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Population Biology |
2Departamento de Biología Vegetal, Universidad de Valencia, Valencia, Spain; 3Instituto Valenciano de Investigaciones Agrarias, Moncada (Valencia), Spain; 4Departamento de Biología Ambiental, Universidad de las Islas Baleares, Mallorca, Spain
Received for publication November 28, 2000. Accepted for publication March 8, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: AMOVA • Digitalis minor • endemic species • population structure • RAPDs • Schrophulariaceae
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Schaal et al., 1998
). All these factors can lead to complex genetic structuring within populations, which is often difficult to resolve. Nevertheless, the development of a number of different DNA markers has provided powerful tools for the investigation of genetic variation within a species and can facilitate understanding of such complexities (Mitton, 1994
).
Random amplified polymorphic DNA (RAPD) technology via the polymerase chain reaction (PCR) has fast become a means of investigating genetic diversity within and between populations and has been applied to many plant species including Digitalis (see Nebauer, Del Castillo Agudo, and Segura, 1999, 2000
, and references therein). In spite of this, the various statistics used to estimate and partition genetic variation in natural populations cannot be applied easily to RAPD data obtained from outcrossing species because complete genotypic determination is largely hampered by their dominant nature (Isabel et al., 1999
). During recent years, however, several strategies have been proposed (Lynch and Milligan, 1994
; Apostol et al., 1996
; Stewart and Excoffier, 1996
) to minimize the effects of RAPD dominance. Of all these approaches, the pruning of fragments with low frequency of null alleles in each population (Lynch and Milligan, 1994
) resulted in FST values concordant with those estimated from haploid sexual tissues (Isabel et al., 1999
) and codominant allozyme markers (Aagaard, Krutovskii, and Strauss, 1998
; Jenczewski, Prosperi, and Ronfort, 1999
). Furthermore, simulation studies on the effects of dominance using allozyme data suggested that RAPDs can reasonably estimate population differentiation (Wu, Krutovskii, and Strauss, 1999
). This also holds true when the FST value was estimated from the analysis of molecular variance (Excoffier, Smouse, and Quattro, 1992
), although simulations were not performed due to the complexity of calculations (Isabel et al., 1999
).
The Mediterranean flora has long been of interest to biologists because of their high level of endemic species richness and complicated patterns of community organization (Cardona and Contandriopoulos, 1979
). In spite of this, only limited information is available on the genetic structure of endemic Mediterranean plant species (for review, see Thompson, 1999
). This is unfortunate because such information is crucial for devising strategies to protect and preserve the genetic resources of the Mediterranean flora.
Digitalis minor L. (Synonym: Digitalis dubia J.J. Rodr.), of the family Scrophulariaceae, is an outcrossing insect-pollinated long-lived herbaceous species with a chromosome number of 2n = 56 (Contandriopoulos and Cardona, 1984
). Although self-incompatibility mechanisms have not been described for D. minor, floral dichogamy (protandry) promotes cross-fertilization of this species (Kampny, 1995
). Digitalis minor shows a high degree of morphological polymorphism, but only two infraspecific taxa are currently recognized according to its differences in pubescence (Hinz, 1987
): D. minor var. minor (pubescent) and D. minor var. palaui (glabrous).
Digitalis minor is an endemic species to the Balearic archipelago (Spain). This species is believed to be an schizoendemic vicariant of D. purpurea ssp. purpurea (Contandriopoulos and Cardona, 1984
) and occurs on three islands of eastern Balearics or Gymnesian Islands (Mallorca, Menorca, and Cabrera). According to paleohistory and present-day geography of the western Mediterranean basin, the differentiation of D. minor seems to be of pre-Messinian origin (Hinz, 1990
). The isolation of the Gymnesian Islands, which constituted on several occasions a single landmass, was probably a more recent event (Cardona and Contandriopoulos, 1979
), since it would date from the last glaciation of the Pleistocene Era (the Würm).
To our knowledge, there is no report on the application of molecular markers to study the population structure of D. minor. As other Digitalis species, it contains cardiac glycosides, widely used in some heart-failure diseases. Thus, an understanding of the extent and distribution of genetic variation within D. minor populations is also essential for devising sampling strategies, which efficiently capture genetic diversity for selection trials and subsequent use of material that fulfils the dual aim of high genetic variation and reasonable performance.
In the present study the genetic diversity in 17 populations of D. minor encompassing the natural distribution of the species in the Mallorca, Menorca, and Cabrera islands was examined using RAPD. The major objectives were to quantify the amount and distribution of genetic variation within and among these populations using genetic diversity measures, F statistics, and spatial-correlation statistics. The possibilities of using the RAPD markers to differentiate the two infraspecific taxa of D. minor were also tested.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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and Nebauer, Del Castillo Agudo, and Segura (2000)
, respectively. RAPD products were separated by size on 1.5% agarose (SeaKem LE, FMC Bioproducts, Rockland, Maine, USA) gel run in TBE buffer (89 mmol/L Tris base, 89 mmol/L boric acid, and 2 mmol/L EDTA, pH 8), stained with ethidium bromide, and photographed in UV light (Nebauer, Del Castillo Agudo, and Segura, 2000
). To aid interpretation of band identity between gels, each contained Gene Ruler DNA ladder mix (Fermentas AB, Vilnius, Lithuania). Primers were initially screened to identify well-amplified, polymorphic bands among populations. Individual Digitalis DNA samples were appropriately diluted and bulked by populations to screen 60 decamer primers (Series OPA, OPB, and OPC from Operon Technologies, Alameda, California, USA). Six primers from the initial screening process (OPA2, OPA20, OPC9, OPC14, and OPC15 from Operon, and OPB7 from Amersham Ibérica, Madrid, Spain) that exhibited a high polymorphism and showed the best readability were chosen for further study of the 162 individual genotypes.
Reproducibility and repeatability of amplification profiles were tested for each primer. Control samples containing all reaction material except DNA were used to test that no self-amplification or DNA contamination occurred. Only those bands consistently reproduced in different analyses were considered. Poor amplifications occurred systematically with individuals from different populations; these were excluded from the analysis and they mainly account for the different sample sizes of this study. At least two replicates per sample were amplified and DNA from all the individuals was extracted twice.
Southern blot of PCR products
To check that the same-size products were homologous among individuals from different populations, hybridizations on Southern transfer of some RAPD gels were performed as described by Nebauer, Del Castillo Agudo, and Segura (2000)
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Statistical analysis
Since RAPD markers are dominant, we assumed that each band represented the phenotype at a single biallelic locus (Williams et al., 1990
). Amplified fragments, named by the primer used and the molecular mass in base pairs (bp), were scored as presence (1) or absence (0) of homologous bands, and a matrix of the different RAPD phenotypes was assembled. Three different types of data were used for the analyses performed in this study for RAPDs: (1) total scorable bands (hereafter referred to as data sets Baleares141, Mallorca141, Menorca141, and Cabrera141); (2) bands that fulfilled the Lynch and Milligan (1994)
criterion (those whose observed frequency in each population is less than 1 – [3/N], where N is the number of sampled plants in the population) among the three islands (hereafter referred to as data sets Baleares39, Mallorca39, Menorca39, and Cabrera39); and (3) bands that fulfilled the Lynch and Milligan criterion within each island (hereafter referred to as Mallorca52, Menorca35, and Cabrera22 data sets; see Table 3). When appropriate, and assuming that all populations are in Hardy-Weinberg equilibrium, we estimated qj(i), the frequency of the null allele a at locus i in population j, as: qj(i) = [xi(i)]1/2, where xi(i) is the frequency of null recessive homozygotes in population j at locus i. The frequencies were computed in RAPDBIOS from RAPDPLOT (Black, 1998
) using Lynch and Milligan's (1994)
correction factor for small sample sizes.
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) and Apostol (Apostol et al., 1993
) coefficients using RAPDistance (Armstrong et al., 1996
) software. A Mantel test (1967)
showed a complete correlation (r = 1) when both matrices were compared in NTSYS-pc (Rohlf, 1997
). The former distance was chosen due to its adequacy for the analysis of molecular variance (see below) and the latter one because it uses both presence and absence of matches, providing more information regarding the phenotypic similarity among pairs of individuals (Apostol et al., 1993
).
The relationships among RAPD phenotypes were assessed as follows. First, the Apostol distance matrices were used to produce dendrograms using the neighbor-joining (NJ) cluster analysis as implemented in NEIGHBOR from the PHYLIP 3.57c package (Felsenstein, 1993
). To give a measure of the variability in the data, bootstrap analysis was conducted and 100 similarity matrices were produced using RAPDPLOT. The NEIGHBOR and CONSENSE programs in PHYLIP were used to generate the 100 trees that were then used to produce a consensus tree. Principal coordinate analyses (PCO) were also performed, using the Apostol distance matrices (DCENTER and EIGEN in NTSYS).
The distance matrices between RAPD patterns were also used to calculate pairwise genetic distances between populations. These distance matrices were used to construct dendrograms using the NJ method. The relationships between matrices of genetic and linear geographic distances were examined with a Mantel (1967)
test in NTSYS. Resulting r values were interpreted as correlation coefficients. Additionally, and for comparative purposes, Nei's unbiased genetic distances (Nei, 1978
) were calculated among populations for each of our data sets with POPGENE software (Yeh et al., 1997
). All dendrograms were displayed and printed using TREEVIEW software (Page, 1996
).
Two different analyses of molecular variance (AMOVA) were performed to study the genetic structure of D. minor populations. First, the selected bands were analyzed directly as phenotypes using the Euclidean distance matrix of Excoffier, Smouse, and Quattro (1992)
. Second, we used the extension of the AMOVA developed by Stewart and Excoffier (1996)
, which allows the estimation of population genetic parameters at the genotypic level with RAPD profile data. In this case, the previously estimated null allele frequencies were used to generate a genotypic distance matrix assuming random mating (S = 0). The nested AMOVAs were used to estimate the partition of total genetic diversity in variance components among individuals within populations, among populations, and among regions (islands). AMOVAs were also performed for the individuals within each of the three islands: Mallorca, Menorca, and Cabrera. The AMOVA procedure was finally implemented to study the relationships between the two infraspecific taxa of D. minor. The whole set of bands as well as the data sets that fulfilled the Lynch and Milligan criterion were employed for these last analyses. The resulting variance components were used as estimates of the genetic divergence among the taxa.
A nonparametric test for homogeneity of molecular variance (HOMOVA), based on the Barlett statistic (Barlett, 1937
), was also applied to test whether populations have different amounts of RAPD variation (see Stewart and Excoffier, 1996
). Both HOMOVA and AMOVA analyses were performed using the WINAMOVA 1.5 program (available from L. Excoffier, Genetics and Biometry Laboratory, University of Geneva, Switzerland).
The WINAMOVA program extracts analogs of F statistics (so-called 
statistics). Then, and for comparative purposes, we used F statistics as a second approach to study population genetics in D. minor. FST was estimated from those RAPD bands that fulfilled Lynch and Milligan's assumptions for the analysis of dominant markers using RAPDFST from RAPDPLOT (the corresponding statistical methods and equations are given in Apostol et al., 1996
). This program estimates FST according to Lynch and Milligan (1994)
, Wright (1951)
, and as 
(Weir and Cockerham, 1984
). When appropriate, a subsequent chi-square value was calculated to determine whether these estimates of FST varied from zero (significant population differentiation). Pairwise FST between populations were also calculated using RAPDDIST from RAPDPLOT, applying the Lynch and Milligan (1994)
correction when estimating allele frequencies. The resulting distance matrices were compared to test their correlation with the ST distance matrices from AMOVAs, as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Relationship among RAPD phenotypes
First, the 141 RAPD phenotypes (the Baleares141 data set) were analyzed using the Apostol coefficient. The unrooted NJ dendrogram based on these data clustered the 162 individuals within distinct groups according to their geographical origin. Three major clusters were formed, each grouping individuals from Mallorca, Menorca, and Cabrera, respectively, and all populations formed subclusters of their own. The bootstrapped (100 replications) dendrogram confirmed the reliability of the differences among islands and the grouping of individuals within their own population (data not shown). This also holds true for the NJ dendrograms performed separately with the 86 individuals from Mallorca, the 57 from Menorca, and the 19 from Cabrera (the Mallorca141, Menorca141, and Cabrera141 data sets), although the individual D9 clustered apart of the other 13 samples from Deià population in Mallorca. It is worth noting that the tree from the Mallorca141 data set revealed a clear differentiation between individuals from var. minor and var. palaui populations. In contrast, the tree from the Menorca141 data set showed that individuals could not be separated as expected from their morphological characters (data not shown).
Individual plants were also separated by their area location using PCO of the Baleares141 data set. The first three coordinate axes accounted for 25.0, 10.5, and 8.4% of the total variance, respectively, and identify three main groups corresponding roughly to Mallorca, Menorca, and Cabrera populations (data not shown). The results showed similar clustering to the NJ analysis and confirmed the grouping of individuals from var. palaui populations in Mallorca, probably due to the more isolated location of these populations (Fig. 1).
When the NJ analysis was restricted to those bands whose observed frequency was <1 – 3/(N) among the three islands (Baleares39 data set), there was no grouping of the individuals into three different geographic regions (islands). Although some individuals from the same population appeared in the same subcluster, the general trend was for individuals to be distributed throughout several subclusters, along with those from other populations (data not shown). This lack of concordance between individuals and their geographical location was also observed when NJ cluster analyses were performed using the Mallorca52, Menorca35, and Cabrera22 data sets. In the three cases, RAPD phenotypes were intermingled in different parts of the dendrograms (data not shown). Trees from Baleares39, Mallorca52, and Menorca35 data sets also failed to differentiate individuals from var. palaui and var. minor populations.
Divergence at the population level
First, interpopulation distances were calculated using the Apostol coefficient based on Baleares141 data set (data not shown). The mean distance among the 17 populations was 0.1799 ± 0.0327 (0.1095–0.2553). The NJ dendrogram obtained from this matrix (Fig. 3A) revealed a good grouping of the D. minor populations into three main clusters, each one corresponding to their respective island location (Mallorca, Menorca, and Cabrera; see Fig. 1). Note that within the main clusters, var. palaui populations formed a distinctive subcluster only in Mallorca, corroborating results from PCO analysis. Interpopulation genetic distances were lower when the Baleares39 data set was employed (mean distance 0.0655 ± 0.0467) and the corresponding NJ dendrogram did not show any relation between populations and their geographic region (data not shown).
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The investigated var. palaui and var. minor populations (Table 1) were genetically very similar. The average Nei's unbiased genetic identity among the 17 populations was 0.8524 ± 0.0388 or 0.9904 ± 0.0053 when either Baleares141 or Baleares39 data sets were employed. Similar values were obtained when the Nei identity was averaged within the 11 var. minor populations or the 6 var. palaui populations.
Population genetic structure
The analyses were exclusively based on those RAPD bands (39 for the 17 sampled populations or 52, 35, and 22 for the 8, 7, and 2 sampled populations from Mallorca, Menorca, and Cabrera islands, respectively) that fulfilled the Lynch and Milligan (1994)
condition for obtaining unbiased estimates of population-genetic parameters.
AMOVA analysis
AMOVA analysis from the phenotypic distance matrix (DP) for the 162 individuals (the Baleares39 data set) permitted a partitioning of the overall variation into three levels (Table 5). Although most of this variation was found within populations (71.4%), there was also evidence for a significant phenotypic structure of the populations (ST = 0.286, P < 0.001). The remaining phenotypic diversity was distributed between islands (2.11%) and between populations (26.47%). Similar results were found for the Mallorca and Menorca analysis, but the Cabrera analysis showed a different partitioning (42.94% among populations and 57.06% among individuals within populations). The bias of these results may be related to the low number of polymorphic bands (six) for the individuals of Cabrera in the Baleares39 data set.
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ST = 0.071, P < 0.001). For the within-region analyses, most of the genetic variation resided between individuals within populations, but again a significant population structure was evident in each island, especially in Cabrera (ST = 0.176 vs. ST = 0.062 or 0.025 in Mallorca and Menorca, respectively). This variance partitioning was corroborated when AMOVAs were performed with DG matrices derived from the Mallorca52, Menorca35, and Cabrera22 data sets (Table 6). Note however, that the three within-region analyses showed a lower among-population differentiation (ST = 0.046, 0.023, and 0.072 for Mallorca, Menorca, and Cabrera, respectively).
Irrespective of the data set used (Baleares39, Mallorca52, or Cabrera22), all pairwise ST values between Mallorca and Cabrera populations derived from genotypic AMOVAs were significant. Pairwise ST values for Menorca populations were also significant, except comparisons among ML-MS, ML-MB, ML-MX, ML-MT, and MB-MT in data set Baleares39, and ML-MB and MB-MT in Menorca35.
It is worth noting that nested AMOVAs (Table 5) showed a nonsignificant partitioning of genetic differentiation among islands (CT = 0.211, P = 0.104; and CT = 0.016, P = 0.082 for both phenotypic and genotypic analyses, respectively).
Except for the two Cabrera populations and the phenotypic HOMOVA from the Menorca35 data set, the computation of the Barlett test for the homogeneity of variance indicated significant levels of RAPD variation (both phenotypic and genotypic) among populations as a whole (Tables 5, 6). Of the 589 pairwise Barlett tests of homogeneity of population variation, 554 tests indicated significant differences between populations (data not shown).
The AMOVA procedure was also implemented to study the relationships between the two infraspecific D. minor taxa (var. minor and var. palaui). The AMOVAs performed with data sets Baleares141 and Baleares39 showed no significant differences between groups (CT = 0.002, P = 0.3247; CT = –0.001, P = 0.3076, respectively), when the 17 populations were subdivided according to their corresponding infraspecific taxon (see Table 1). These results were corroborated when the Menorca141 (CT = –0.021, P = 0.7103), Menorca35 (CT = –0.01, P = 0.7612), and Mallorca52 (CT = 0.0, P = 0.3077) data sets were analyzed. Nevertheless, the AMOVA performed with the Mallorca141 data set showed a significant variance among var. minor and var. palaui populations (CT = 0.079, P = 0.005), which was in agreement with results from the NJ dendrogram of this data set.
Estimation of FST
Estimates of FST and averaged over the 39 loci for the 162 individuals and those derived from separate analyses performed for each island with the appropriate set of bands are shown in Table 7. The correlations across loci between all the values were high (>91%, P = 0.0000). Irrespective of the method employed, the estimated FST and were significantly different from zero, indicating among-population genetic differentiation. Interpopulation distances obtained using ST from AMOVA and Lynch and Milligan's FST or showed a significant positive correlation (r > 0.94, P = 0.0002). This correlation was slightly lower with Wright's FST pairwise distances (r = 0.80, P = 0.02). All these results corroborated those above described in the extension of the AMOVA analysis (Stewart and Excoffier, 1996
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), the number of RAPD markers used in our study seems to be enough to produce reliable estimates of genetic structure. Furthermore, the high level of conformity among the different statistical approaches used for RAPD data analysis demonstrates the suitability of these molecular markers to accurately resolve the population structure of D. minor.
Both phenotypic and genotypic nested AMOVA showed a nonsignificant partitioning of genetic diversity among groups (islands). Since the current distribution of D. minor is relatively recent (Cardona and Contandriopoulos, 1979
), the absence of among-island differentiation may only be a reflection of historical gene flow between D. minor populations when the Gymnesian Islands constituted a single landmass. Within species, genetic exchange rather than historical relationships (e.g., persistent ancestral polymorphisms) has traditionally been emphasized as the determinant of genetic structure. Nevertheless, shared common ancestry and similar selective regimes could also account for the observed genetic cohesion of plants (Schaal et al., 1998
). Thus, in many groups, genetic exchange across the species range is severely restricted, either by wide geographical distribution of populations or by limited pollen and seed dispersal. In these cases, historical events such as range expansion, range fragmentation, and population bottlenecks will be strong determinants of population genetic structure (Schaal et al., 1998
). Thus, the observed genetic similarity between D. minor populations belonging to different islands probably owes more to recent common ancestry than to any ongoing process of genetic exchange. Nevertheless, further studies based on cpDNA markers can help discriminate between limited contemporary gene flow and patterns caused by ancestral polymorphism (Avise, 1994
; Schaal et al., 1998
).
Our RAPD-based AMOVA studies show that most genetic variation in D. minor is distributed within populations rather than between them, indicating a relatively restricted population differentiation as expected in outcrossing species. Such a pattern of population genetic structure has been previously reported for the mainland congener species D. obscura (Nebauer, Del Castillo Agudo, and Segura, 1999
) and many other outcrossing species (Hamrick and Godt, 1996
). Furthermore, the percentages of within-population variability found in our study (92.87% for the among-islands analysis and 82.45–97.72% for the within-island analyses) were either similar or somewhat higher than those reported in other outcrossing plant species using RAPD-based AMOVA analysis (see Tables 5 and 6 from Busell, 1999
, and Bartish, Jeppsson and Nybom, 1999
, respectively).
Our estimates of the fixation indices (ST or FST) also demonstrate that some structure can be discerned among D. minor populations. Genotypic AMOVA analyses resulted in ST estimates lower than those given by RADPLOT. Note, however, that all ST or FST pairwise distance matrices showed a significant positive correlation. Our estimates of ST and FST are close to those given in the literature for analysis of population structure in mixed and outcrossing species: 0.1–0.24 (Loveless and Hamrick, 1984
); 0.099–0.216 (Hamrick and Godt, 1990
); and 0.03–0.31 (Heywood, 1991
). The relatively low FST values corroborate that most of the genetic diversity resides within D. minor populations and that these populations have experienced gene flow in recent history or diverged a relatively short time ago. Except for the interpopulation genetic distances derived from the Mallorca141 data set, there was no significant correlation between the genetic and the actual geographical distances among populations. This is an indication that isolation by distance is not the process accounting for the distribution of genetic variation among populations within the Mallorca or Menorca islands.
The NJ and PCO analyses revealed a similar, overall geographical structuring of the RAPD variation in D. minor when the 141 markers were used. These analyses, however, seem to overestimate the relatedness among individuals since all analyses based on RAPD markers that fulfilled the Lynch and Milligan (1994)
criterion failed to follow a consistently geographical pattern either among islands or within each of the islands. The lack of separation in the dendrograms reflects a weak genetic differentiation among populations, corroborating results from AMOVA and traditional F statistics.
The most accepted systematic positioning of D. minor takes into account the plasticity of the species and considers two infraspecific taxa, varieties minor and palaui, in virtue of their differences in pubescence (Hinz, 1987
). According to this character, six out the 17 sampled populations (see Table 1) would belong to D. minor var. palaui. Our RAPD data do not support, however, this taxonomic differentiation of D. minor. First of all, our cluster analysis from the data set that fulfilled the Lynch and Milligan criterion does not show a closer relationships among var. palaui populations than among any other of the sampled populations; thus, the different RAPD phenotypes of var. palaui and var. minor plants were intermingled in different parts of the NJ and PCO analyses. Also, genetic differences among populations are small. Crawford (1989)
stated that the mean identity for populations belonging to the same taxon is often above 0.90. We calculated population mean identities of 0.8201 ± 0.0327 and 0.9345 ± 0.0467 (for the Baleares141 and Baleares39 data sets, respectively) using the Apostol coefficient (Apostol et al., 1993
). Note, however, that the population mean identities derived from Nei's unbiased distance (Nei, 1978
) were higher (0.8524 ± 0.0388 and 0.9904 ± 0.0053 for the Baleares141 and Baleares39 data sets, respectively). The Apostol coefficient measures the similarity of pairs of individuals by examining both the shared presence and the shared absence of bands and then provides a clearer separation than the Nei distance when very related individuals are compared (Apostol et al., 1993
). Finally, the AMOVA procedure, implemented as an alternative approach to study the relationships between var. minor and var. palaui populations, demonstrates that there were no significant differences between groups when the populations were subdivided according to their corresponding infraspecific taxon. The reliability of this result is endorsed by the accuracy of RAPD-based AMOVA to detect dissimilarities among several plant species, including Digitalis (see Nebauer, Del Castillo Agudo, and Segura, 2000
, and references therein). Corroborating all these findings, analyses of the sequences of the nuclear rDNA internal transcribed spacers (ITS region) did not discriminate var. minor and var. palaui (J. A. Rosselló, personal communication).
In conclusion, our results indicate that RAPDs are sufficiently informative and powerful to assess genetic variability in D. minor. Estimates of genetic variation reported herein provide a basis for the in situ conservation and exploitation of genetic resources in this species. With knowledge of the available genetic structure, an appropriate strategy for sampling and propagation of D. minor may be easily formulated when ex situ conservation is required. Also, information on the spatial structure of natural populations of D. minor provides important insights into the colonization history, isolation, and diversification of this species.
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FOOTNOTES |
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5 Author for reprint requests: Departamento de Biología Vegetal, Facultad de Farmacia, Universidad de Valencia, Avda. Vicent A. Estellés s/n. Burjassot 46100, Valencia, Spain (phone: +34 963 864 922; FAX: + 34 963 864 926; e-mail: juan.segura{at}uv.es
).
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LITERATURE CITED |
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, D. minor var. minor populations; 
, D. minor var. palaui populations
