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Section of Plant Biology, University of California, Davis, California 95616; and United States Department of Agriculture - Agricultural Research Service, 2021 South Peach Avenue, Fresno, California 93727
Received for publication May 28, 1997. Accepted for publication June 28, 1998.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLiterature Cited |
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Key Words: Asteraceae • clonal perennials • chaparral • gene flow • genetic diversity • plant congeners • population membership • Wyethia
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLiterature Cited |
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Various studies (Hamrick, Linhart, and Mitton, 1979
; Loveless and Hamrick, 1984
; Hamrick and Godt, 1990
) have used the isozyme data from hundreds of investigations to examine the relationship between genetic variation and various aspects of life history. Geographic range was positively correlated with genetic diversity at the species level, but had no bearing on the distribution of genetic variation among populations. The factors most highly associated with the development of genetic structure were those related to the vagility of pollen and seeds. Reduced pollen exchange due to inbreeding, localized pollination, and/or short life span, and dispersal of seed by gravity were usually associated with pronounced genetic structure.
These general associations have been examined in specific studies that control for disparate phylogenetic relationships by comparing congeners and that control for differing selection regimes by examining the same isozymes (Karron, 1987
and references therein; Karron et al., 1988
; Pleasants and Wendel, 1989
; Sherman-Broyles et al., 1992
; Linhart and Premoli, 1993
; Baskauf, McCauley, and Eickmeier, 1994
; Edwards and Wyatt, 1994
; Purdy, Bayer, and MacDonald 1994
; Purdy and Bayer, 1995
, 1996; Young and Brown, 1996
). No central idea has emerged from these studies; differences between congeners in the amount of genetic variation and degree of genetic structure arise from a variety of factors. To understand genetic differences between endemic species and their more widespread congeners, it is necessary to sample populations of the two species over the same range, to evaluate the potential for directional selection, to establish the systematic relationship between the species, to determine population size and persistence, and to identify clonal expanse.
Traditionally, researchers have employed allozyme markers to understand some of these issues (references in Hamrick and Godt, 1990
). With the advent of DNA-based techniques, it is timely to compare and combine traditional allozyme analysis with the new types of markers. Random amplified polymorphic DNA (RAPD)-based assays can yield an abundance of markers. Particularly when allozyme polymorphisms are few, RAPD markers allow more precise identification of clones (e.g., Stewart and Porter, 1995
), allow determination of the genetic relationships among individuals (van Heusden and Bachmann, 1992
), and provide an estimate of population structure (Apostol et al., 1996
; Ayres and Ryan, 1997
; Gabrielsen et al., 1997
; Martin, Gonzalez-Benito, and Iriondo, 1997
).
Phenetic analyses of individuals using binary data from allozymes and RAPDs can potentially identify related groups of individuals and show the relationship between genetic and geographical distance. Population analyses using allozyme and RAPD frequency data yield quantitative estimates of population divergence and effective migration rates (Swofford and Selander, 1989
; Black, 1995
; Apostol et al., 1996
). Population genetic analysis using allozymes permits estimation of inbreeding and departure from Hardy-Weinberg equilibrium within populations and assessments of amounts of genetic variation within populations and species. An added advantage of isozyme-based genetic analyses is that the results can be placed into larger context by comparison with similar data from other studies.
The goals of this study were to analyze and compare the phenetic and genetic results obtained from allozymes with those obtained from RAPD markers in describing the clonal and population structure of two species of Wyethia and to evaluate these comparisons with reference to differences between the species in distribution, abundance, and life history. This is the first study to compare a congeneric pair of species by contrasting and combining isozyme and RAPD methodologies. Specifically, we asked, (1) What is the population clonal diversity of each species of Wyethia and how effective is each marker type is identifying each genet? (2) How large are populations of each species? (3) For each species, how do the genetic and spatial population structures revealed by each marker type vary? (4) Can distribution, abundance, and/or life history differences between the congeners explain differences in genetic and spatial population structure?
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLiterature Cited |
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). Wyethia reticulata is an edaphic endemic, restricted to the 14-km long gabbro soil chaparral community of western El Dorado County. The gabbro soils in this area are derived from a gabbrodiorite rock intrusion that occurred 175 million years ago (Hunter and Horenstein, 1991
), creating an edaphic island; surrounding soils are derived from metasedimentary rock at lower elevations and from granitic rocks at upper elevations (Rogers, 1974
). Dominated by the shrubs Arctostaphylos viscida and Adenostoma fasciculatum, the area is known for the richness of the flora (Stebbins, 1993
). Seven species, in addition to W. reticulata, are mostly restricted to the gabbro plant communities. Five are listed as threatened or endangered under the federal Endangered Species Act; all are considered rare or endangered in California by the California Native Plant Society (Smith and Berg, 1988
). The principal threats to the rare species are the loss of habitat due to residential development and the suppression of fire (EIP Associates, 1991
). Wyethia bolanderi occurs in a narrow band that extends 275 km along the foothills. (Fig. 1) and overlaps the distribution of W. reticulata, with some sites containing both species (Weber, 1946
). Both species have underground stems that survive periodic fires in the chaparral.
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). In the spring, leafy upright stems arise from the rhizome. In open sites these annual stems are up to 1-m tall and can be topped by one to a few inflorescences (Weber, 1946;
Ayres, 1997
). Plants flower late in the spring, are pollinated chiefly by native bees, and are self-incompatible (Ayres, 1997
). The fruits, epappus achenes, are dispersed by gravity.
Wyethia bolanderi is a clonal perennial with annual stems that arise from a taprooted caudex (Hickman, 1993
). The leafy stems are <3 dm in height with a single terminal inflorescence (Weber, 1946
). Plants flower early in the spring and appear to be insect pollinated (Ayres, 1997
). The epappus achenes are likely dispersed by gravity.
Sampling methods
To permit comparison of genetic diversity between Wyethia reticulata and W. bolanderi over the same spatial scale, one population of each species was sampled from four sites in El Dorado County, California, where both species occur. The sites, chosen to span the entire 14 km range of W. reticulata, were Cameron Park (CP) in the south, Rescue (R) in the center, and two populations separated by 
0.5 km near Salmon Falls Road (SFA and SFB), located at the northern limit of its range (Fig. 1). El Dorado County is near the geographical center of the range of W. bolanderi. An additional population of W. bolanderi in Yuba County, located 73 km north of the Salmon Falls sites, was also surveyed to examine genetic diversity over a wider scale.
The sampling goal was to survey 10–20 genetic individuals from each population using molecular markers. However, extensive clonal growth (discovered by genetic analysis) and small population numbers in W. reticulata limited the sample sizes for three of the four populations. In addition, the Salmon Falls populations of W. bolanderi contained only three individuals each. Fifty-two individuals of W. reticulata were analyzed with both allozyme and RAPD markers; 55 individuals of W. bolanderi were analyzed for allozymes, 52 individuals were also analyzed using RAPD markers (three individuals had incomplete RAPD data). The sampling methods used for W. reticulata were as described previously (Ayres and Ryan, 1997
). Samples from populations of W. bolanderi were taken at 1–2-m intervals along a transect through the largest dimension of the patch. Apical buds were collected for DNA extraction in February and March, while fully expanded leaves were collected later in the spring for isozyme analysis.
Allozyme analysis
To compare genetic diversity between the two species over the same loci, three clearly resolved and interpretable enzyme systems were used: alcohol dehydrogenase (ADH - EC 1.1.1.1), aspartate amino transferase (AAT - EC 2.6.1.1), and phosphoglucomutase (PGM - EC 2.7.5.1). Two additional isozymes of phosphogluconate dehydrogenase (PGD - EC 1.1.1.44) were resolvable for W. reticulata; these were used for calculating total genetic diversity of polymorphic loci (HTP) and in the single-species phenetic analysis. Protein extraction, conditions of electrophoresis on acrylamide gels, and interpretation of gels have been described previously (Ayres and Ryan, 1997
). Extracts of both species were run on the same gels to facilitate comparison. Allozymes were evaluated as presence/absence data, yielding 22 monomorphic and polymorphic bands for each species; genetic analyses were conducted as well, based on the known structure of the isozymes (see below). Allozymes that were resolved only for W. reticulata (PGM-1, PGD-1, and PGD-2) were included in the single-species phenetic analyses of the binary data. Both species had 20 polymorphic bands that were used as binary data for their respective phenetic analyses.
Genetic analysis of allozyme data
Allozyme data were analyzed using BIOSYS-1 version 1.7 (Swofford and Selander, 1989
) as modified by Black and Krafsur (1985)
using the calculations of Weir and Cockerham (1984)
for small population size and number. BIOSYS-1 was used to calculate gene frequencies, the proportion of polymorphic loci (P), the mean number of alleles per locus (A), the observed and expected heterozygosity (Ho and He, respectively), departure from Hardy-Weinberg equilibrium for each population/locus combination, and F statistics for each species. Total variation (HT) was calculated using both monomorphic and polymorphic loci, while total variation of polymorphic loci (HTP) was calculated using only polymorphic loci. The number of migrants per generation (Nm) was calculated from FST according to Hartl and Clark (1989)
. A cluster diagram was generated from Nei's (1978)
genetic distance using the UPGMA (unweighted pair group method arithmetic average) of NTSYS-pc version 1.80 (Rohlf, 1993
).
RAPD analysis
DNA extraction, primer screening, amplification, and analysis of amplified products were similar for both species and have been described for W. reticulata (Ayres and Ryan, 1997
). DNA from W. bolanderi was amplified using an Ericomp thermocycler (San Diego, California) set for a cycling regime similar to that of the Perkin-Elmer thermocycler (Perkin-Elmer, Norwalk, Connecticut) used for the earlier work with W. reticulata.
Sixty primers (Operon kits C, G, and R; Operon Technologies, Alameda, California) were used to screen DNA from W. reticulata, as described previously (Ayres and Ryan, 1997);
nine primers produced 21 clear, reproducible polymorphic bands (C6, G2, G9, G10, G11, G12, G13, G16, R11). Eighty primers were screened for W. bolanderi DNA (Operon primer kits C, F, G, and R); eight primers produced 20 clear polymorphic bands (C16, F12, G7, G8, R4, R5, R6, R11). Each DNA sample was amplified at least twice with all primers. Polymorphic bands were scored as present or absent.
Genetic analysis of RAPD data
Estimation of the reduction in heterozygosity due to drift in a subdivided population (FST) (Hartl and Clark, 1989
) and effective migration rates (Nm) from RAPD data requires two assumptions. First, null bands, which are used to estimate the frequency of the recessive allele, are assumed to be homologous in all individuals. Second, it is assumed that populations are in Hardy-Weinberg equilibrium, so that expected heterozygosities can be calculated. Investigations of the first assumption have not yet been reported in the literature; the second assumption can be evaluated if corresponding allozyme data are available. FST and Nm were estimated by three different methods using the RAPDFST program of Black (1995)
. Two methods were derived from the equations of Wright (1931)
and Weir and Cockerham (1984)
and the third employed the method described in Lynch and Milligan (1994)
. For the first two methods the data sets were unaltered. However, the Lynch and Milligan method requires that null bands with a frequency of <3/N (where N is the sample size of a population) be removed from the data set. RAPD markers are unable to distinguish heterozygotes, therefore it is not possible to estimate FIS from this data.
Binary data analyses
The presence/absence data from the RAPD and allozyme samples were used to identify clones and examine patterns of genetic similarity among individuals. Phenetic analysis of banding patterns requires band homology between samples and is not dependent on Hardy-Weinberg equilibrium. A genet-by-genet similarity matrix was calculated from the binary data using the simple matching coefficient, which gives equal weight to shared band presence and absence. The similarity matrices based on allozymes, RAPDs, and the combination of both marker types, were analyzed using UPGMA cluster analysis. All analyses and the significance of matrix correlations, evaluated using the Mantel test (Mantel, 1967
), were performed by the NTSYS computer program (Rohlf, 1993
). Samples with incomplete data were deleted from phenetic analyses and comparisons.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLiterature Cited |
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, table 2). Wyethia bolanderi exhibited seven alleles not found in W. reticulata (Aat-1 c, Aat-3 c, Aat-4 c and d, Adh-2 b, Adh-3 b and c) while W. reticulata possessed one unique allele (Pgm-2 c). The UPGMA dendrogram of Nei's (1978)
genetic distance between populations of the two species (Fig. 2) separates each species at a genetic distance of 0.51. Populations of W. bolanderi separate at a genetic distance of 0.16; populations of W. reticulata separate at 0.18.
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The more widespread congener, W. bolanderi, had a higher proportion of polymorphic loci, a greater number of alleles per locus, a higher expected heterozygosity, whether single populations or their averages were compared, and on the scale of species (Table 4), and had greater total genetic diversity, HT, than W. reticulata (Table 5). Despite this greater genetic variation, W. bolanderi had, on average, the same observed heterozygosity as its narrowly distributed congener.
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FST, reduction of heterozygosity due to random genetic drift within populations, has been estimated according to various methods using both allozymes and RAPDs (Table 5). Estimates of FST based on RAPD loci were generally higher than estimates based on allozymes. Estimates using the Lynch and Milligan (1994)
method for RAPDs were up to two times higher than other estimates based on RAPDs and two to three times higher than some estimates based on allozymes.
FST is inversely related to Nm, the effective migration rate; a high FST means that populations are genetically differentiated, the result of a low migration of genotypes. When both species are considered at the same spatial scale (i.e., in El Dorado County), the restricted congener, W. reticulata, by any measure for both allozyme and RAPD data, showed greater population genetic differentiation than W. bolanderi. Averaging the RAPD- and allozyme-based estimates employing the Weir and Cockerham (1984)
method resulted in FST values of 0.25 and 0.10, respectively, for each species among El Dorado County populations. The corresponding estimate of migration rate for W. reticulata was 0.8 individuals per generation, compared to a migration rate of 2.2 for W. bolanderi. Increasing the geographic range of sampled W. bolanderi populations to include Yuba County resulted in a doubling of the average FST to 0.22 and a reduction in the estimated effective migration rate to less than one individual per generation.
Phenetic analyses of binary data
The two species were analyzed together phenetically, using the eight isozyme loci common to both species, by UPGMA cluster analysis (Fig. 3). As in the genetic analysis of distance between populations (Fig. 2), the phenetic analysis of distance between individuals also results in the clear separation of species.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLiterature Cited |
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; Purdy and Bayer, 1996; Young and Brown, 1996
). Explanations of this phenomenon have been put forth at various levels, although the history of each species pair is unique and represents the interaction of effects at several levels. Genetic endowment at speciation (Crawford, Ornduff, and Vasey, 1985
; Pleasants and Wendel, 1989
; Linhart and Premoli, 1993
; Purdy, Bayer, and MacDonald, 1994
; Purdy and Bayer, 1996) will interact with "niche width" (Van Valen, 1965
) or other selective pressures (Purdy, Bayer, and MacDonald, 1994
; Purdy and Bayer, 1996) to produce different levels of genetic diversity between members of the pair. Genetic drift and bottleneck and founder effects, particularly in unstable or changing environments, may reduce genetic variation (Sherman-Broyles et al., 1992
; Baskauf, McCauley, and Eickmeier, 1994
; Purdy and Bayer, 1995
).
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Combined markers of both types allowed the identification of clones and thus provided reliable estimates of population sizes. Populations of W. reticulata were small, 10–50 individuals, dominated by a few large clones. In contrast, populations of W. bolanderi (except at the Salmon Falls sites) were larger, with 100–300 individuals and small, uniformly sized clones. The loss of diversity through genetic drift is consonant with these characteristics of the populations of W. reticulata. The level of genetic variation is high in consideration of the size of the population and suggests that clonal longevity contributes to the maintenance of diversity.
The sizes of some W. reticulata individuals, coupled with the slow lateral growth observed in the field (Ayres, 1997
), suggest that some individuals are extremely old. Genetic drift will effectively occur only upon the death of the parental generation, hence the maintenance of relatively high levels of genetic variation may be expected in populations of long-lived individuals. Similar conclusions have been drawn concerning the population structure of Erythronium propullans, a narrow, clonal endemic (Pleasants and Wendel, 1989
).
Gene flow
Genetic analyses using both RAPDs and isozymes provide alternative estimates of gene flow rates between populations. Estimates derived independently from isozymes and RAPDs were in good agreement with each other (except for the Lynch and Milligan [1994]
method) for W. reticulata. RAPD-based migration estimates were lower than isozyme-based estimates for W. bolanderi; however, since an FIS of 0.22 implies that this species is inbred, violating the assumption of Hardy-Weinberg equilibrium, RAPD-based estimates of gene flow may be invalid. Differentiation among populations of W. bolanderi, as measured by FST , was doubled when the Yuba County population was included. While this may have occurred in our study if the Yuba population was atypical, increasing the sampled area also resulted in higher estimates of FST in Achillea millefolium ssp. lanulosa (Purdy and Bayer, 1996), Aletes humilis (Linhart and Premoli, 1993
), Daviesia mimosoides (Young and Brown, 1996
), Rhus glabra and R. copallina (Sherman-Broyles et al., 1992
), and Stellaria longipes (Purdy, Bayer, and MacDonald, 1994
) (Table 8). The substantial effect of range substructure on indirect gene flow estimates was reported by Husband and Barrett (1995)
for Eichhornia paniculata. These results suggest that when gene flow is distance dependent, as it is with many sedentary organisms, comparisons of FST between congeners must be made over the same range.
It might be argued that the potential for gene flow is similar for the two species since both are pollinated primarily by native bees (Ayres, 1997
; Ayres, personal observation), and have seeds that lack apparent dispersal mechanisms. However, in El Dorado County, FST in W. reticulata is two to three times higher than in W. bolanderi. One explanation for this apparent contradiction is that W. bolanderi populations have had more opportunities to exchange pollen and seed than W. reticulata populations. On burned chaparral sites, W. reticulata plants only flower heavily for a few years after fire (Ayres, 1997
), and the fire return interval is 20–70 yr (Rundel, 1986
). In contrast, all chaparral populations of W. bolanderi have flowered every year, without diminishment, during the 5-yr course of this study (Ayres, personal observation). Freely flowering W. bolanderi populations (e.g., Yuba County) also occur in permanent natural openings within other vegetation types. The greater flower production of W. bolanderi, spatially and temporally, may provide a greater opportunity for gene exchange to occur.
Gene flow between populations occurs when migrant genes arriving by pollen or seed become established in new genets. Low seed set was a common occurrence in W. reticulata, with some clones never setting seed (Ayres, 1997
). Seedling establishment and growth within stands of W. reticulata were also very limited (Ayres, 1997
). While comparable assessments of seed set and survivorship were not made for W. bolanderi, larger population densities suggest that recruitment of new genets is more frequent in W. bolanderi than in W. reticulata. Greater opportunities for pollen exchange, seed production, and seeding establishment suggest that gene flow has been more extensive in W. bolanderi. On the other hand, temporal constraints on flowering, limited seed set, and low seedling survival could reduce the opportunity for gene flow between populations and result in the genetic divergence of populations of W. reticulata. The phenetic analysis of genetic similarity and geographical separation (discussed below) supports this conclusion.
The extensive clonal growth of W. reticulata may also reduce gene flow rates. Large clones can reproductively dominate within a population and reduce the genetic diversity of pollen exported to other populations. In contrast, clones of W. bolanderi are composed of only a handful of shoots; one individual does not contribute the majority of seed or pollen. Exported pollen will reflect the genetic diversity of the population, and imported pollen will be distributed more evenly throughout the population.
Wyethia bolanderi showed more inbreeding within populations than the restricted congener W. reticulata. Limited pollen and seed movement may be expected to result in a reduction in heterozygosity due to self-pollination and breeding between related individuals (biparental inbreeding). Various authors have found that inbred species have more highly differentiated populations than their outbreeding congeners (e.g., Schoen, 1982
; Crawford, Ornduff, and Vasey, 1985
). Purdy, Bayer, and MacDonald (1994)
have suggested that biparental inbreeding has resulted in an increase in population differentiation in Stellaria arenicola. Hamrick and Godt (1990)
found that breeding system (degree of outcrossing) was one of the factors that accounted for most of the explainable variation in GST, a measure analogous to FST, the relative magnitude of genetic differentiation among subpopulations (Nei, 1987, p. 190). Selfing species generally have higher GST values than outcrossing species.
In the present study, W. bolanderi, with a low FST, exhibited a 22% reduction in heterozygosity due to inbreeding, measured by FIS, while W. reticulata, with a much higher level of among-population differentiation, evidenced a complete absence of inbreeding. The lack of inbreeding is due to self-incompatibility (Ayres, 1997
), and, further, paternity analyses suggest that matings between neighboring, hence possibly genetically related individuals, may have limited success. The breeding system of W. bolanderi is unknown, but the high FIS will be due to biparental inbreeding rather than selfing if self-incompatibility is a generic trait, as might be expected.
Phenetic analyses
Each marker type provided different estimates of genetic similarity between individuals, and, within El Dorado County, revealed disparate patterns of variation in multivariate phenetic analyses for each species. A moderate, but significant, correlation between marker types was found when all individuals of W. bolanderi from Yuba and El Dorado counties were analyzed. The lack of accord between RAPDs and allozymes on a local scale and concordance of marker types over regional scales have also been found in Buffalograss by Peakall, Smouse, and Huff (1995)
. They concluded that the correlation between marker types at regional scales was due to the genetic divergence of widely separated populations and not to concordance of marker types in estimating genetic similarity between individuals. Lack of correlation in estimates of genetic similarity between individuals based on these two marker types, they note, may be a general result.
Neither marker type alone was particularly accurate at resolving geographically defined populations of either species of Wyethia within El Dorado County using phenetic data. However, the combination of markers produced a dendrogram that reflected geographically defined populations for both species (Fig. 6; Ayres and Ryan, 1997
, fig. 3). This illustrates the value of phenetic analyses of individual genotypes; interindividual comparisons of genetic similarity allow population membership to emerge from empirical data. The results demonstrated that the two Salmon Falls populations of W. reticulata, separated by only 0.5 km, were genetically distinct populations. Inaccuracies in genetic differentiation among geographically separate populations of W. bolanderi in El Dorado County (Fig. 5) is in accord with the finding of widespread gene flow from genetic analyses.
Phenetic analysis of individuals also allows for the estimation of the relationship between genetic similarity and spatial separation of individuals, and, by inference, indicates the degree to which gene flow is spatially dependent. Significant inverse correlations were found at the larger geographical scale for W. bolanderi, reflecting a basic dissimilarity between Yuba County and El Dorado County individuals. Within El Dorado County, the correlation between genetic similarity and spatial separation was significant for W. reticulata throughout the range, and within the Cameron Park population, but was not significant over a similar range or within populations of W. bolanderi. This disparity between the two species argues for marked differences in gene flow rates and concurs with the results from the genetic analyses.
Comparison of congeneric species is a useful approach to determine which ecological or life history factors contribute to genetic diversity and structure. Ample opportunities for gene flow among W. bolanderi populations in El Dorado County apparently result in a low FST, in the mixing of genets from spatially separate populations seen in Fig. 6, and in the lack of correlation between genetic similarity and spatial proximity. The high FST found in W. reticulata results from limited opportunities for interpopulation gene flow, local movement of pollen and seed, and the reproductive dominance of large, long-lived individuals. The extensive clonality of W. reticulata apparently has allowed the species to retain a high level of genetic diversity despite small population sizes and infrequent gene flow.
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FOOTNOTES |
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2 Author for correspondence, current address: University of California, Bodega Marine Laboratory, Box 247, Bodega Bay, CA 94923 (e-mail: drayres@ucdavis.edu).
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Literature Cited |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLiterature Cited |
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Peakall, R., P. E. Smouse, and D. R. Huff. 1995 Evolutionary implications of allozyme and RAPD variation in diploid populations of dioecious buffalograss Buchloe dactyloides. Molecular Ecology 4: 135–147.
Pleasants, J. M., and J. F. Wendel. 1989 Genetic diversity in a clonal narrow endemic, Erythronium propullans, and its widespread progenitor, Erythronium albidum. American Journal of Botany 76: 1136–1151.
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{[ri(ri - 1)}/{R(R - 1)]}, where ri is the number of ramets of genotype i, and R is the number of sampled ramets).
