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(American Journal of Botany. 2003;90:85-92.)
© 2003 Botanical Society of America, Inc.

Population Biology

Genetic structure of an endangered plant, Antirrhinum microphyllum (Scrophulariaceae): allozyme and RAPD analysis1

Elena Torres, José M. Iriondo2 and César Pérez

Departamento de Biología Vegetal, Universidad Politécnica de Madrid, Ciudad Universitaria, E-28040, Madrid, Spain

Received for publication April 16, 2002. Accepted for publication July 12, 2002.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Thirteen allozyme loci and 68 random amplified polymorphic DNA (RAPD) markers were analyzed to assess the genetic diversity and population structure of threatened Antirrhinum microphyllum (Scrophulariaceae), a narrow endemic of central Spain known from only four populations. According to allozyme data, species genetic diversity (p = 46.15%, A = 2.61, and He = 0.218), as well as within-population genetic diversity (p = 44.23%, A = 2.10, and He = 0.204), were high when compared to average estimates for other narrowly distributed plant species. Ninety-four percent of species genetic diversity corresponded to within-population genetic diversity. Nevertheless, significant differences were found among populations in allele frequencies of four of the six polymorphic loci, and three private alleles were detected. Inbreeding coefficients (FIS) suggest that populations are structured in genetic neighborhoods. The RAPDs also showed high levels of genetic diversity (p = 89.71% and He = 0.188 at the species level, and p = 67.65% and He = 0.171 at the population level). Nei's genetic distances estimated both from allozymes and RAPDs indicated low differentiation among populations. In spite of this, the low frequencies of certain alleles and the presence of private alleles indicate that efforts should be made to conserve all four remaining populations.

Key Words: allozyme • Antirrhinum microphyllum • conservation management • genetic diversity • population structure • RAPDs • Scrophulariaceae • snapdragon • threatened species


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
The preservation of genetic diversity in endangered species is a main goal in conservation planning, since long-term species survival depends on the maintenance of sufficient genetic variability within and among populations to accommodate new selection pressures brought about by environmental change (Barrett and Kohn, 1991 ). Although some authors have questioned the importance of genetic studies with regard to demographic approaches (Lande, 1988 ; Schemske et al., 1994 ), many others think that assessing genetic diversity and understanding how diversity is structured is not only a prerequisite in designing suitable conservation strategies (Falk and Holsinger, 1991 ; Avise, 1995 ), but, furthermore, this knowledge helps to resolve taxonomic (Van Buren et al., 1994 ; Cole and Kuchenreuther, 2001 ), phylogenetic (Smith and Pham, 1996 ), demographic, and ecological (Bachmann, 1994 ; Cruzan, 1998 ) questions of great relevance for conservation.

Frequently, managers responsible for the conservation and recovery of threatened plant species need to make choices in matters such as the area that needs to be protected, the populations that should be preserved, the minimum number of individuals required to avoid inbreeding problems and to sustain quantitative genetic variation, or the way germplasm should be collected from a population in order to capture most of the genetic diversity for ex situ conservation. In published literature it is possible to find recommendations and guidelines based on theoretical aspects and on generalizations extracted from studies that link species genetic diversity and its partitioning within and among populations to factors such as geographic range or breeding system. Thus, according to surveys of genetic variation in plants made by Hamrick and Godt (1990) and Gitzendanner and Soltis (2000) , narrow endemics tend to have lower genetic diversity than widespread species and outcrossing species have higher levels of variability within populations but lower differentiation among populations than selfing species. However, several examples of highly polymorphic endemic species have been found (e.g., Gottlieb, Warwick, and Ford, 1985 ; Linhart and Premoli, 1993 ; Ranker, 1994 ; Lewis and Crawford, 1995 ; Young and Brown, 1996 ), as well as cases of outcrossing plants with great variation among populations (Chalmers et al., 1992 ). Thus, it is clear that the genetic diversity and structure of a species cannot always be predicted on the basis of distribution size and life history and that, therefore, they must be independently studied for each species.

Allozyme markers have been extensively used to describe the genetic structure of plant populations. They require little plant material, alleles exhibit simple Mendelian inheritance and codominant expression in most cases, and comparisons of homologous loci across populations are straightforward. Nevertheless, allozymes have well-known limitations, the most serious being the limited number of alleles and loci available for study and the bias in the portion of the genome sampled (only genes encoding soluble enzymes are surveyed) (Fritsch and Rieseberg, 1996 ). Therefore, they may underestimate genetic diversity (e.g., Sun et al., 1999 ) and lead to inappropiate conservation recommendations (Les, Reinartz, and Esselman, 1991 ). An additional limitation is the existence of occasional differences between tissues or ontogenetic stages (Lee, Ledig, and Johnson, 2002 ). In the last decade, random amplified polymorphic DNA (RAPD) markers (Williams et al., 1990 ) have been increasingly employed for population studies, especially in endangered plants (e.g., Martín, González-Benito, and Iriondo, 1997 ; Hogbin, Ayre, and Whelan, 1998 ; Fischer et al., 2000 ). Random amplified polymorphic DNA analysis has the advantage of assaying a greater number of potential polymorphic loci and a more random sample of genome than allozymes (Fritsch and Rieseberg, 1996 ). In addition, when compared to other DNA-based markers, the procedure is cheaper and technically simpler and it does not require any prior knowledge of the target genome. However, most RAPD loci show dominant expression and are assumed to possess only two alleles per locus, which may bias some population genetic parameters if selection is occurring or if the population is not randomly mating (Lynch and Milligan, 1994 ; Isabel, Beaulieu, and Bousquet, 1995 ; Szmidt, Wang, and Lu, 1996 ). Another disadvantage is the lower reproducibility of RAPDs between laboratories as compared to other molecular markers. As the abovementioned techniques have different properties, studies that simultaneously analyze allozyme and RAPD markers are very valuable because they provide a larger picture of existing genetic diversity and may avoid misleading results and conclusions for conservation derived from the use of a single type of marker.

In this paper we studied the genetic variation of a threatened species, Antirrhinum microphyllum, through allozyme electrophoresis and RAPD analysis and addressed the following questions: (1) How much genetic variability exists in this species? (2) Are all populations equally diverse? (3) What proportion of the species genetic variation is represented within populations? (4) Is there gene flow among populations? (5) Do allozyme and RAPD analysis provide similar results? This work is part of a wider project in which the status of the species was also assesed through ecological and demographic approaches. The results and conclusions deriving from the different approaches are being integrated and used for the design of a recovery plan for the species.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Species
Antirrhinum microphyllum Rothm. (Scrophulariaceae) is an herbaceous perennial listed as "vulnerable" (Gómez-Campo, 1987 ; VVAA, 2000 ) according to IUCN (International Union for Conservation of Nature) categories, and protected by the regional legislation of Junta de Castilla-La Mancha (Anonymous, 1998 ) due to its narrow distribution, an area of approximately 30 km2. Plants grow in rocky outcrops and dolomitic cliffs located in the banks of the Tajo and Guadiela rivers in the north half of Sierra de Altomira (Central Spain). This taxon belongs to Antirrhinum section Sempervirentia, a small group of rupicolous species that grow on limestone substrates and have discontinuous and restricted distributions (Fernández-Casas, 1997 ) that may have originated in the climatic and topographic changes that occurred during the Pleistocene (Davis, 1951 ). At present, four populations are known (Fig. 1). The most distant populations (15 km) are "Entrepeñas" and "Bolarque" found along the Tajo river. The "Anguix" population is between them, whereas the "Buendía" population is located 3.5 km east of Anguix on the banks of the Guadiela river and separated by the Enmedio mountain. Population sizes range between 400 for Anguix and 2000 for Buendía. Entrepeñas and Bolarque may have experienced a recent reduction in population size due to the building of two dams at these two sites in the 1960s. Antirrhinum microphyllum is a self-incompatible species (Torres, Iriondo, and Pérez, 2002 ), and flowers are pollinated mainly by a solitary bee, Rhodanthidium sticticum (Megachilidae) (Torres et al., 2001 ). The fruit is a poricide capsule, with 200 small (0.5–0.8 mm) seeds in each capsule. Its chromosome number is 2n = 16 (Boscaiu et al., 1997 ; Torres, 1999 ).



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  		Fig. 1. Locations of the four known populations of Antirrhinum microphyllum (Entrepeñas, Anguix, Bolarque, and Buendía) and the closest population of Antirrhinum pulverulentum (Durón)

 
Collection of samples
In 1998, samples of leaf tissue of A. microphyllum were collected from Anguix, Bolarque, Buendía, and Entrepeñas populations for allozyme analysis. Forty-six individuals were randomly sampled within each population. Since populations have an essentially linear spatial distribution of individuals along the banks of the river, a transect that crossed each population parallel to the river was established. In each population 46 random numbers were obtained with the aid of a calculator. They were forced to be within the distance range between both ends of the population along the transect. The individuals located the nearest to the 46 points along the transect were selected as samples. Plant material was placed in microcentrifuge tubes and stored on ice until reaching the laboratory. Thereafter it was stored at –80°C until being homogenized. To allow for the comparison of results obtained from allozymes with those obtained from RAPD markers, young leaves of A. microphyllum were collected from the same 184 plants.

Allozyme electrophoresis
Homogenates were prepared by grinding one or two adult leaves in buffer containing Tris-HCl buffer (0.1 mol/L, pH 8.0), sucrose (200 mmol/L), PVP-40 (1% m/v), and 2-mercaptoethanol (1% v/v). Electrophoresis was carried out in 10% starch gels following standard electrophoretic procedures (Acquaah, 1992 ). Three electrode/gel buffer systems were used to resolve 11 enzyme systems: 0.223 mol/L Tris and 0.069 mol/L citric acid, pH 7.2/0.008 mol/L Tris and 0.002 mol/L citric acid, pH 7.2 was used for resolving isocitrate dehydrogenase (IDH), phosphoglucoisomerase (PGI), and phosphogluconate dehydrogenase (6-PGD); 0.3 mol/L boric acid, pH 8.6/0.015 mol/L Tris and 0.004 mol/L citric acid, pH 7.8 for alcohol dehydrogenase (ADH), aspartate amino transferase (AAT), catalase (CAT), leucine aminopeptidase (LAP), peroxidase (PRX), and superoxide dismutase (SOD); and 0.065 mol/L l-histidine and 0.015 mol/L citric acid, pH 5.7/0.009 mol/L l-histidine and 0.002 mol/L citric acid, pH 5.7 for dihydrolipoamide dehydrogenase (DDH) and phosphoglucomutase (PGM). Enzyme activity staining was carried out following Wendel and Weeden (1989) for ADH, Murphy et al. (1996) for DDH, and Soltis et al. (1983) for all other enzyme systems. Genetic interpretation of band patterns followed standard principles (Weeden and Wendel, 1989 ). Putative loci and alleles were numbered or labeled sequentially from the cathode.

Genetic analysis of allozyme data
Allele frequencies were calculated for the species and for each of the four populations. Chi-square ({chi}2) tests were carried out to test for homogeneity in allele frequencies among populations (Workman and Niswander, 1970 ). Measures of genetic diversity were also calculated for the species and for each population. These statistics included the percentage of polymorphic loci (Pp) (we considered that a locus was polymorphic when the most common allele ocurred at a frequency ≤0.95 [Nei, 1987 ]), the mean number of alleles per locus (A), the mean number of alleles per polymorphic locus (Ap), the observed heterozygosity (Ho), and the expected heterozygosity assuming Hardy-Weinberg equilibrium (He). Departure from Hardy-Weinberg equilibrium for each population/polymorphic locus combination was examined in two ways. First, chi-square goodness-of-fit tests were perfomed to compare observed and expected genotypic frequencies. When some of the expected frequencies were below 0.05, we pooled genotypes in three classes: homozygotes for the most common allele, heterozygotes for the most common allele and one of the other alleles, and all other genotypes. Secondly, Wright's fixation index (FIS) was calculated for each polymorphic locus according to Nei and Chesser (1983) . We tested for significant deficit or excess from expected levels of heterozygosity at each locus using a chi-square test, {chi}2 = N(r – 1)FIS2 with r(r – 1)/2 degrees freedom, where N is sample size and r is the number of alleles at the locus (Li and Horvitz, 1953 ). The FIS values were averaged across polymorphic loci to obtain an overall estimate of the degree of inbreeding within populations. These values were tested to estimate if they were significantly different from zero using a two-tailed Student t test (Sokal and Rohlf, 1995 ) based on jackknife-generated standard deviation values.

In order to assess the distribution of genetic variation within and among populations, the coefficient of genetic differentiation (GST) (Nei, 1973 ) was calculated for each polymorphic locus. According to Chakraborty and Leimar (1987) , for GST values greater than 0.01, this measure of differentiation is more appropiate than Weir and Cockerham's FST estimate (1984). The statistical significance of each GST was calculated using a chi-square test, {chi}2 = 2NGST(r – 1) with (r – 1)(s – 1) degrees freedom, where N is the total sample size, r is the number of alleles at the locus, and s is the number of populations (Workman and Niswander, 1970 ). The GST values were averaged across loci to obtain an overall estimate of population divergence. Mean values of GST were tested by a Student t test (Sokal and Rohlf, 1995 ) based on jackknife-generated standard deviation values.

Genetic distances between pairs of populations (D) were calculated according to Nei (1972) . To examine the genetic relationship among populations, a cluster diagram was generated from distance values using the unweighted pair-group method of arithmetic averages (UPGMA) of NTSYS-pc version 2.02c (Rohlf, 1997 ).

DNA isolation and PCR amplification
DNA was extracted using the procedure reported by Torres, Weeden, and Martin (1993) with some modifications. Leaf material (50 mg) was ground to a fine powder in liquid nitrogen, mixed with 800 µL of extraction buffer (CTAB [hexadecyltrimethyl-ammonium bromide] [2% m/v], Tris-HCl [100 mmol/L pH 8.0], EDTA [20 mmol/L pH 8.0], NaCl [1.4 mol/L], PVP-40 [1% m/v], sodium bisulfite [0.1% m/v], and 2-mercaptoethanol [0.4% v/v]), and incubated at 60°C for 45 min. The samples were mixed with an equal volume of chloroform-isoamyl alcohol (24 : 1) and centrifuged at 9000 x g for 5 min. The resulting supernatants were removed to new microcentrifuges tubes. The DNA was precipitated by adding one volume of pure ethanol at –20°C for 1 h. The crude DNA was spooled out with a glass rod and washed in sodium acetate (0.2 mol/L)/ethanol (75%) and ammonium acetate (0.01 mol/L)/ethanol (75%). Finally, the DNA pellet was air dried and suspended in an appropriate volume of TE buffer (10 mmol/L Tris-HCl pH 8.0 and 1 mmol/L EDTA pH 8.0). DNA concentration for all samples was estimated using a spectrophotometer at 260 and 280 nm.

Amplification reaction volumes were 20 µL, each containing 1x buffer (Tris-HCl [20 mmol/L pH 8.55], (NH4)2SO4 [50 mmol/L], EDTA [0.1 mmol/L], 2-mercaptoethanol [10 mmol/L], Thesit [50%], glycerol, MgCl2 [1.5 mmol/L]), 0.1 mmol/L dNTP (Ecogen, Madrid, Spain), 0.2 µmol/L 10-base primer, 0.7 unit Taq DNA polymerase (BIOTAQ, Ecogen, Madrid, Spain), and 2 ng/µL genomic DNA. Amplifications were performed in a thermalcycler (model 480, Perkin Elmer, Foster City, California, USA) programmed as follows: 3 cycles of 94°C (2 min)/36°C (1 min 30 s)/72°C (2 min), and 41 cycles of 94°C (20 s)/36°C (40 s)/72°C (2 min). Control samples containing all reaction material except DNA were used, in order to check that no self-amplification or DNA contamination occurred. We first tested 50 primers from the University of British Columbia Biotechnology Laboratory (UBC) and 20 from Operon Technologies (Alameda, California, USA) series O (Op-O) on three individuals per population. Twelve primers that showed relatively clear RAPDs were included for further experiments. Repeatability was checked for each primer on 50 samples chosen at random. Furthermore, samples that had rare bands (<0.3) or did not have very frequent bands (>0.95) were also repeated. Fragments generated by amplification were separated according to size on 1.5% agarose gel run in Tris-borate EDTA buffer (1x), stained with ethidium bromide (0.5 µg/mL), and visualized with ultraviolet light. A "100-bp ladder" of DNA fragments (Pharmacia, Peapack, New Jersey, USA) was included in the gels as a size reference.

Genetic analysis of RAPD data
The percentage of polymorphic loci and expected heterozygosity among individuals at each locus/population were calculated. To estimate the frequency of recessive alleles we assumed that (1) null bands are homologous in all individuals and (2) that populations are in Hardy-Weinberg equilibrium. The RAPD data were analyzed using POPGENE version 1.31 (Yeh et al., 1997 ). To avoid biased parameter estimates in the studied loci (Lynch and Milligan, 1994 ), bands with a frequency of <3/N (where N is the sample size, 184 plants) were removed from analyses. Nevertheless, this pruning of loci tends to reject loci that are highly homozygous and may affect average within- and between-population gene diversities (Lynch and Milligan, 1994 ). The RAPD frequencies were also calculated using the estimates of departure from Hardy-Weinberg equilibrium (FIS mean values) obtained from the allozyme analysis and applying the algorithm given by Chong, Yang, and Yeh (1994) . Total gene diversity (HT) and coefficient of gene differentiation (GST) were computed at the species level following the same procedure as with allozymes.

Genetic distances between pairs of populations (D) were calculated according to Nei (1972) . In a similar way to the allozyme analysis, a UPGMA cluster analysis of distance values was generated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Allozyme analysis
Thirteen loci (Aat-2, Aat-3, Adh, Cat, Ddh, Pgi-1, Pgi-2, Idh, Lap-2, Per, Pgd, Pgm, and Sod) coding for the 11 enzymes were resolved. Seven loci were found to be monomorphic, while six loci showed considerable polymorphism (Aat-2, Aat-3, Adh, Ddh, Pgi-2, and Lap) in at least one of the populations. Allele frequencies for each population and for the species are shown in Table 1. Most alleles were shared among populations, but very highly significant differences in allele frequencies (P < 0.001) were detected for all polymorphic loci except for Ddh and Pgi-2. Moreover, three private alleles (sensu Slatkin, 1985 ) were present (Table 1).


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  		Table 1. Summary of allele frequencies for six polymorphic loci in four populations of Antirrhinum microphyllum. Values in italic type indicate the most frequent allele in each population

 
Genetic diversity estimates for each population and for the species are presented in Table 2. For every population, mean observed heterozygosity was lower than expected. Genotype frequencies differed significantly (P < 0.05) from Hardy-Weinberg expectations at six of the 23 loci tested (26%). These deviations were found at Adh locus in Anguix, Bolarque, and Buendía and at Lap-2 in Anguix, Buendía, and Entrepeñas. Five of the 23 fixation indices (22%) were significantly different from zero (Table 3). In all five cases the values were positive, indicating an excess of homozygotes. All mean values of the fixation indices across loci for each population were positive and significantly different from zero except for Bolarque. The highest mean FIS value was obtained in Buendía.


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  		Table 2. Estimates of genetic diversity based on allozyme data in four populations of Antirrhinum microphyllum. Pp = percentage of polymorphic loci; A = mean number of alleles per locus; AP = mean number of alleles per polymorphic locus; Ho = observed heterozygosity; He = expected heterozygosity. Standard deviations are in parentheses

 

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  		Table 3. Fixation index, FIS, for six polymorphic loci in four populations of Antirrhinum microphyllum. For each locus and population, a chi-square test was performed to test for deviations from Hardy-Weinberg equilibrium (***P < 0.001; *P < 0.05)

 
Only 6% of the total genetic variation of A. microphyllum can be attributed to variation among populations (GST = 0.056) (Table 4). This result is in accordance with the low interpopulational genetic distances. Average genetic distance between populations was 0.025 ± 0.015 (mean ± 1 SD) and ranged from 0.013 (Anguix to Buendía) to 0.054 (Bolarque to Entrepeñas). The relationships among populations derived from the UPGMA phenogram are shown in Fig. 2a.


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  		Table 4. Gene diversity statistics. HT = total gene diversity; HS = gene diversity within populations; and GST = the proportion of total gene diversity found among populations. ***P < 0.001; **P < 0.01

 


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  		Fig. 2. UPGMA phenograms of mean genetic distance (Nei, 1972 ) among Antirrhinum microphyllum populations based on (a) allozyme data and (b) RAPD data

 
The RAPD analysis
From a total of 68 bands that were analyzed, 61 (89.71%) were polymorphic at the species level. The percentages of polymorphic loci (Pp) for a single population ranged from 72.06% (Entrepeñas) to 64.71% (Anguix and Buendía) with an average of 67.65 ± 3.6%. The population with the highest gene diversity value was Entrepeñas (He = 0.185 ± 0.177), followed by Anguix (0.176 ± 0.183), Bolarque (0.172 ± 0.168), and Buendía (0.149 ± 0.171). Assuming Hardy-Weinberg equilibrium, the average gene diversity within populations (HS) was 0.171 ± 0.022, whereas the adjusted estimate, which took into account the mean fixation index obtained from allozymes in each population, was slightly lower (HS = 0.168 ± 0.021). As in the allozyme analysis, a low percentage of total diversity (HT = 0.188 ± 0.027) corresponded to diversity among populations (GST = 0.076). Only two population specific bands were found, one with primer UBC-507 in Entrepeñas and other with primer UBC-540 in Buendía.

Genetic distances at the RAPD level varied from 0.015 (Bolarque to Buendía) to 0.039 (Bolarque to Entrepeñas) with a mean of 0.028 ± 0.009. The relationships among populations are shown in Fig. 2b.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Genetic diversity
Species genetic diversity can be interpreted under two criteria. One is the allelic richness, which is measured as the total number of different alleles at each locus in a population or species. A second factor that also accounts for diversity is the evenness of allele frequencies in each locus, and this is measured along with allelic richness by the coefficient of gene diversity (Nei, 1973 ). Under the two criteria A. microphyllum has higher levels of diversity than other plant species with similar life history characteristics (Hamrick and Godt, 1990 ), namely, short-lived, herbaceous perennials (A = 1.70, He = 0.116, N = 152); endemics (A = 1.80, He = 0.096, N = 81) and species with an animal-pollinated, outcrossing system (A = 1.99, He = 0.167, N = 172). Mateu-Andrés (1999) also found high levels of diversity in A. microphyllum through allozyme analysis (range of population values A = 2.17–2.83, He = 0.335–0.470). However, she used different enzyme systems and included individuals from Durón, a small locality 15 km north of Entrepeñas, which is nowadays considered to belong to Antirrhinum pulverulentum (J. Güemes, Jardín Botánico de Valencia, personal communication).

Diversity estimated from RAPD data is difficult to compare with that from other species since authors differ in the way they use monomorphic and polymorphic loci in their calculations and in the criteria used to select polymorphic loci to avoid biased results (Nybom and Bartish, 2000 ). Nevertheless, taking into account that every studied plant had a different RAPD phenotype and the high percentage of polymorphic bands (88.41%) available as a measure of variability, results show that A. microphyllum has a high level of diversity.

Factors contributing to the maintenance of this variation may be the persistence of multiple generations within populations and population sizes that are not critically small. According to Holsinger and Gottlieb (1991) , rare species such as A. microphyllum or Tradescantia hirsuticaulis (Godt and Hamrick, 1993 ) that have restricted distributions but are locally common may resemble more widespread congeners in their ecological and genetic characteristics than other narrow endemics with depauperate populations. In addition, selective biotic and abiotic factors unique to different rock outcrops may lead to the maintenance of variation within the species through microdifferentiation processes (Snaydon and Davies, 1976 ; Dyer and Rice, 1997 ).

Genetic diversity within populations
At the population level, we expected the Entrepeñas and Bolarque populations to be genetically depauperate, compared to the rest of the populations because the size of their populations was probably seriously affected by the construction of the dams in the 1960s. Diversity estimates obtained with allozymes indicate that these populations have lower allelic richness but similar average heterozygosity (Table 2). This result agrees with theoretical models that indicate that these parameters are differently affected by population bottlenecks. Although both allelic diversity and average heterozygosity decrease with a reduction in population size, the loss of allelic diversity is greater than the loss of heterozygosity when compared to the original parental population (Nei, Maruyama, and Chakraborty, 1975 ; Allendorf, 1986 ). According to RAPD data, diversity was also similar among populations as measured by the proportion of polymorphic loci and expected heterozygosity.

The levels of heterozygosity observed in all populations were lower than expected from Hardy-Weinberg equilibrium values. Two possible explanations can explain these results. First, significant amounts of selfing could be occurring within A. microphyllum populations. However, experimental crossings have shown that A. microphyllum is strictly self-incompatible (Torres, Iriondo, and Pérez, 2002 ). Second, genetic diversity may be structured in neighborhoods, and mating may mainly take place among genetically related and geographically close individuals. Field observations of Rhodanthidium sticticum, the main pollinator of this species, have shown that males have a territorial behavior and that females fly to the nearest plant while they collect pollen and nectar (Torres, 1999 ). The effects of nearest-neighbor pollination on genetic structure were simulated by Turner, Stephens, and Anderson (1982) . They concluded that it increases inbreeding, homozygosity, and patchiness in the spatial distribution of genotypes. Thus, pollinator behavior coupled with the lack of specialized seed dispersal mechanisms could be favoring the establishment of neighborhoods of related individuals. Ongoing spatially explicit studies of the genetic structure of these populations along with spatial analysis techniques (Legendre and Legendre, 1998 ) will provide more detailed information on this matter.

Intra- and interpopulational variation
Although most alleles were shared among populations, significant differences were detected primarily in allele frequency for four polymorphic loci. Nevertheless this accounted for only 6% of total variation (mean GST = 0.056). Similar levels of population differentiation were estimated by RAPD analysis (mean GST = 0.076), which indicates most genetic variation occurs within populations. This result was expected taking into account that the breeding system is the main ecological factor that conditions genetic structure (Hamrick, 1982 ; Loveless and Hamrick, 1984 ) and that A. microphyllum is a self-incompatible species. Predominantly outcrossing species have higher levels of variability within populations but a lower degree of differentiation among populations than selfing species (Hamrick and Godt, 1990 ; Schoen and Brown, 1991 ). However, these values are fairly low when compared with those of other animal-pollinated outcrossers. In their allozyme compilation Hamrick and Godt (1990) reported an average GST of 0.197 for outbreeding species (N = 124). Similarly, Nybom and Bartish (2000) reported an average RAPD-based GST value of 23% (N = 24). One possible explanation for this low degree of differentiation between populations may be that the populations have diverged only recently, and there simply has not been sufficient time to differentiate through drift.

Different population clusterings were obtained based on Nei's genetic distance generated from allozyme and RAPD data (Fig. 2). The RAPD markers revealed more geographically concordant groupings than allozyme markers, because Bolarque and Buendía, which are adjacent populations, were grouped together by the former but not by the latter. The matrix of genetic distances showed that the two most geographically distant populations (Entrepeñas and Bolarque, 15 km), also had the longest genetic distances. This result suggests that these populations may be partially isolated by distance. Gene flow via pollen between Entrepeñas and Bolarque seems improbable since the geographic distance is too great when compared to the flight distances of R. sticticum.

Allozyme and RAPDs data
Several authors have stressed the importance of using more than one class of molecular markers to estimate genetic diversity of endangered species (Chase, Kesseli, and Bawa, 1996 ; Fritsch and Rieseberg, 1996 ; Esselman et al., 1999 ). However, few studies have directly compared genetic diversity estimates based on allozyme and other DNA markers, such as RAPDs (e.g., Peakall, Smouse, and Huff, 1995 ; Ayres and Loveless, 1997 ; Aagaard, Krutovski, and Strauss, 1998 ; Ayres and Ryan, 1999 ). The advantages of studying both allozymes and RAPDs are evident because they analyze geographically and functionally different genome regions. In the case of allozymes, the detection of genetic variation is limited to protein coding loci while RAPDs are presumed to result from amplification from noncoding regions of DNA (Williams et al., 1990 ). Furthermore, the regions of DNA sampled by the RAPD technique are expected to be less responsive to selection and to have a higher tolerance to mutation than DNA coding for enzymes. Thus, it has been suggested that allozymes and DNA markers may experience different evolutionary dynamics and reveal different patterns of genetic variation (Ayres and Loveless, 1997 ).

Overall, the allozyme and RAPD techniques gave similar results in this study. Both of them detected a relatively large amount of genetic variation, and the partition of genetic diversity within and among populations was comparable between the two data sets. Low estimates of GST based on RAPDs confirmed the existence of little genetic differentiation among populations, as revealed by allozyme data. Nei's genetic distance estimate based on RAPDs (D = 0.028) also corroborates the allozyme estimate (D = 0.025). Most of the studies that have compared allozymes and RAPD report the same pattern of population genetic structure for the two sets of data, and greater diversity estimates from RAPDs than from allozymes in terms of percentage of polymorphic loci and gene diversity (e.g., Liu and Furnier, 1993 ; Lannér-Herrera et al., 1996 ; Diaz et al., 1999 ; Wong and Sun, 1999 ; Virk et al., 2000 ). However, these comparisons of genetic diversity estimates between RAPDs and allozymes must be taken with extreme caution. Comparisons of the percentage of polymorphic loci are hindered by the fact that in RAPDs primers are usually selected for their ability to produce a high rate of polymorphic bands. Moreover, the comparison of gene diversity estimates with Nei's H index is not appropriate because its range of variation depends on the number of alleles per locus. Since RAPDs can only have two alleles at each locus, its He value cannot exceed 0.50. However, in allozymes, the number of alleles per locus is not restricted to two and the maximum value of He can reach up to 1. Only when every allozyme loci had two alleles per locus, would both estimates be comparable.

Implications for conservation
The ability of a population to respond to selection is directly related to the level of genetic variation available for relevant adaptative characters (Huenneke, 1991 ). Therefore it is expected that species with a narrow genetic base will not be able to respond as well to changes in abiotic or biotic environmental conditions as species with a broad genetic base. According to our estimates of allelic diversity and average heterozygosity, A. microphyllum maintains a significant level of genetic variation in the studied regions of the genome. However, we must be cautious with the interpretation of data because allozymes and RAPDs are thought to be neutral or nearly neutral genetic markers, and variation at allozyme and RAPD loci does not necessarily correlate with the levels of variation of loci affecting traits of present or future adaptive importance (Lewontin, 1984 ; Ennos et al., 1997 ). Furthermore, a large proportion of the alleles identified (24–30%) occurs at frequencies below 0.05 at Anguix, Bolarque, and Buendía. Therefore, A. microphyllum is especially vulnerable to the loss of allelic richness due to fluctuations in population size (Nei, Maruyama, and Chakraborty, 1975 ).

Data on the distribution of genetic variation among populations have direct implications for the management of endangered species, for instance in suggesting what sampling strategy should be adopted to efficiently sample the gene pool for ex situ conservation (e.g., Ceska, Affolter, and Hamrick, 1997 ; Ferguson et al., 1998 ) or in deciding which populations should be enhanced and/or preserved (e.g., Kress, Maddox, and Roesel, 1994 ; Richter, Soltis, and Soltis, 1994 ; Fischer et al., 2000 ). The detection of three private alleles (see Table 1) and two unique RAPD bands in some populations suggest that a similar pattern of rare alleles may also exist in loci of adaptive value. Taking this into consideration, as well as the small number of existing populations in A. microphyllum, efforts should be made to protect the four populations and to collect seeds in all them. However, due to the small differentiation among the populations and following criteria suggested by Hamrick et al. (1991) , we estimate that the sampling of two populations of A. microphyllum could provide 99% of the genetic diversity found in the species. Pairwise comparisons between populations show that Anguix and Bolarque populations have the highest genetic diversity when combined (Pp = 61.54%, A = 2.54, He = 0.218). In addition, 23 of the 25 alleles observed in the four populations occur in these two populations. Therefore, if we had to restrict our sampling or decide which populations are more valuable in genetic terms, Anguix and Bolarque would probably be the best choice.


   FOOTNOTES
 
1 The authors thank Dra. I. Aguinagalde for her supervision with the allozyme analysis and Mrs. Lori De Hond for her linguistic assistance. This work was supported by the Spanish Government CICYT project no. AMB-1021-C02-01. Back

2 Author for reprint requests (iriondo{at}ccupm.upm.es ) Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Aagaard J. E. K. V. Krutovski S. H. Strauss 1998 RAPDs and allozymes exhibit similar levels of diversity and differentiation among populations and races of Douglas-fir. Heredity 81: 69-78[CrossRef][ISI]

Acquaah G. 1992 Practical protein electrophoresis for genetic research. Dioscorides Press, Portland, Oregon, USA

Allendorf F. W. 1986 Genetic drift and the loss of alleles versus heterozygosity. Zoo Biology 5: 181-190

Anonymous. 1998 Decreto 33/1998, 5 de mayo, por el que se crea el Catálogo Regional de Especies Amenazadas de Castilla-La Mancha. Diario Oficial de Castilla-La Mancha 22: 3391-3398

Avise J. C. 1995 Introduction: the scope of conservation genetics. In J. C. Avise and J. L. Hamrick [eds.], Conservation genetics, 1–9. Chapman and Hall, New York, New York, USA

Ayres D. R. M. D. Loveless 1997 The clonal and population structure of a rare endemic plant, Wyethia reticulata (Asteraceae): allozyme and RAPD analysis. Molecular Ecology 6: 761-772[CrossRef][ISI]

Ayres D. R. F. J. Ryan 1999 Genetic diversity and structure of the narrow endemic Wyethia reticulata and its congener W. bolanderi (Asteraceae) using RAPD and allozyme techniques. American Journal of Botany 86: 344-353[Abstract/Free Full Text]

Bachmann K. 1994 Molecular markers in plant ecology. New Phytologist 126: 403-418[CrossRef][ISI]

Barrett S. C. H. R. Kohn 1991 Genetic and evolutionary consequences of small population size in plants: implications for conservation. In D. A. Falk and K. E. Holsinger [eds.], Genetics conservation of rare plants, 1–30. Oxford University Press, New York, New York, USA

Boscaiu M. J. Riera E. Estrelles J. Güemes 1997 Números cromosómicos de plantas occidentales, 751–776. Anales del Jardín Botánico de Madrid 55: 430-431

Ceska J. F. J. M. Affolter J. L. Hamrick 1997 Developing a sampling strategy for Baptisia arachnifera based on allozyme diversity. Conservation Biology 11: 1133-1139[CrossRef][ISI]

Chakraborty R. O. Leimar 1987 Genetic variation within a subdivided population. In N. Ryman and F. Utter [eds.], Population genetics and fishery management, 89–120. University of Washington Press, Seattle, Washington, USA

Chalmers K. J. R. Waugh J. I. Sprent A. J. Simons W. Powell 1992 Detection of genetic variation between and within populations of Gliricidia sepium and G. maculata using RAPD markers. Heredity 69: 465-472

Chase M. W. R. V. Kesseli K. Bawa 1996 Microsatellite markers for population and conservation genetics of tropical trees. American Journal of Botany 83: 51-57[CrossRef][ISI]

Chong D. K. X. R. C. Yang F. C. Yeh 1994 Nucleotide divergence between populations of trembling aspen (Populus tremuloides) estimated with RAPDs. Current Genetics 26: 374-376[CrossRef][ISI][Medline]

Cole C. T. M. A. Kuchenreuther 2001 Molecular markers reveal little genetic differentiation among Aconitum noveboracense and A. columbianum (Ranunuculaceae) populations. American Journal of Botany 88: 337-347[Abstract/Free Full Text]

Cruzan M. B. 1998 Genetic markers in plant evolutionary ecology. Ecology 79: 400-412[CrossRef][ISI]

Davis P. H. 1951 Cliff vegetation in the Eastern Mediterranean. Journal of Ecology 39: 63-93[CrossRef]

Diaz O. G.-L. Sun B. Salomon R. Von Bothmer 1999 Levels and distribution of allozyme and RAPD variation in populations of Elymus fibrosus (Schrenk) Tzvel. (Poaceae). Genetic Resources and Crop Evolution 47: 11-24[CrossRef][ISI]

Dyer A. R. K. J. Rice 1997 Evidence of spatial genetic structure in a California bunchgrass population. Oecologia 112: 333-339[CrossRef][ISI]

Ennos R. A. N. R. Cowie C. J. Legg C. Sydes 1997 Which measures of genetic variation are relevant in plant conservation? A case study of Primula scotica. In T. E. Tew, T. J. Crawford, J. W. Spencer, D. P. Stevens, M. B. Usher, and J. Warren [eds.], The role of genetics in conserving small populations, 73–79. Joint Nature Conservancy Council (JNCC), Peterborough, UK

Esselman E. J. L. Jianqiang D. J. Crawford J. L. Winduss A. D. Wolfe 1999 Clonal diversity in the rare Calamagrostis porteri ssp. insperata (Poaceae): comparative results for allozyme and random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) markers. Molecular Ecology 8: 443-451[CrossRef][ISI]

Falk D. A. K. E. Holsinger 1991 Genetics and conservation of rare plants. Oxford University Press, New York, New York, USA

Ferguson M. E. B. V. Ford-Lloyd L. D. Robertson N. Maxted H. J. Newbury 1998 Mapping the geographical distribution of genetic variation in the genus Lens for the enhanced conservation of plant genetic diversity. Molecular Ecology 7: 1743-1755[CrossRef]

Fernandez-Casas J. 1997 De Antirrhinis notulae. Fontqueria 48: 195-202

Fischer M. R. Husi D. Prati M. Peintinger M. Van Kleunen B. Schmid 2000 RAPD variation among and within small and large populations of the rare clonal plant Ranunculus reptans (Ranunculaceae). American Journal of Botany 87: 1128-1137[Abstract/Free Full Text]

Fritsch P. L. H. Rieseberg 1996 The use of random amplified polymorphic DNA (RAPD) in conservation genetics. In T. B. Smith and R. K. Wayne [eds.], Molecular genetics approaches in conservation genetics, 54–73. Oxford University Press, New York, New York, USA

Gitzendanner M. A. P. S. Soltis 2000 Patterns of genetic variation in rare and widespread plant congeners. American Journal of Botany 87: 783-792[Abstract/Free Full Text]

Godt M. J. W. J. L. Hamrick 1993 Genetic diversity and population structure in Tradescantia hirsuticaulis (Commelinaceae). American Journal of Botany 80: 959-966[CrossRef][ISI]

Gómez-Campo C. 1987 Libro rojo de especies vegetales amenazadas de España Peninsular e Islas Baleares. ICONA, Madrid, Spain

Gottlieb L. D. S. I. Warwick V. S. Ford 1985 Morphological and electrophoretic divergence between Layia discoidea and L. glandulosa. Systematic Botany 10: 484-495[CrossRef][ISI]

Hamrick J. L. 1982 Plant population genetics and evolution. American Journal of Botany 69: 1685-1693[CrossRef][ISI]

Hamrick J. L. M. J. W. Godt 1990 Allozyme diversity in plant species. In A. H. D. Brown, M. T. Clegg, A. L. Kahler, and B. S. Weir [eds.], Plant population genetics, breeding and genetic resources, 43–63. Sinauer, Sunderland, Massachusetts, USA

Hamrick J. L. M. J. W. Godt D. A. Murawski M. D. Loveless 1991 Correlation between species traits and allozyme diversity: implications for conservation biology. In D. A. Falk and K. E. Holsinger [eds.], Genetics and conservation of rare plants, 75–86. Oxford University Press, New York, New York, USA

Hogbin P. M. D. J. Ayre R. J. Whelan 1998 Genetic variation and reproductive success of road verge populations of the rare shrub Grevillea barklyana (Proteaceae). Heredity 80: 180-186[CrossRef][ISI]

Holsinger K. E. L. D. Gottlieb 1991 Conservation of rare and endangered plants: principles and prospects. In D. A. Falk and K. E. Holsinger [eds.], Genetics and conservation of rare plants, 195–208. Oxford University Press, New York, New York, USA

Huenneke L. F. 1991 Ecological implications of genetic variation in plant populations. In D. A. Falk and K. E. Holsinger [eds.], Genetics and conservation of rare plants. Oxford University Press, New York, New York, USA

Isabel N. J. Beaulieu J. Bousquet 1995 Complete congruence between gene diversity estimates derived from genotypic data at enzyme and random amplified polymorphic DNA loci in black spruce. Proceedings of the National Academy of Sciences, USA 92: 6369-6373[Abstract/Free Full Text]

Kress W. J. G. D. Maddox C. S. Roesel 1994 Genetic variation and protection priorities in Ptilimnium nodosum (Apiaceae), an endangered plant of the Eastern United States. Conservation Biology 8: 271-276[CrossRef][ISI]

Lande R. 1988 Genetic and demography in biological conservation. Science 241: 1455-1460[Abstract/Free Full Text]

Lannér-Herrera C. M. Gustfsson A.-S. Fält T. Bryngelsson 1996 Diversity in natural populations of wild Brassica oleracea as estimated by isozyme and RAPD analysis. Genetic Resources and Crop Evolution 43: 13-23[CrossRef][ISI]

Lee S. W. F. T. Ledig D. R. Johnson 2002 Genetic variation at allozyme and RAPD markers in Pinus longaeva (Pinaceae) of the White Mountains (California). American Journal of Botany 89: 566-577[Abstract/Free Full Text]

Legendre P. L. Legendre 1998 Spatial analysis: developments in environmental ecology, 707–785. Elsevier Science, Amsterdam, The Netherlands

Les D. H. J. A. Reinartz E. J. Esselman 1991 Genetic consequences of rarity in Aster furcatus (Asteraceae), a threatened, self-incompatible plant. Evolution 45: 1641-1650[CrossRef][ISI]

Lewis P. O. D. J. Crawford 1995 Pleistocene refugium endemics exhibit greater allozymic diversity than widespread congeners in the genus Polygonella (Polygonaceae). American Journal of Botany 82: 141-149

Lewontin R. C. 1984 Detecting population differences in quantitative characters as opposed to gene frequencies. American Naturalist 123: 115-124[CrossRef][ISI]

Li C. C. D. G. Horvitz 1953 Some methods of estimating the inbreeding coefficient. American Journal of Human Genetics 5: 107-117[ISI][Medline]

Linhart Y. B. A. C. Premoli 1993 Genetic variation in Aletes acaulis and its relative, the narrow endemic A. humilis (Apiaceae). American Journal of Botany 80: 598-605[CrossRef][ISI]

Liu Z. G. R. Furnier 1993 Comparison of allozyme, RFLP, and RAPD markers for revealing genetic variation within and between trembling aspen and bigtooth aspen. Theoretical and Applied Genetics 87: 97-105[ISI]

Loveless M. D. J. L. Hamrick 1984 Ecological determinants of genetic structure in plant populations. Annual Review of Ecology and Systematics 15: 65-95

Lynch M. B. G. Milligan 1994 Analysis of population genetic structure with RAPD markers. Molecular Ecology 3: 91-99[Medline]

Martín C. M. E. González-Benito J. M. Iriondo 1997 Genetic diversity within and among populations of a threatened species: Erodium paularense Fern. Gonz. & Izco. Molecular Ecology 6: 813-820[CrossRef]

Mateu-Andrés I. 1999 Allozymic variation and divergence in three species of Antirrhinum L. (Scrophulariaceae-Antirrhineae). Botanical Journal of the Linnean Society 131: 187-199[CrossRef]

Murphy R. W. J. W. Sites D. G. Buth C. H. Haufler 1996 Proteins: isozyme electrophoresis. In D. M. Hillis, C. Moritz, and B. K. Mable [eds.], Molecular systematics, 51–120. Sinauer, Sunderland, Massachusetts, USA

Nei M. 1972 Genetic distance between populations. American Naturalist 106: 283-292[CrossRef][ISI]

Nei M. 1973 Analysis of gene in subdivided populations. Proceedings of the National Academy of Sciences, USA 70: 3321-3323[Abstract/Free Full Text]

Nei M. 1987 Genetic variation within species. In M. Nei [ed.], Molecular evolutionary genetics, 176–207. Columbia University Press, New York, New York, USA

Nei M. R. K. Chesser 1983 Estimation of fixation indices and gene diversities. Annals of Human Genetics 47: 253-259[ISI][Medline]

Nei M. T. Maruyama R. Chakraborty 1975 The bottleneck effect and genetic variability in populations. Evolution 29: 1-10

Nybom H. I. V. Bartish 2000 Effects of life history traits and sampling strategies on genetic diversity estimates obtained with RAPD markers in plants. Perspectives in Plant Ecology, Evolution and Systematics 3/2: 93-114

Peakall R. P. E. Smouse D. R. Huff 1995 Evolutionary implications of allozyme and RAPD variation in diploid populations of buffalograss (Buchloë dactyloides (Nutt.) Engelm). Molecular Ecology 4: 135-137

Ranker T. A. 1994 Evolution of high genetic variability in the rare hawaiian fern Adenophorus periens and implications for conservation management. Biological Conservation 70: 19-24

Richter T. S. P. S. Soltis D. E. Soltis 1994 Genetic variation within and among populations of the narrow endemic, Delphinium viridescens (Ranunculaceae). American Journal of Botany 81: 1070-1076[CrossRef][ISI]

Rohlf F. J. 1997 NTSYS-pc: numerical taxonomy and multivariate analysis system. Exeter Software, Setauket, New York, USA

Schemske D. W. B. C. Husband M. H. Ruckelshaus C. Goodwillie I. M. Parker J. G. Bishop 1994 Evaluating approaches to the conservation of rare and endangered plants. Ecology 75: 584-606[CrossRef][ISI]

Schoen D. J. A. H. D. Brown 1991 Intraspecific variation in population gene diversity and effective population size correlates with the mating system in plants. Proceedings of the National Academy of Sciences, USA 88: 4494-4497[Abstract/Free Full Text]

Slatkin M. 1985 Rare alleles as indicators of gene flow. Evolution 39: 53-65[CrossRef][ISI]

Smith J. F. T. V. Pham 1996 Genetic diversity of the narrow endemic Allium aaseae (Alliaceae). American Journal of Botany 83: 717-726[CrossRef][ISI]

Snaydon R. W. M. S. Davies 1976 Rapid population differentiation in a mosaic experiment. IV. Populations of Anthoxanthum odoratum at sharp boundaries. Heredity 37: 9-25[ISI]

Sokal R. R. F. J. Rohlf 1995 Biometry: the principles and practice of statistics in biological research. W. H. Freeman, New York, New York, USA

Soltis D. E. C. H. Haufler D. C. Darrow G. J. Gastony 1983 Starch gel electrophoresis of ferns: a compilation of grinding buffers, gel and electrode buffers, and staining schedules. American Fern Journal 73: 9-27[CrossRef][ISI]

Sun G.-L. O. Diaz B. Salomon R. Von Bothmer 1999 Genetic diversity in Elymus caninus as revealed by isozyme, RAPD, and microsatellite markers. Genome 42: 420-431[Medline]

Szmidt A. E. X.-R. Wang M.-Z. Lu 1996 Empirical assessment of allozyme and RAPD variation in Pinus sylvestris L. using haploid tissue analysis. Heredity 76: 412-420[ISI]

Torres A. M. N. F. Weeden A. Martín 1993 Linkage among isozyme, RFLP and RAPD markers in Vicia faba. Theoretical and Applied Genetics 85: 937-945[ISI]

Torres M. E. 1999 Estudio de la ecología, biología reproductiva y diversidad genética de Antirrhinum microphyllum. Evaluación del estado actual de conservación. Ph.D. dissertation, Universidad Politécnica de Madrid, Madrid, Spain

Torres M. E. J. M. Iriondo C. Pérez 2002 Vulnerability and determinants of reproductive success in the narrow endemic Antirrhinum microphyllum (Scrophulariaceae). American Journal of Botany 89: 1171-1179[Abstract/Free Full Text]

Torres M. E. C. Ruiz J. M. Iriondo C. Pérez 2001 Pollination ecology of Antirrhinum microphyllum Rothm. Bocconea 13: 543-547

Turner M. E. J. C. Stephens W. W. Anderson 1982 Homozygosity and patch structure in plant populations as a result of nearest-neighbor pollination. Proceedings of the National Academy of Sciences, USA 79: 203-207[Abstract/Free Full Text]

Van Buren R. K. T. Harper W. R. Andersen D. J. Stanton S. Seyoum J. L. England 1994 Evaluating the relationship of autumn buttercup (Ranunculus acriformis var. aestivalis) to some congeners using random amplified polymorphic DNA. American Journal of Botany 81: 514-519[CrossRef][ISI]

Virk P. S. J. Zhu H. J. Newbury G. J. Bryan M. T. Jackson B. V. Ford-Lloyd 2000 Effectiveness of different classess of molecular marker for classifying and revealing variation in rice (Oryza sativa) germplasm. Euphytica 112: 275-284[CrossRef][ISI]

VVAA. 2000 Lista roja de flora vascular española (valoración según las categorías UICN). Conservación Vegetal 6: 11-38

Weir B. S. C. C. Cockerham 1984 Estimating F-statistics for the analysis of population structure. Evolution 38: 1358-1370[CrossRef][ISI]

Weeden N. F. J. F. Wendel 1989 Genetics of plant isozymes. In D. E. Soltis and P. S. Soltis [eds.], Isozymes in plant biology, 46–72. Dioscorides Press, Portland, Oregon, USA

Wendel J. F. N. F. Weeden 1989 Visualization and interpretation of plant isozymes. In D. E. Soltis and P. S. Soltis [eds.], Isozymes in plant biology, 5–45. Dioscorides Press, Portland, Oregon, USA

Williams J. G. K. A. R. Kubelik K. J. Livak J. A. Rafalski S. V. Tingey 1990 DNA polymorphism amplified polymorphic by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18: 6531-6535[Abstract/Free Full Text]

Wong K. C. M. Sun 1999 Reproductive biology and conservation genetics of Goodyera procera (Orchidaceae). American Journal of Botany 86: 1406-1413[Abstract/Free Full Text]

Workman P. L. J. D. Niswander 1970 Population studies on southwestern Indian tribes. II. Local differentiation in the papago. American Journal of Human Genetics 22: 24-49[ISI][Medline]

Yeh F. C. R.-C. Yang T. J. B. Boyle Z.-H. Ye J. X. Mao 1997 POPGENE, the user-friendly shareware for population genetic analysis. Molecular Biology and Biotechnology Centre, University of Alberta, Edmonton, Alberta, Canada

Young A. G. A. H. D. Brown 1996 Comparative population genetic structure of the rare woodland shrub Daviesia suaveolens and its common congener D. mimosoides. Conservation Biology 10: 1220-1228[CrossRef][ISI]




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