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Systematics and Phytogeography |
2 Departamento de Biología Vegetal II, Facultad de Farmacia, Universidad Complutense 28040 Madrid, Spain 3 Département de Biologie, Faculté des Sciences, Université Mohammed V-Agdal, Rabat, Morocco
Received for publication 16 August 2007. Accepted for publication 13 November 2007.
ABSTRACT
The tribe Genisteae includes genera of great ecological importance in Mediterranean countries because they are dominant elements of many plant communities. Genetic variation and diversification patterns in Stauracanthus (Genisteae) provide information relevant for the study of the processes of diversification in relation to the environmental history of the western Mediterranean. Nineteen populations of S. boivinii and S. genistoides were assessed by 11 chloroplast microsatellite markers, revealing 44 haplotypes. Both species had different haplotypes and contrasting patterns of karyological, morphological, and genetic variation. In the minimum spanning tree of the haplotypes, AMOVA analysis, and nested clade analysis, S. boivinii had high levels of differentiation and restricted gene flow among populations. Allopatric differentiation occurred between the Moroccan and Iberian populations of S. genistoides, although S. genistoides subsp. spectabilis and subsp. vicentinus had high levels of differentiation among populations (FST), whereas S. genistoides subsp. genistoides had a low FST. Genetic patterns are discussed in relation to the Messinian salinity crisis (MSC): hard conditions drove plants to refuge habitats along the Atlantic coast and higher altitude areas in the Moroccan mountains (S. genistoides subsp. spectabilis and S. boivinii). After the MSC, S. boivinii underwent polyploidization and expansion, whereas S. genistoides expanded and continued diversifying into S. genistoides subspp. genistoides and vicentinus.
Key Words: chloroplast microsatellites • cpSSRs • Leguminosae • Messinian salinity crisis • northern Morocco • phylogeography • southern Spain • Stauracanthus
The Genisteae (Adans.) Benth. is a tribe of Fabaceae, basically circum-Mediterranean, with two maxima of diversity located in the west (Morocco–Spain and surrounding countries) and in the east (Balkanic region). The tribe also extends to the Canary Islands, northern Europe (Cristofolini, 1991
), and America (Lupinus L.). The Genisteae have great ecological importance in the western Mediterranean countries not only for the high species diversity but also for colonization of degraded forests and deforested areas (López González, 2001) and dominating many plant communities that characterize the landscape (e.g., Spanish Central System). Different palaeoclimatic and geological processes have been involved in the evolution of Mediterranean plant communities, including those characterized by Genisteae, i.e., the isolation of microplates resulting from Tertiary tectonic movements; the Messinian salinity crisis (MSC) at the Miocene–Pliocene boundary 5.96–5.33 million years ago (Ma) (Hsü et al., 1977; Krijgsman et al., 1999), the establishment of a Mediterranean climate type at Late Pliocene 3.2–2.8 Ma (Suc, 1984), and sea level changes associated with Pleistocene glaciations (Hewitt, 2000). For the Iberian and northern African plants, an important event affecting the present distribution and vicarism of many plants was the opening of the Strait of Gibraltar in the Early Pliocene (Loget and Van Den Driessche, 2006), which constituted from there on an important barrier of gene flow, at least for plants with short-distance dispersal mechanisms (Valdés, 1991). Since the end of the Miocene, two other historical factors have been decisive in the shaping of plant species distribution and endemism around the Mediterranean: the onset of a summer drought and, more recently, the development of human activities (Thompson, 2005).
The tribe is complex cytologically in having high chromosome numbers, which suggests that recent polyploidy and aneuploidy have played an enormous role in the evolution of the group (Goldblatt, 1981). The present distribution of Genisteae has been explained by assuming the cladogenesis above the generic level was achieved before the end of the Paleogene, whereas diversification within the genera and sections should be largely due to later evolution, after the aridity crisis during the Late Miocene (Cristofolini, 1991). However, few detailed studies have measured the genetic diversification of Genisteae populations on both sides of the Strait of Gibraltar. This information is relevant for understanding the diversification of western Genisteae.
Stauracanthus Link is a small genus of Genisteae, restricted to the southwest of the Iberian Peninsula and the northwest of Africa: Morocco and Algeria (Sampaio, 1924; Guinea and Webb, 1968a). These plants are small shrubs that produce numerous flowers visited by Hymenoptera (Apoidea; Herrera, 1988). The carunculate seeds are released violently from pods and then may be gathered by foraging ants (Herrera, 1999). Seeds germinate easily after the hard seed coat is broken.
Stauracanthus comprises two species: S. genistoides (Brot.) Samp. and S. boivinii (Webb) Samp. [= Nepa boivinii (Webb) Webb]. Stauracanthus genistoides grows on littoral and inland sandy soils in the southwest of the Iberian Peninsula (Portugal and Spain), and on the Atlantic coast of Morocco (Fig. 1). Morphological differences within S. genistoides led to the recognition of three subspecies (Rothmaler, 1941): S. genistoides subsp. genistoides, throughout the range of the species in the Iberian Peninsula, extending inland into sandy areas around rivers in Portugal; S. genistoides subsp. vicentinus (Daveau ex Cout.) Rothm., restricted to sandy littoral sectors of western Portugal; and S. genistoides subsp. spectabilis (Webb) Rothm, with two disjunct areas in southwest Portugal and western Morocco. Morphological characters used for differentiating the three subspecies are size and shape of the bracteoles, standard petal indumentum, and calyx size. However, intermediate states are often found, which blur the limits of the taxa. Stauracanthus genistoides is karyologically homogeneous, with 2n = 48 chromosomes (De Castro, 1941, 1943; Cubas, 1987; Tahiri et al., 2004).
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). Different chromosome numbers have been reported for this species (2n = 48, 96, ± 128, and 144 chromosomes; De Castro, 1941; Cubas, 1987; Talavera and Arista, 1995; Tahiri et al., 2005, 2006). Hybridization between populations with different chromosome complements has not been reported. Based on minor morphological differences such as spine size and branching pattern, several infraspecific taxa (e.g., S. boivinii f. escayracii (Webb) Samp.; S. boivinii f. luridus (Webb) Samp.; S. boivinii f. vaillantii (Webb) Samp.; S. boivinii f. webbianus (Coss.) Samp.) were described by Webb, 1852, Sampaio, 1924, and Maire, 1987, among others. However, morphological variation between specimens can be found even at the scale of single populations. Besides, no relationships between morphology, chromosome number, geography, or ecology have been established. Thus, we follow the taxonomy adopted in modern floras, such as Flora Iberica (Paiva and Coutinho, 1999), which includes all these forms as synonyms under S. boivinii.
Stauracanthus is closely related to Ulex L., a genus that also grows in Morocco and Algeria but extends northward along western Europe. Both genera have reduced leaves (small scale or spine-tipped phyllodes in the mature plant), short spines, and a two-lipped calyx. However, Stauracanthus has a short tube at the base of the calyx (at least in bud) and the legume is conspicuously exserted from the calyx, while Ulex has a calyx divided to the base into two lips and scarcely exserted legumes (Sampaio, 1924; Guinea and Webb, 1968a, b). Serologic and phylogenetic analyses show that Stauracanthus and Ulex form a monophyletic group (Feoli-Chiapella and Cristofolini, 1981; Aïnouche et al., 2003; Pardo et al., 2004) closely related to a predominantly north African group that includes species of Genista section Cephalospartum (Gibbs, 1966). Ulex and Stauracanthus are good examples of cytological diversification within Genisteae: both genera form euploid series, based on n = 12 in Stauracanthus and on n = 16 in Ulex (De Castro, 1941; Cubas, 1987; Misset, 1990; Misset and Gourret, 1996; Talavera and Arista, 1995; Tahiri et al., 2004, 2005, 2006, among others).
Chloroplast microsatellites (cpSSRs) are abundant polymorphic elements of the chloroplast genome that consist of tandem repetitions of mono-, di-, or tetranucleotide units. Polymorphisms on microsatellite size have been used to detect genetic diversity (Powell et al., 1995; Ennos et al., 1999; Provan et al., 2001) and differentiation and spatial structure among and within populations (Petit et al., 1997; Gómez et al., 2003; Vettori et al., 2004; Walter and Epperson, 2004; Robledo-Arnuncio et al., 2005; Terrab et al., 2006). These markers reveal polymorphisms in Ulex population growing on both sides of the Betic-Rif arc and allow exploration of the genetic relationships and divergence processes of closely related populations (Cubas et al., 2005). Conservation of microsatellites among closely related genera and species has been found among other Fabaceae (Dayanandan et al., 1997; Peakall et al., 1998). Therefore, to explore the relationships and genetic divergence between the Stauracanthus populations on both sides of the Strait of Gibraltar (southwestern Iberia and northern Africa), we investigated the utility of the cpSSR markers used for Ulex in the study of variability in Stauracanthus.
Stauracanthus is a good model for studying how the complex environmental history of the western part of the Mediterranean Basin (geographical and climatic changes) has affected the processes of diversification in Genisteae. Stauracanthus (1) forms a very small polyploid series whose evolutionary history is not yet well established, (2) is restricted geographically to both sides of the Strait of Gibraltar, and (3) grows in habitats under heavy human pressure (e.g., Algarve, Portugal), which could be eroding the genetic diversity of the populations. The two species of Stauracanthus have different geographical, karyological, and morphological patterns. Stauracanthus boivinii forms a euploid series without clearly associated morphological variation, while S. genistoides has morphological variation without karyological differentiation. Besides, S. genistoides subsp. spectabilis has a contemporary disjunct Moroccan–Portuguese distribution. Main issues to be addressed are whether (1) morphological differentiation in S. genistoides and karyological differentiation in S. boivinii correlate with genetic variability, (2) gene flow via seeds can be detected between African and Iberian populations on both species, (3) Iberian S. genistoides populations have diverged through population contiguous range expansion, long distance colonization, or population fragmentation, leading to a strong decrease in the effective population sizes. Plausible historical factors for reduction of the area and fragmentation are the harsh climatic conditions during the MSC and the posterior isolation of both sides after the opening of the Strait of Gibraltar.
This work continues the search for molecular markers that could be used to investigate evolutionary patterns in Ulex, Stauracanthus, and other related genera in the Genisteae tribe. This study aims to (1) investigate the usefulness of cpSSRs for assessing the genetic variability at the population level in the genus Stauracanthus, (2) detect intraspecific polymorphisms and measure genetic variability and gene flow between the populations of Stauracanthus boivinii and S. genistoides from the Iberian Peninsula and northern Africa, (3) discuss the relationships of the populations of Stauracanthu s on both sides of the Gibraltar Strait, particularly the Moroccan–Iberian disjunct populations of S. genistoides subsp. spectabilis, and (4) compare the history of S. boivinii and S. genistoides in relation to the geological and historical factors potentially influencing contemporary patterns of variation in Stauracanthus.
MATERIALS AND METHODS
Samples
Nineteen natural populations (150 individuals) of Stauracanthus were analyzed at 11 cpSSR loci. The samples were collected in the field at different altitudes and from different substrates. The names, provenance, ecology, and voucher number of the samples are indicated in Table 1 and Fig. 1. Based on morphological criteria, they were ascribed to: Stauracanthus genistoides subsp. genistoides, subsp. spectabilis, or subsp. vicentinus (Rothmaler, 1941), and S. boivinii.
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. Another cpSSR, named 108 and previously used for Ulex samples, was used. This microsatellite region is included in the trnL-trnF IGS region (cpDNA) and was amplified using primer e (Taberlet et al., 1991) as forward and primer 108 as reverse (Cubas et al., 2005).
Simple PCR amplifications for individual loci were initially run, and then multiplex reactions were performed combining two (ccmp1 and ccmp6) or three primer pairs (ccmp2, ccmp4, and ccmp7; ccmp3, ccmp5, and ccmp10). The microsatellite 108 of the trnL-trnF IGS region was amplified separately. PCR amplifications and sizing were performed following the procedure of Cubas et al. 2005. The amplified products were sized at the Unidad de Genómica (Parque Científico de Madrid-Universidad Complutense, Madrid) with an automated laser fluorescence DNA sequencer (Applied Biosystem 3730 DNA analyzer; Applied Biosystems, Foster City, California). Fragment sizes expressed in base pairs (bp) were calculated using the program GeneMapper TM, version 3.5 (Applied Biosystems) by comparison to an internal molecular marker standard. Several runs of sizing at different concentration and from different PCR runs were performed to confirm the obtained fragment length.
Statistical analysis
Because the chloroplast genome does not recombine, a unique combination of size variants (alleles) across the microsatellite regions (loci) was defined as a different haplotype. Differences in allele size are due to an increase or decrease in the number of repeats, except in some cases where differences are also related to indels in the flanking regions. We then calculated statistic parameters and analyzed the molecular diversity of the samples according to the infinite allele model (IAM) that only takes into account the number of different alleles and does not assume specific microsatellite mutation mechanism. Haplotype variation within populations was calculated by estimating the number of polymorphic loci (Pn), the total number of haplotypes (No), the effective number of haplotypes (Ne), and the unbiased haplotype diversity (He) (Nei, 1987). Genetic distance among individuals within populations was calculated based on the IAM model (average gene diversity over loci, 
n; Nei, 1987). A minimum spanning tree (MST, Fig. 2A) was computed from the matrix of pairwise distances between all pair of haplotypes using the number of different alleles between two haplotypes.
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), and the significance of the fixation indices was computed using nonparametric permutation procedures with 1000 permutations, implemented and described in Arlequin version 3.1 (Excoffier et al., 2005). Distances between haplotypes were calculated from the number of different alleles (IAM model). When genetic structures indices are estimated, these distances lead the fixation indices of genetic differentiation among populations FST (Weir and Cockerham, 1984).
The AMOVA analysis was supplemented by a nested clade analysis (NCA) to test the null hypothesis of no geographical association of genetic variation. We built a nested clade design in which haplotypes (0-step clades) are grouped into 1-step clades (haplotypes differing by one change), and these into 2-steps clades (haplotypes differing by two changes), and so on, until the next level of nesting includes the whole tree. The program GEODIS 2.5 (Posada et al., 2000, 2006) was used to test for significant association between haplotypes and their geographical distribution and to separate population structure from population history (Templeton et al., 1995; Posada et al., 2000, 2006). The permutational 
2 statistic was calculated by randomly permuting the lower level clade categories within the nesting clade vs. geographical locality 1000 times. Two major statistics were calculated: the clade distance (Dc), a measure of how geographically spread are the individuals that bear haplotypes of this clade; and the nested clade distance (Dn), a measure of how far individual of a clade are from all individuals that bear haplotypes of higher-level nesting category (Templeton et al., 1995). Clade contributions to the estimated distances are weighted by their relative abundance at each location. These measures of geographical distribution were used to infer historical processes (Templeton, 2004), using the upgraded biological inference key indicated in GEODIS 2.5 (Posada et al., 2006).
An unrooted phylogram was constructed by neighbor joining with the program PAUP* version 4.0b10 for Macintosh (Swofford, 2002), based on the matrix of pairwise genetic distances between populations calculated by the number of different alleles (Nei, 1987).
RESULTS
Morphological and taxonomic considerations
Stauracanthus genistoides and S. boivinii differ by the branching pattern of young twigs (mostly opposite in S. genistoides, alternate in S. boivinii), the arrangement of the spines (opposite to subopposite in S. genistoides, subopposite to alternate in S. boivinii), the length of the standard petal (about equal to the calyx in S. genistoides, much longer in S. boivinii), and the shape and size of the legumes (long and linear-oblong in S. genistoides, short and broadly ovoid to linear-oblong in S. boivinii).
Stauracanthus boivinii samples vary slightly in morphology but are easily identified. Chromosome data available for the studied populations or for plants collected in close proximity revealed three different ploidy levels as indicated in Fig. 2A. The distribution of the known cytotypes of S. boivinii follows a northward pattern, i.e., plants with 2n = 48 have only been detected in the Middle Atlas of Morocco (Talavera and Arista, 1995; Tahiri et al., 2005), plants with 2n = 96 grow in northern Morocco (around Tangier; Tahiri et al., 2006), while plants with 2n = 96, ± 128, and 144 chromosomes have been reported from the Iberian Peninsula (De Castro, 1941; Cubas, 1987).
The taxonomy of S. genistoides is still controversial, and different infraspecific taxa have been proposed (Sampaio, 1924; Rothmaler, 1941; Guinea and Webb, 1968a; Díaz et al., 1990). The most conspicuous morphological features to distinguish between infraspecific taxa are the shape and size of the bracteoles, and the size of the calyx. Other characters that have been used, such as the indumentum of the standard petal, are more difficult to diagnose. Plants at the extremes of the range of variation are easily identified as S. genistoides subsp. genistoides (bracteoles minute, less than 1 mm wide, linear to linear-lanceolate; calyx 9–11 mm long) and S. genistoides subsp. spectabilis (bracteoles more than 3 mm wide, more or less orbicular, and calyx longer than 11 mm). However, bracteole morphology and calyx size are often variable, even within populations. Plants with morphologies overlapping the range of S. genistoides subsp. genistoides and S. genistoides subsp. spectabilis are not uncommon, which blurs the limits of taxa. These "intermediate" plants have been included in (1) a third subspecies, S. genistoides subsp. vicentinus (Rothmaler, 1941), (2) S. genistoides subsp. genistoides (Guinea and Webb, 1968a), (3) S. genistoides subsp. spectabilis (Paiva and Coutinho, 1999), or (4) as a subspecies of S. spectabilis Webb [S. spectabilis subsp. vicentinus (Daveau ex Cout.) T. E. Díaz et al.; Díaz et al., 1990]. We named the intermediate plants according to Rothmaler, 1941, which has no further taxonomic implications. An example of the morphological variability is provided by populations SVGR and SVBA (see Fig. 1 for location), which show a wide range of individual variation in the diagnostic features.
Characterization and levels of polymorphism shown by cpSSRs
Nine of the 11 chloroplast loci initially assayed were successfully amplified. Loci ccmp2, ccmp3, ccmp4, ccmp6, ccmp7, ccmp10, and 108 were polymorphic, giving 38 different alleles (Table 2) among 150 individuals from 19 populations. Loci ccmp8 and ccmp9 failed in all the assays and, consequently, were discarded. Locus ccmp5 amplified successfully but gave a multiband pattern and so was excluded from the analysis. Loci ccmp1 was monomorphic in all the samples. The range in size of the alleles is similar to that in Ulex baeticus group (Cubas et al., 2005), and differences are due to expansion or contraction of the nucleotide repeat region. Two exceptions were found for loci ccmp2 and 7, where size differences reflected repeat length variation and indels in flanking regions. Locus ccmp2 is 12 bp longer in S. genistoides than in Ulex because of a 12-nucleotide insertion. This insertion is present in S. boivini once or thrice (population SBBA). Locus ccmp7 is shorter in S. boivinii than in S. genistoides and Ulex because of a 17-nucleotide deletion.
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0.041, seven or more individuals). Fourteen haplotypes were found in seven populations of S. boivinii; none were shared between populations. Three populations are monomorphic for all loci (SBBB, SBMO, and SBRU; haplotypes D, H, and K, respectively). The five populations of S. genistoides subsp. genistoides have 16 haplotypes. Population SGNA has only one haplotype (AB) whereas the others have 3–5 haplotypes (SGSJ; haplotypes AF, AG, AH, AI, and AJ). There are nine haplotypes in the four populations of subsp. spectabilis. Population SPSI has only one haplotype (AP), while SPBR has one private haplotype (AK), one (R) shared by S. genistoides subsp. vicentinus (SVGR), and two others (AB, AF) shared by S. genistoides subsp. genistoides populations (SGNA and SGSJ, respectively). The three populations of S. genistoides subsp. vicentinus have eight haplotypes, all population-specific, except P (shared by SVBA and SVGR) and R (shared by SVGR and SPBR).
Table 3 shows the estimates of genetic variation based on the seven cpSSR loci in the 19 studied populations. Haplotype diversity He has a high range of variation in all the taxa: 0.0–0.61 in S. boivinii, 0.0–0.84 in S. genistoides subsp. genistoides, 0.0–0.75 in S. genistoides subsp. spectabilis, and 0.25–0.75 in subsp. vicentinus. The values of average gene diversity over loci (n) are higher in S. genistoides subsp. genistoides populations than in all the other taxa. The overall genetic divergence among populations (FST) also differs among the taxa. Stauracanthus boivinii, S. genistoides subsp. spectabilis, and S. genistoides subsp. vicentinus have high values (0.89, 0.89, and 0.73, respectively), while S. genistoides subsp. genistoides has a lower value (0.33).
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The AMOVA analysis, using as distance the number of different alleles, showed differences in the apportionment of genetic diversity (Table 4). When samples are grouped into two species (S. boivini and S. genistoides), the analysis showed that 33% of the variation is due to differences between groups and that 48% is attributable to differences among populations within groups. At the specific level, most of the variation detected is due to differences among populations both in S. boivinii (89%), and S. genistoides (64%). However, the apportionment of the genetic diversity within S. genistoides is not homogeneous. When S. genistoides samples are grouped into subspecies, taxonomic ascription accounts for little (negative values) of the total genetic variation, whereas 66% is attributable to among populations differentiation. Differentiation among populations accounts for 33% in S. genistoides subsp. genistoides, 73% in subsp. vicentinus, and 89% in subsp. spectabilis.
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). This lack of genetic and geographical variation is the case for most of the lower level clades of S. boivinii, for which only clade 1–2 includes different haplotypes found in separate areas. The case contrasts with the genetic and geographic variation at lower level clades in S. genistoides, especially in clades 1–10 and 1–14. Interestingly, haplotypes of S. genistoides subsp. genistoides are at the tips of both clades, linked to different haplotypes of S. genistoides subsp. spectabilis.
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). We followed a similar 1, 2, 3, 5, 6, 7, YES path for nesting clades 1–10, 1–14, 2–4, 2–5, and 3–1, and the path 1, 19, NO for clades 1–2 and 3–2. The S. boivinii clades and haplotypes nested in clade 3–1 approach the expected pattern for a population with restricted gene flow/dispersal but with some long distance dispersal. The distribution of the S. genistoides haplotypes at higher-level nesting (clade 3–2) shows allopatric fragmentation, which corresponds with the disjunction of the Moroccan–Iberian haplotypes. Lower level clades which include the Iberian populations (clade 2–5, 1–10, and 1–14) show restricted gene flow/dispersal but with some long distance dispersal. In the case of Stauracanthus, long-distance dispersal via seed is not very plausible. Alternatively, the shared haplotypes could reflect past ancestral relationships and/or reticulate evolution that NCA fails to distinguish statistically, a situation that occurs in some cases (Knowles and Maddison, 2002). The unrooted neighbor joining (NJ) phylogram, based on pairwise comparison of population samples using the number of different alleles as a measure of genetic distance (Fig. 3), depicted several groups. Stauracanthus boivinii populations stand at one end of the tree, separated by very short internal branches and long terminal ones. This pattern indicates an old, shared ancestry and a posterior high differentiation among populations. The populations of S. genistoides subsp. spectabilis diverged from very close nodes by long branches, except for population SPBR that connected by a very short branch to a genistoides population. Populations of S. genistoides subspp. genistoides and vicentinus are interspersed in the tree: vicentinus populations had medium to long branches, and genistoides populations had medium to shorter ones. Geographic distribution and subspecific ascription seemed not to form coherent population clades, suggesting shared ancestry and more extensive chloroplast exchange among these populations than in the case of S. genistoides subsp. spectabilis. Interestingly, one population of S. genistoides subsp. vicentinus (SVSA) is linked to SPFU (subsp. spectabilis), and another (SPBR; subsp. spectabilis) is connected by a very short branch to SGNA (subsp. genistoides).
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Genetic divergence of Stauracanthus populations
Similar to the case of Ulex, the studied cpSSR markers have proved useful for detecting genetic within-population variation in Stauracanthus. In most populations, 2–5 haplotypes have been detected, and only five populations are monomorphic for all the loci. Microsatellites reveal a high level of genetic differentiation at the population level in Stauracanthus. However, our study shows different patterns of divergence in the Stauracanthus taxa.
Stauracanthus boivinii has a strong differentiation among populations, indicated by the high value of the structure parameter (FST = 0.89). The MST showed that (1) most of the haplotypes of the same population are connected, (2) the populations are linearly related through long branches, (3) the Iberian haplotypes are drawn from different Moroccan ones (and populations), and (4) haplotypes from populations with the higher ploidy levels are not linked together. The NCA results match the expected pattern under a restricted gene flow/dispersal model. Haplotypes recovered in diploid and tetraploid populations are interior clades and can be considered as the older ones. However, there is not enough karyological information to establish a clear relationship between haplotype variation and ploidy level. The NJ phylogram, based on the matrix of pairwise genetic distance, also shows long terminal branches separating the S. boivinii populations. These data suggest an old, shared ancestry between the African and European plants, followed by long-term genetic diversification of the populations associated with a restricted gene flow by seed dispersal. All these factors would account for the lack of correlation between genetic, morphological, and karyological variability.
In the case of S. genistoides, the MST depicted that (1) the haplotypes of the three subspecies are interconnected; (2) those of Portuguese subspecies spectabilis and vicentinus form the central part of the tree (interior clades), while the genistoides ones link at the ends (tip clades); (3) subsp. vicentinus shares haplotypes with subsp. genistoides and subsp. spectabilis; and (4) the private haplotypes of the Moroccan population SPMA separate by a long branch from the rest. The genetic diversity among populations of S. genistoides subspp. spectabilis and vicentinus has high FST values (0.89 and 0.73, respectively). In contrast, the FST in subsp. genistoides is significantly lower (0.33), and most of its haplotypes are connected by short branches. The NCA reflects a pattern that matches population structure and historical events associated with allopatric differentiation of spectabilis populations from Morocco and the Iberian Peninsula. The Iberian haplotypes reflect shared ancestry of all the populations and differentiation through a restricted gene flow/dispersal model. Some long-distance dispersal events are not precluded but seem less plausible. The population range expansion model (Templeton et al., 1995) was discarded by the significantly large averaged differences in distances between interior and tip clades in clade 2–5, which comprises the Iberian populations. The NJ phylogram also shows long terminal branches that separate the spectabilis populations, and shorter ones for the vicentinus and genistoides populations.
The combination of all these data suggests that S. genistoides subsp. spectabilis is an old taxon that might have had a more extended distribution on both sides of the Strait of Gibraltar. The taxon became later restricted to small populations in Morocco and Portugal, which eventually resulted in strong allopatric genetic divergence of the Iberian and Moroccan populations without morphological differentiation. On the other hand, S. genistoides subsp. genistoides is currently restricted to the Iberian Peninsula, and no data suggest that it might have had a more extensive area in the past. The MST indicates that its haplotypes are linked to haplotypes from different populations of S. genistoides subsp. spectabilis of Portugal. The tip position of the haplotypes (Posada et al., 2000), the lower FST values, and the shorter branches separating the populations in the NJ tree indicate that S. genistoides subsp. genistoides is younger and has diverged less than S. genistoides subsp. spectabilis. There is no conclusive evidence indicating whether separated residual populations of spectabilis differentiated into genistoides populations in the Iberian Peninsula, or whether an ancestral "genistoides" taxon (already separated from spectabilis) existed before. At present, populations of S. genistoides subsp. genistoides can be morphologically differentiated from those of subsp. spectabilis. However, the morphological intermediacy of subsp. vicentinus can be viewed as the result of introgressive processes between spectabilis populations and the genistoides. This idea is supported by the links of the haplotypes in the MST and the connections of the populations in the NJ tree.
A potential evolutionary scenario for Stauracanthus populations in Spain and Morocco
Stauracanthus is restricted to an area that has been considered one of the 10 hotspots of plant biodiversity in the Mediterranean Basin (Médail and Quézel, 1997). Stauracanthus is phylogenetically related to Ulex, a genus also present on both sides of the Strait of Gibraltar and highly differentiated in southern Iberia and northern Morocco. Like Stauracanthus, Ulex avoids continental climate but has a much wider distribution and reaches northern Europe (Guinea and Webb, 1968b). The genetic variation detected in this study suggests a plausible scenario for the differentiation of S. genistoides and S. boivinii, and for the present restricted distribution of Stauracanthus.
In Morocco, S. genistoides subsp. spectabilis grows in a very restricted area on the Atlantic coast, in one of most extended oak–cork (Quercus suber L.) forests of the world (Fôret de Mamora). This area is controlled by a Mediterranean climate, with temperatures in the range of 11–34°C and mean annual precipitation of 450–600 mm. The summer drought is buffered in the coastal areas by a strong and constant humidity (Aafi et al., 2005). Similarly, in Portugal S. genistoides subsp. spectabilis is restricted to the Atlantic coastal area around Cabo de Sines, where oceanic influence moderates the summer drought (Instituto de Meteorologia, 2005). The data suggest an old origin for these populations, which might have had a larger distribution that contracted because of ecological factors; according to this inference, the contraction was followed by allopatric differentiation that separates the Moroccan from the Portuguese populations. The large-scale desiccation of the Mediterranean basin during the Messinian (Late Miocene) (Hsü et al., 1973, 1977) could be a useful working hypothesis for explaining the present disjunct distribution, and its refugee presence in the western oceanic areas (Atlantic coast).
Before the opening of the Strait of Gibraltar, the Betics to the north and the Moroccan Rif to the south were part of a single orogenic system, which formed during the Alpine compressions in Early Miocene time (African–Iberian collision) (Doblas and Oyarzun, 1989). During this time, spectabilis populations may have been widespread in southern Iberia and northern Morocco, a single land mass during Lower Miocene times. The closure of the Betic and Rif gateways resulted in the Messinian salinity crisis (MSC) (Hsü et al., 1973, 1977). The MSC must have altered the climate and affected the vegetation of southern Iberia and northern Africa. Hot and dry conditions might have forced the populations of spectabilis on both sides to retreat to isolated spots in the westernmost sectors of Iberia and Morocco, where the influence of the Atlantic Ocean must have maintained milder climatic conditions. The MSC did not last more than 600 Ky (Krijgsman et al., 1999), and the opening of the Strait of Gibraltar in the Early Pliocene (Loget and Van den Driessche, 2006) allowed reconnection of the Mediterranean with the Atlantic and at least partial restoration of milder climatic conditions in the surroundings areas, probably a few degrees warmer and slightly more humid that at the present time (Thompson, 2005). However, the Strait of Gibraltar from there on constituted an important barrier for gene flow between the populations of both sides, at least for plants with short-distance dispersal mechanisms (Valdés, 1991). The isolation of the spectabilis populations on different sides of the Strait would have resulted in diminishing population size and increased genetic divergence between the Moroccan and Portuguese plants. On the other hand, when milder climatic conditions where restored in the Iberian Peninsula, other remaining genistoides populations would have rapidly began diversification by genetic divergence, extending inland and maintaining gene flow via seeds that partially erased the geographic structure. Contact of these expanding populations with different spectabilis populations would have generated genetically and morphologically intermediate populations that resulted in subsp. vicentinus.
A different process might account for the genetic diversification of S. boivinii. This species grows in the Tangier area, the Atlantic coast of Morocco, and southern Iberia. This region is influenced by both the Atlantic Ocean and the Mediterranean Sea, which reduce temperature oscillations and hence diminish the risk of frost in winter and severe drought in summer (Ojeda et al., 1996; Marañón et al., 1999). While excluding the most arid coastal places, the distribution of S. boivinii also extends inland throughout the Rif Mountains and Middle Atlas (Morocco) to the Massif of Tlemcen and Ghar Rouban Mountains (Algeria) at higher altitudes. These areas have a high rate of endemism due to long-lasting isolation of these massifs and the higher altitudes (Médail and Quézel, 1997). The relationships between the haplotypes of the northern Morocco and southern Iberia polyploid populations suggest that the species possibly had a continuous distribution along the Betic-Rifean floristic region (Valdés, 1991) on both sides of the Gibraltar Arc. This area of distribution might have been broken up during the hard climatic conditions of the MSC. Thus, we envisage two locations where S. boivinii might have found refuge: the Rif and Middle Atlas mountains in Morocco and the Atlantic coast of southern Iberia. Thus, the diploid Moroccan populations might represent the ancient groups that remained at higher altitudes, while those that persisted under oceanic influence diverged through polyploidization, a limited range of seed dispersal, and historical human influences.
Conclusions
The data suggest that the diploid populations of S. boivinii from the Middle Atlas Mountains represent old populations that survived the MSC crisis and recolonized when climatic condition permitted. During this advance, polyploidization and diversification took place, thus explaining the genetic and karyological differences between the populations. The populations today recognized as S. genistoides subsp. spectabilis are the remains of an old taxon that went through hard environmental conditions, distribution was greatly reduced, and the populations became genetically isolated. Some of these populations, or closely related ones, managed to endure these conditions, and when climatic conditions ameliorated, they recovered, expanded, and diversified into S. genistoides subsp. genistoides. When they came into contact with the western refugee spectabilis populations, introgression processes took place, and plants with intermediate morphology were formed. These populations are now recognized as S. genistoides subsp. vicentinus. The data also indicate that the scarce populations of subsp. spectabilis both in Morocco and Portugal should be protected because introgression and hybridization between the three taxa could be occurring at present.
The studied cpSSR markers have proved useful for studying genetic variation within populations in Stauracanthus and have allowed us to estimate the variation (33%) attributable to differences between species. Data from the markers corroborate that S. boivinii and S. genistoides are two phylogenetically different units and do not share cpSSR haplotypes. The question of whether they should be ranked as two separate genera (Nepa and Stauracanthus) or a single one (Stauracanthus) must be addressed from a broader perspective and requires the analysis of the origin of the related genus Ulex.
Stauracanthus and Ulex have similar patterns of variation, and in most cases, have similar size ranges of microsatellites. However, interspecific differences between alleles in Stauracanthus involve not only change in number of repeats but also indels in the flanking regions, and therefore they are more complex than in Ulex. The results indicate that the two species of Stauracanthus evolved through different processes of diversification. Genetic, spatial, and morphological patterns of variation also indicate that Stauracanthus has a different history than Ulex species growing in the same area. Ulex has diploid and tetraploid populations on both sides of the Strait of Gibraltar and has morphological diversification. This diversification has been associated with colonization of new habitats (dolostones and peridotites; U. baeticus) and most probably with genetic drift (U. scaber). Hybridization between related diploid populations, followed by polyploidization, explains the origin of tetraploid populations (U. congestus-borgiae) (Cubas et al., 2005).
The cross-genus amplification of cpSSRs between Ulex and Stauracanthus opens further possibilities for combining these markers with other molecular information (e.g., nuclear DNA sequences). This approach should provide a deeper insight into the patterns and magnitudes of phylogenetic differentiation in these two closely related genera and will surely contribute to a significant advance of their systematics and taxonomy. Extension of these studies to key species of Genisteae, which differentiated in the Iberian Peninsula and North Africa, would contribute to a significant advance of the systematic and taxonomy of the tribe and to a better understanding of the processes of diversification in relation to the environmental history of the western Mediterranean Basin.
FOOTNOTES
1 The authors thank S. Talavera (Sevilla, Spain) for providing material of S. genistoides and M. García (Unidad de Genómica, Parque Científico de Madrid-Universidad Complutense) for invaluable technical assistance. Funding for this work was provided by the Ministry of Education and Science of Spain (Projects REN2002-00225 and CGL2006-10392BOS) and the Spanish Agency of International Cooperation (Ministerio de Asuntos Exteriores, 83/04/P/E and A/2818/05). This paper benefited from the comments of anonymous reviewers and Editor-in-Chief J. Jernstedt. 
4 Author for correspondence (e-mail: cubas{at}farm.ucm.es)
LITERATURE CITED
Aafi, A., A. Achhal El Kadmiri, A. Benabid, AND M. Rochdi. 2005. Richesse et diversité floristique de la suberaie de la Mamora (Maroc). Acta Botanica Malacitana 30: 127–138.
Aïnouche, A., R. J. Bayer, P. Cubas, AND M.-Th. Misset. 2003. Phylogenetic relationships within tribe Genisteae (Papilionoideae) with special reference to genus Ulex. In B. Klitgaard, and A. Bruneau [eds.], Advances in legume systematics, part 10, Higher level systematics, 239–252. Royal Botanic Gardens, Kew, UK.
Cristofolini, G. 1991. The biodiversity of the Leguminosae-Genisteae and its genesis. Lagascalia 19: 121–128.
Cubas, P. 1987. Números cromosomáticos en Ulex L. y Stauracanthus Link (Genisteae-Papilionaceae). Anales del Jardín Botánico de Madrid 43: 217–233.
Cubas, P., C. Pardo, AND H. Tahiri. 2005. Genetic variation and relationships among Ulex (Fabaceae) species in southern Spain and northern Morocco assessed by chloroplast microsatellite (cpSSR) markers. American Journal of Botany 92: 2031–2043.
Dayanandan, S., K. S. Bawa, AND R. Kesseli. 1997. Conservation of microsatellites among tropical trees (Leguminosae). American Journal of Botany 84: 1658–1663.[Abstract]
De Castro, D. 1941. Algumas contagens de cromosomas no genero Ulex L. (sensu lato). Agronomia Lusitanica 3: 103–141.
De Castro, D. 1943. Contribuïçao para o conhecimiento cariológico dos generos Ulex L., Stauracanthus Link e Nepa Webb. Agronomia Lusitanica 5: 243–249.
Díaz, T. E., S. Rivas Martínez, AND F. Fernández González. 1990. Stauracanthus Link (Leguminosae) en la Península Ibérica. Itinera Geobotanica 3: 131–135.
Doblas, M., AND R. Oyarzun. 1989. Neogene extensional collapse in the western Mediterranean (Betic-Rif Alpine orogenic belt): Implications for the genesis of the Gibraltar Arc and magmatic activity. Geology 17: 430–433.
Ennos, R. A., W. T. Sinclair, X.-S. Hu, AND A. Langdon. 1999. Using organelle markers to elucidate the history, ecology and evolution of plant populations. In P. M. Hollingsworth, R. M. Bateman, and R. J. Gornall [eds.], Molecular systematics and plant evolution, 1–19. Taylor & Francis, New York, New York, USA.
Excoffier, L., P. E. Smouse, AND J. M. Quattro. 1992. Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131: 479–491.[Abstract]
Excoffier, L., G. Laval, AND S. Schneider. 2005. Arlequin ver. 3.0: An integrated software package for population genetics data analysis. Evolutionary Bioinformatics Online 1: 47–50.[Medline]
Feoli-Chiapella, L., AND G. Cristofolini. 1981. Serological contributions to the systematics of Ulex (Genisteae-Fabaceae) and allied genera. Nordic Journal of Botany 1: 723–729.[CrossRef]
Gibbs, P. E. 1966. A revision of the genus Genista L. Notes Royal Botanical Garden Edinburgh 27: 11–99.
Goldblatt, P. 1981. Cytology and the phylogeny of Leguminosae. In R. M. Polhill, and P. H. Raven [eds.], Advances in legume systematics, part 2, 427–463. Royal Botanic Gardens, Kew, UK.
Góómez, A., S. C. González-Martínez, C. Collada, J. Climent, AND L. Gil. 2003. Complex populations genetic structure in the endemic Canary Island pine revealed using chloroplast microsatellite markers. Theoretical and Applied Genetics 107: 1123–1131.[CrossRef][Web of Science][Medline]
Guinea, E., AND D. A. Webb. 1968a. Stauracanthus Link. In T. G. Tutin, V. H. Heywood, N. A. Burges, D. M. Moore, D. H. Valentine, S. M. Walters, and D. A. Webb [eds.], Flora Europaea, vol. 2, 103–104. Cambridge University Press, Cambridge, UK.
Guinea, E., AND D. A. Webb. 1968b Ulex L. In T. G. Tutin, V. H. Heywood, N. A. Burges, D. M. Moore, D. H. Valentine, S. M. Walters, and D. A. Webb [eds.], Flora Europaea, vol. 2, 102–103. Cambridge University Press, Cambridge, UK.
Herrera, J. 1988. Pollination relationships in southern Spanish mediterranean shrublands. Journal of Ecology 76: 274–287.[CrossRef][Web of Science]
Herrera, J. 1999. Fecundity above the species level: Ovule number and brood size in the Genisteae (Fabaceae: Papilionoideae). International Journal of Plant Sciences 160: 887–896.[CrossRef][Web of Science][Medline]
Hewitt, G. 2000. The genetic legacy of the Quaternary ice ages. Nature 405: 907–913.[CrossRef]
Hsü, K. J., W. B. F. Ryan, AND M. B. Cita. 1973. Late Miocene desiccation of the Mediterranean. Nature 242: 240–244.[CrossRef][Web of Science]
Hsü, K. J., L. Montadert, D. Bernoulli, M. B. Cita, A. Erickson, R. E. Garrison, R. B. Kidd, F. Melieres, C. Müller, AND R. Wright. 1977. History of the Mediterranean salinity crisis. Nature 267: 399–403.[CrossRef][Web of Science]
Instituto De Meteorologia. 2005. Perfil climático. Portugal continental, IM, 07-07-2005, http//www.meteo.pt/resources/im/pdfs/clim_ac_00_00.pdf [December 2006]
Knowles, L. L., AND W. P. Maddison W. 2002. Statistical phylogeography. Molecular Ecology 11: 2623–2635.[CrossRef][Medline]
Krijgsman, W., F. J. Hiigeni, I. Raffi, F. J. Sierro, AND D. S. Wilson. 1999. Chronology, causes and progression of the Messinian salinity crisis. Nature 400: 652–655.[CrossRef]
Loget, N., AND J. Van Den Driessche. 2006. On the origin of the Strait of Gibraltar. Sedimentary Geology 188-189: 341–356.
López González, G. 2001. Los árboles y arbustos de la Península Ibérica e Islas Baleares. Ed. Mundi Prensa, Madrid, Spain.
Maire, R. 1987. Flore de l’Afrique du Nord, vol 16, Editions Lechevalier, S.A.R.L., Paris, France.
Marañón, T., R. Ajbilou, F. Ojeda, AND J. Arroyo. 1999. Biodiversity of woody species in oak woodlands of southern Spain and northern Morocco. Forest Ecology and Management 115: 147–156.[CrossRef][Web of Science]
Médail, F., AND P. Quezel. 1997. Hot-spots analysis for conservation of plant biodiversity in the Mediterranean basin. Annals of the Missouri Botanical Garden 84: 112–127.[CrossRef][Web of Science]
Misset, M. T. 1990. Données caryologiques chez le genre Ulex L. (Papilionidae) dans le Massif Armoricain. Taxon 39: 630–635.[CrossRef][Web of Science]
Misset, M. T., AND J. P. Gourret. 1996. Flow cytometric analysis of the different ploidy levels observed in the genus Ulex L. Faboideae-Genisteae in Brittany (France). Acta Botanica 109: 72–79.
Nei, M. 1987. Molecular evolutionary genetics. Columbia University Press, New York, New York, USA.
Ojeda, F., T. Marañón, AND J. Arroyo. 1996. Patterns of ecological, chorological and taxonomic diversity at both sides of the Strait of Gibraltar. Journal of Vegetation Science 7: 63–72.[CrossRef][Web of Science]
Paiva, J., AND A. X. P. Coutinho. 1999. Stauracanthus Link. In S. Talavera, C. Aedo, S. Castroviejo, C. Romero Zarco, L. Sáez, F. J. Salgueiro, and M. Velayos [eds.]. Flora Iberica, vol. 2, 240–245, CSIC, Madrid, Spain.
Pardo, C., P. Cubas, AND H. Tahiri. 2004. Molecular phylogeny and systematics of Genista (Leguminosae) and related genera based on nucleotide sequences of nrDNA (ITS region) and cpDNA (trn L-trn F intergenic spacer). Plant Systematics and Evolution 244: 93–119.[CrossRef][Web of Science]
Peakall, R., S. Gilmore, W. Keys, M. Morgante, AND A. Rafalsky. 1998. Cross-species amplification of soybean (Glycine max) simple sequence repeats (SSRs) within the genus and other legume genera: Implications for the transferability of SSRs in plants. Molecular Biology and Evolution 15: 1275–1287.[Abstract]
Petit, R. J., E. Pineau, B. Demeseure, R. Bacilieri, A. Ducousso, AND A. Kremer. 1997. Chloroplast DNA footprints of postglacial recolonization by oaks. Proceedings of the National Academy of Sciences, USA 94: 9996–10001.
Posada, D., K. A. Crandall, AND A. R. Templeton. 2000. GeoDis: a program for the cladistic nested analysis of the geographical distribution of genetic haplotypes. Molecular Ecology 9: 487–488.[CrossRef][Medline]
Posada, D., K. A. Crandall, AND A. R. Templeton. 2006. Nested clade analysis statistics. Molecular Ecology Notes 6: 590–593.[CrossRef][Web of Science]
Powell, W., M. Morgante, R. McDevitt, G. G. Vendramin, AND J. A. Rafaslki. 1995. Polymorphic simple sequence repeat regions in chloroplast genomes: Applications to the population genetics of pines. Proceedings of the National Academy of Sciences, USA 92: 7759–7763.
Provan, J., W. Powell, AND P. M. Hollingsworth. 2001. Chloroplast microsatellites: new tools for studies in plant ecology and evolution. Trends in Ecology & Evolution 16: 142–147.[CrossRef][Web of Science][Medline]
Robledo-Arnuncio, J. J., C. Collada, R. Alia, AND L. Gil. 2005. Genetic structure of montane isolates of Pinus sylvestris L. in a Mediterranean refugial area. Journal of Biogeography 32: 595–605.[CrossRef][Web of Science]
Rothmaler, W. 1941. Revision der Genisteen. I. Monographien der gattungen um Ulex. Botanische Jahrbücher für Systematik 72: 69–116.
Sampaio, G. 1924. Revisão das Ulicineas portuguesas. Brotéria, série. Botanica 21: 142–168.
Suc, J. P. 1984. Origin and evolution of the Mediterranean vegetation and climate in Europe. Nature 307: 429–432.[CrossRef]
Swofford, D. L. 2002. AUP*: Phylogenetic analysis using parsimony (*and other methods), version 4.0 b10. Sinauer, Sunderland, Massachusetts, USA.
Taberlet, P., L. Gielly, G. Pautou, AND J. Bouvet. 1991. Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Molecular Biology 17: 1105–1109.[CrossRef][Web of Science][Medline]
Tahiri, H., P. Cubas, AND C. Pardo. 2004. Reports (1376–1381). In G. Kamari, C. Blanché, and F. Garbari [eds.], Mediterranean chromosome number reports 14. Flora Mediterranea 14: 424–428.
Tahiri, H., P. Cubas, AND C. Pardo. 2005. Reports (1428–1437). In G. Kamari, C. Blanché, and F. Garbari [eds.], Mediterranean chromosome number reports 15. Flora Mediterranea 15: 702–710.
Tahiri, H., P. Cubas, C. Pardo, AND A. Crespo. 2006. Reports (1604–1612). In G. Kamari, C. Blanché, and F. Garbari [eds.], Mediterranean chromosome number reports 16. Flora Mediterranea 16: 443–449.
Talavera, S., AND M. Arista. 1995. Números cromosomáticos de plantas occidentales, 712–717. Anales Jardín Botánico de Madrid 53: 101–782.
Templeton, A. R., E. Routman, AND C. A. Phillips. 1995. Separating population structure from population history: a cladistic analysis of the geographical distribution of mitochondrial DNA haplotypes in the tiger salamander, Ambystoma tigrinum. Genetics 140: 762–782.
Templeton, A. R. 2004. Statistical phylogeography: methods of evaluating and minimizing inference errors. Molecular Ecology 13: 789–809.[CrossRef][Medline]
Terrab, A., O. Paun, S. Talavera, K. Tremetsberger, M. Arista, AND T. F. Stuessy. 2006. Genetic diversity and populations structure in natural populations of Moroccan Atlas cedar (Cedrus atlantica; Pinaceae) determined with cpSSR markers. American Journal of Botany 93: 1274–1280.
Thompson, J. D. 2005. Plant evolution in the Mediterranean. Oxford University Press, New York, New York, USA.
Valdéés, B. 1991. Andalucía and the Rif. Floristic links and a common flora. Botanika Chronika 10: 117–124.
Vettori, C., G. G. Vendramin, M. Anzidei, R. Pastorelii, D. Paffetti, AND R. Giannini. 2004. Geographic distribution of chloroplast variation in Italian populations of beech (Fagus sylvatica L.). Theoretical and Applied Genetics 109: 1–9.[CrossRef][Web of Science][Medline]
Walter, R., AND B. K. Epperson. 2004. Microsatellite analyses of spatial structure among seedlings in populations of Pinus strobus (Pinaceae). American Journal of Botany 91: 549–557.
Webb, P. B. 1852. Observations sur le groupe des ulicinées et énumération de ses espèces. Annales des Sciences Naturelles, Botanique, série 3, 17: 280–291.
Weir, B. S., AND C. C. Cockerham. 1984. Estimating F-statistics for the analysis of population structure. Evolution; International Journal of Organic Evolution 38: 1358–1370.
Weising, K., AND R. C. Gardner. 1999. A set of conserved PCR primers for the analysis of simple sequence repeat polymorphisms in chloroplast genomes of dicotyledoneous angiosperms. Genome 42: 9–19.[Medline]
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