules, were also roughly similar in the three regions in 2000.
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11 (= the mean number of ovules per carpel) increased from 48.0% in 2000 to 60.4% in 2001.
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2 = 180.4, df = 21, P < 0.0001, for 2000; 2 = 527.0, df = 21, P < 0.0001 for 2001; Kruskal-Wallis analysis of variance, data from all individuals in each population pooled). Population means ranged between 6.9 and 13.2 tubes in 2000, and between 1.9 and 15.5 tubes in 2001 (Fig. 3), and there was a statistically significant increase in the variability of population means from 2000 to 2001 (P < 0.0001, Levene's test).
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In the 22 Cazorla populations, the variance partition profile differed between years in significant ways. The component due to variation among populations increased considerably from 2000 to 2001 in both absolute (1.2–16.7) and relative (from 13.6–39.5%) terms, which reflects the marked annual differences in the degree of variability of population means noted earlier (Fig. 3). The within-plant variance component more than doubled from 2000 to 2001 in absolute terms (7.1–18.7) and remained roughly constant in relative terms (51.9–44.4%).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Despite this, however, fruit set of naturally pollinated plants reaches relatively high levels (60–80%), quite similar to the average value reported by Sutherland and Delph (1984)
for a large sample of hermaphroditic self-compatible species (72.5%).
The high fruit set of H. foetidus has been interpreted in relation to the long duration of their flowers, since even with flower visitation probabilities as low as those observed, the probability of individual flowers receiving at least one effective pollinator visit during its 6–15 d long female receptive stage is far from negligible (Herrera et al., 2001
). Supplemental hand pollinations of open-pollinated flowers conducted in 1998 and 1999 consistently resulted in modest increases of fruit set at the three study regions, although results reached statistical significance only in Cazorla (Herrera et al., 2001: Table 2). Two results of the present study corroborate these earlier findings and suggest that H. foetidus experiences a weak, chronic pollination deficit at the three study regions. First, the number of pollen tubes was inferior to the number of ovules per carpel in a large proportion of styles. And second, the number of seeds produced was closely and lineary related to the number of microgametophytes. Annual fluctuation in the frequency of styles with <11 pollen tubes in the Cazorla region (52 and 40% in 2000 and 2001, respectively) suggests, however, that the magnitude of the pollination deficit may vary between years, most likely in connection with variation in weather conditions, as discussed below.
A similar combination of high natural fruit set, weak but consistent pollen limitation, and slight but significant annual fluctuation in the magnitude of the limitation, has been also found in Narcisus longispathus (Amaryllidaceae), another early-blooming, insect-pollinated perennial herb from the Cazorla region characterized also by the extended longevity of its flowers (Herrera, 1995
). Combinations of relatively high fruit set levels, moderate to weak or no pollen limitation, and long floral durations have been likewise reported for other winter- and early-spring flowering herbs of disparate taxonomic affiliation (Schemske, 1977
, 1978
; Schemske et al., 1978
; Motten et al., 1981
; Motten, 1986
; Murphy and Vasseur, 1995
; Vesprini, Nepi, and Pacini, 1999
; Ishii and Sakai, 2000
; Vesprini and Pacini, 2000
). Contrary to intuition, therefore, winter and early spring flowering possibly is not an "inferior choice" for insect-pollinated plants of the forest understory insofar as floral durations are long enough to cope with infrequent and irregular pollinator visitation.
Spatiotemporal mosaic of microgametophyte populations
Pollinator censuses conducted in 1998 and 1999 in Caurel, Mágina, and Cazorla revealed extensive regional similarity in pollinator composition, the same species of bumble bees (Bombus terrestris and B. pratorum) being the main pollinators at all sites (Herrera et al., 2001
). Regions did differ significantly, however, in pollinator service, as measured by the probability per time unit of plants and individual flowers receiving a pollinator visit. The relatively minor differences between the three regions in mean microgametophyte numbers found in this study are consistent with these earlier results, as both pollinator service and mean number of microgametophytes per style were distinctly lower in Mágina than in either Caurel or Cazorla.
Elevational differences in mean size of microgametophyte populations in Cazorla may be interpreted in relation to differential pollinator activity caused by abiotic environmental factors. Bumble bees, the main pollinators of H. foetidus, are endotherms that can forage at relatively low ambient temperatures, but they still require a minimum threshold temperature for foraging (Heinrich, 1979
), and their activity is quite susceptible to inclement weather, particularly rainy periods (Teräs, 1976
; Lundberg, 1980
). The range of elevations sampled in Cazorla was sufficiently broad for average weather conditions to deteriorate significantly with increasing elevation, which would lead to a reduction in bumble bee activity and a decline in microgametophyte numbers. This prediction was confirmed for 2001 but not for 2000, a discrepancy that may also be explained in terms of weather variations. The year 2001 was characterized by the decade's most rainy (at low and middle elevations) and snowy (at high elevations) January–February period, whereas the year 2000, in contrast, was characterized by the driest January–February period in the decade. With clear skies and sunny weather prevailing almost uninterruptedly during the whole flowering period of H. foetidus in 2000 at all populations, the absence of any elevational gradient in microgametophyte numbers that year was not surprising.
In all data sets analyzed, most variance in microgametophyte numbers occurred within local populations. Within-population variance, in turn, was apportioned quite unequally among and within individual plants. Although individual plants of the same population did differ significantly in mean microgametophyte numbers, the magnitude of these differences was relatively minor in comparison to the extent of variation occurring within plants, which was the most important source of variance. In those Cazorla and Caurel populations where within-plant variance could be partitioned into its among- and within-flower (i.e., among styles) components, variation among styles of the same flower turned out to be the most important source of within-plant variation. These results indicate that there was considerably more variability in microgametophyte numbers over the few centimeters separating the flowers of the same H. foetidus plant and the few millimeters separating the styles of the same flower than among the means of populations and regions dozens or hundreds of kilometers away.
There are few comparable investigations providing data on microgametophyte variation in populations of naturally pollinated plants, but these suggest that patterns similar or even more extreme than those found in this study may be widespread. Within-plant variation was also the predominant source of variation in microgametophyte numbers in Lindera benzoin (85.4% of total regional variance; Niesenbaum, 1994
) and Lavandula latifolia (78.0% of total regional variance; 15 populations sampled in the Cazorla region; C. M. Herrera, personal observations). The studies of Levin (1990)
on Phlox drummondii and Honig, Linder, and Bond (1992)
on the wind-pollinated Staberoha banksii showed as much or more variation among pistils of single plants as among plants, although no formal variance partitioning analyses were conducted in these investigations. Taken together, results from these investigations and those reported here for H. foetidus suggest that, in naturally pollinated populations, variation in microgametophyte numbers is a phenomenon that predominantly takes place at the within-plant scale and is thus presumably due to stochastic variations among flowers and styles in the size and composition of pollen loads. On the practical side, marked fine-graininess in the spatial structure of variance in microgametophyte populations implies that an adequate understanding of patterns of natural variation requires carefully designed sampling schemes that allow for precise partitioning of variability at the scale of individual plants and below. This finding also has implications in relation to the frequency of occurrence and ecological and evolutionary significance of gametophyte competition in natural populations, as discussed in the next section.
Fine-grained mosaics and microgametophyte competition
For all populations and years combined, styles of H. foetidus contained an average of 0.95 microgametophytes per ovule (M/O ratio hereafter). This figure is inferior to all the M/O values reported by the few comparable investigations on naturally pollinated plants: 1.5 in Lindera benzoin (Niesenbaum, 1994
), 2 in Epilobium canum (Snow, 1986
), 3 in Polemonium viscosum (Galen and Newport, 1988
), 4.7 and 3.7 in Phlox drummondii (Levin, 1990
; Plitmann and Levin, 1996
), and 5.2 in Staberoha banksii (Honig, Linder, and Bond, 1992
). Pooled average values of this kind have been used sometimes as rough indications of the possibilities of gametophyte competition in natural populations, and the M/O ratio <1 found here for H. foetidus would tend to rule out the possibility of gametophyte competition in this species. Nevertheless, extensive spatiotemporal variability in microgametophyte numbers revealed by this and other studies (Levin, 1990
; Plitmann and Levin, 1996
) suggest that average M/O figures may convey little information on the possible occurrence of gametophyte competition and should be interpreted with caution. At least in H. foetidus, there is evidence that conditions conducive to gametophyte competition were far from unusual despite a low M/O ratio.
A close relationship was found in this study between microgametophyte numbers and number of seeds produced, and consideration of the slopes of the seeds/microgametophytes regressions leads to a projected requirement of 14 pollen tubes for full seed set of individual carpels. For all populations and years combined, 21% of styles had >14 microgametophytes, hence an excess over the maximum number required for full seed set. For gametophyte competition to occur, a fairly synchronous arrival and germination of pollen grains is also necessary (Snow, 1986
; Bertin, 1990
; Spira et al., 1992
). Both of these conditions most likely apply generally in H. foetidus, because (1) average pollinator visitation rates are so low in my study populations (Herrera et al., 2001
) that the time between consecutive pollinator visits to the same flower is expected to range from many hours to several days, hence pollen grains will generally arrive as temporally distinct, widely separated cohorts; (2) only 12 h elapse between pollen deposition and pollen tubes reaching the ovules (Vesprini and Pacini, 2000
); and (3) stigma receptivity generally ceases 1 d after pollination (C. M. Herrera, personal observation). It may then be concluded that, despite an average M/O < 1, broad variability around this mean generates conditions conducive to gametophyte competition in about one-fifth of H. foetidus styles.
Spatial structures of variance in microgametophyte numbers similar to that documented here for H. foetidus have also been reported in a few previous investigations (Levin, 1990
; Niesenbaum, 1994
; Plitmann and Levin, 1996
). The present study also found a significant temporal component superimposed on a fine-grained spatial mosaic, such that differences between populations and the spatial apportionment of regional variance fluctuated between years. On one side, these findings support the conclusion, already anticipated by Snow (1986)
for Epilobium canum, that the frequency of occurrence of gametophyte competition in nature will ordinarily vary in complex ways between years, regions, populations, individual plants, flowers on the same plant, and even carpels within the same gynoecium. Simple assessments based on short-term studies of one or a few populations should therefore be abandoned in favor of more extensive sampling of natural ranges of variation. In addition to this practical corollary, the particular structure of spatial variability in microgametophyte numbers found here for H. foetidus, whereby within-plant variation is the predominant component of variance in local populations (see also Niesenbaum, 1994
), suggests few opportunities for selective scenarios derived from gametophyte competition and nonrandom fertilization in this species. Even if local pollen donors differed in pollen-tube growth rate and seed-siring ability and their differences remained consistent across maternal parents (Snow and Spira, 1996
; Marshall, 1998
), marked stochasticity among and within maternal parents in the characteristics of the competitive environments found within styles would lead to: (1) a considerable reduction in the opportunities of selection among pollen donors based on pollen performance and (2) the "protection" from the eroding action of natural selection of additive genetic variation underlying differences in male competitive ability. This would provide a parsimonious explanation for the apparent paradox, noted by some authors (Snow and Spira, 1996
; Delph and Havens, 1998
), of long-term persistence in natural populations of broad individual variation in pollen competitive ability despite consistent individual differences in male fitness. This example, along with other findings of this study, provide support to the claims of authors that have so far emphasized (although apparently with little success) that empirical field data are an indispensable complement to manipulative experiments if we are to understand the ecological and evolutionary roles of gametophyte competition and, more generally, sexual selection in natural plant populations (Snow, 1986
; Levin, 1990
; Niesenbaum, 1994
; Plitmann and Levin, 1996
).
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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