Annals of Botany 89: 321-327, 2002
© 2002 Annals of Botany Company
Testing Taxonomic and Biogeographical Relationships in a Narrow Mediterranean Endemic Complex (Hippocrepis balearica) using RAPD Markers
1Jardín Botánico de Valencia, Universidad de Valencia, c/Quart 80, E-46008, Valencia, Spain and 2CREAF, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Barcelona, Spain
* For correspondence. Fax +34 963156826, e-mail rossello@uv.es
Received: 18 May 2001; Returned for revision: 2 September 2001; Accepted: 3 December 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Analyses of RAPD profiles from 17 populations of the Hippocrepis balearica complex revealed a highly structured geographic pattern, not only among continental–insular areas but also within the eastern Balearic islands. In marked contrast to previous morphometric results, a clear separation between continental and insular samples was found, and intermediates between H. balearica and H. valentina samples were not detected. Molecular data indicated that western and eastern Balearic populations of the complex (H. grosii and H. balearica) were more closely related to each other than to continental populations (H. valentina). Multivariate analyses of the RAPD data clearly indicated that the similarities between continental and eastern Balearic samples of the H. balearica complex recovered by morphometric methods are due either to parallel evolution or to retention of plesiomorphic features.
Key words: Hippocrepis balearica, Hippocrepis grosii, Hippocrepis valentina, interspecific variation, species boundaries, Mediterranean flora, vicariant taxa, DNA fingerprinting.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Tracing the origin of plant taxa inhabiting islands has been one of the most exciting topics in insular biogeography. This field of research has benefited greatly from the application of molecular techniques to studies of the source of the ancestral immigrant taxa, to estimate the minimum number of colonizations involved, and to infer patterns of inter-island dispersal after colonization (Stuessy and Ono, 1998). Unlike continental islands, oceanic islands have never been connected to continents and usually result from volcanic activity. Thus, it is not by chance that the model islands used for studying colonization patterns and processes belong to oceanic archipelagos including Juan Fernández (Sang et al., 1994), Galápagos (Wendel and Percival, 1990), Hawaii (Baldwin et al., 1990), the Micronesian islands (Sheely and Meagher, 1996), the Macaronesian islands (Kim et al., 1996), and St Helena and Tristan da Cunha (Richardson et al., 2001a, b).
The search for the origins of plants endemic to continental islands suffers from some biological constraints, making the research goals more difficult to achieve. First, continental island taxa do not show prolific diversification through adaptative radiation processes, and a low species/genus ratio is thus expected for groups with endemic taxa. Secondly, island taxa could not have originated in situ, being the remnants of widespread continental populations that may now be extinct. In addition, the task is made more difficult if islands are situated in areas with a highly complex palaeogeographic history, such as the Mediterranean basin, where the isolation of some archipelagos has occurred relatively recently and where there is a long history of human disturbance.
The Balearic archipelago has a rich endemic element. Nearly 100 non-apomictic endemic taxa, representing approx. 7 % of the flora (Cardona and Contandriopoulos, 1979), are believed to occur in the Balearic islands (Alomar et al., 1997). Among many of the biogeographic connections inferred from plant endemism, one long-recognized pattern concerns the presence of schizoendemic taxa inhabiting the eastern Iberian peninsula and the Balearic islands. Schizoendemic taxa are vicariant endemics with identical chromosome numbers (Favarger and Contandriopoulos, 1961). Balearic schizoendemism has been invoked as a paradigm for vicariance and gradual plant speciation in the western Mediterranean (Cardona and Contandriopoulos, 1977; Contandriopoulos and Cardona, 1984).
The Hippocrepis balearica Jacq. complex (Fabaceae) has been one of the most studied cases of Iberian–Balearic schizoendemism (Cardona and Contandriopoulos, 1977, 1979; Cardona, 1979; Contandriopoulos and Cardona, 1984). This complex (Table TB1) comprises diploid shrubby plants that mainly inhabit calcareous cliffs and littoral scrubs. Traditionally, H. valentina Boiss., the mainland species, was considered to be the vicariant taxon of H. balearica, the insular element (Fig. 1). This picture was later modified slightly (Mus et al., 1990), with H. balearica being split into two allopatric taxa growing either in the eastern (H. balearica subsp. balearica) or western islands (H. balearica subsp. grosii (Pau) Mus, Rosselló and N. Torres). Using multivariate analyses it has recently been shown (Llorens et al., 1995) that the mainland samples of the complex (H. valentina) are more closely related to those from the eastern (H. balearica) than the western Balearics (H. grosii). However, these authors did not detect clear phenetic gaps between H. valentina and H. balearica samples using clustering methods and they treated the mainland and eastern Balearic populations as a single species, H. balearica subsp. balearica and H. balearica subsp. valentina (Boiss.) Hrabêtova-Uhrová. Moreover, western Balearic populations of the complex appeared to be phenetically distinct and were considered to be an independent taxon at the specific rank, H. grosii (Pau) Boira, Gil and L. Llorens.
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The patterns of vicariance between H. valentina and H. balearica, as inferred from the results of Llorens et al. (1995) could compromise the classical views that (1) a substantial period of effective isolation has occurred between shared Iberian and Balearic endemic biota; (2) sibling Balearic endemic taxa (such as the pair H. balearica–H. grosii) are more closely related to each other than other mainland taxa; and (3) biogeographic connections between the Iberian mainland and the Balearic islands involved plants from the western rather than the eastern islands.
Biogeographic hypotheses should rely on model species showing well-resolved taxonomic and evolutionary relationships. Therefore, we decided to elucidate relationships in the H. balearica complex to substantiate the inferred vicariance patterns between the Iberian mainland and the eastern Balearic archipelago, using the PCR fingerprinting technique random amplified polymorphic DNA (RAPD; Williams et al., 1990) to explore the extent to which morphological similarities are mirrored by molecular relatedness within the H. balearica complex.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Plant sampling and DNA extraction
Since our study focused primarily on geographic patterns rather than population genetic variation, we analysed a small number of plants (usually five) per locality. Seventeen populations were selected for study (Fig. 1, Table 2). The sampling strategy was designed to span the distribution range and to cover adequately the morphological variability reported in each area. Leaves were harvested directly from field populations or from adult plants grown in a glasshouse. Healthy young leaves were placed in polyethylene bags filled with silica gel and stored at –20 °C until required. A small fragment of dried leaf (approx. 50 mg) was extracted using the CTAB protocol of Doyle and Doyle (1987) scaled down for use in microfuge tubes. The total DNA concentration of all samples was adjusted by dilution to approx. 50 ng µl–1. Pooled DNA samples were obtained by mixing DNA from five individuals (200 ng from each individual). Thus, each pooled sample (1 µg DNA) was composed of five diploid genomes, representing five genotypes.
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RAPD amplifications
RAPD reactions were performed in 20 µl reaction mixtures containing 2·5 mM MgCl2, 2 µl 1 x Taq polymerase buffer, 1 unit Taq DNA polymerase (Ecogen), 0·1 mM each of dATP, dCTP, dGTP and dTTP, 0·2 µM primer and 50 ng genomic DNA. All PCR reactions were prepared as master mixes for each primer to minimize measurement deviations. A control containing all components except genomic DNA was included in each set of reactions to check for contamination. Amplifications were carried out in a GeneE (Techne) thermal cycler using 40 cycles at 92 °C for 1 min 15 s, 40 °C for 1 min, and 72 °C for 1 min. A final cycle at 72 °C for 10 min was included. Sixty primers (kits B, C, H; Operon Technologies, Alameda, CA, USA) were surveyed in a pilot study using three plants (one each from the mainland, Ibiza and Mallorca), of which 16 were chosen for RAPD fingerprinting. Duplicate amplifications with the selected primers were then conducted on each sample, and only bands present in both runs were considered in the analysis. Amplification products were separated on horizontal 1·4 % agarose gels in 1 x TAE buffer at 100 V for 2 h, stained with ethidium bromide and observed under transmitted UV light. The gels were scanned and fragment sizes were determined by comparison with molecular weight standards using a gel analysis program (Quantity One, BioRad). To ensure that the amplification profiles from single plants were additive in the composite DNA sample, we conducted several PCR experiments using a single population and all the selected primers.
Data analysis
Each RAPD band was coded as present or absent (1 or 0, respectively) and a binary data matrix was constructed. Bands showing the same gel mobilities were assumed to be homologous, a rationale widely used in RAPD studies. Following the suggestions of Grosberg et al. (1996), no attempts were made to code for band intensity. DNA bands showing quantitative variation in brightness were scored as present, regardless of their intensities, and absent if they were undetectable. Phenetic similarity was analysed using Principal Coordinate Analysis (PCOA) and cluster analysis applied to similarity matrices constructed using the Dice coefficient (Dice, 1945). The Unweighted Pair Group Method Algorithm (UPGMA) was used to construct the phenograms. Other similarity index (Jaccard) and building-tree algorithms (Weighted Pair Group Method, nearest neighbour, farthest neighbour) were also used to compute similarity matrices and draw phenograms. The NTSYS-PC (Rohlf, 1993) and the Multivariate Statistical Package (MVSP version 3·12d; http://www.kovcomp.com/mvsp) software were used in the analyses.
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RESULTS |
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Primers were selected on the basis of the number and intensity of polymorphic amplified bands. RAPD bands obtained from DNA from single individuals were always present in the PCR amplifications from pooled DNA samples derived from five individuals. Furthermore, no band absent in any individual amplification appeared in the PCR reactions from the mixed DNAs. Sometimes, lower amplification yields were obtained for some bands in the pooled DNA when compared with single amplification profiles. However, this did not have any effect on the results because bands were scored on a qualitative rather than on a quantitative basis. Accordingly, pooled DNA (hereafter samples) representing 85 genotypes were shown to portray adequately the RAPD profiles of 17 populations.
Overall, 168 reproducible RAPD bands were scored, ranging from 370 to 3160 bp. Approx. 85 % of the amplified products were polymorphic, allowing the differentiation of all 17 samples on the basis of their multilocus RAPD profiles. The distribution of polymorphic bands and phenotypes within taxa is shown in Table 3. Phenetic relationships among samples of the H. balearica complex based on a Dice similarity matrix portrayed a clear geographic pattern (Fig. 2). Congruent results were also obtained with other similarity index and dendrogram algorithms, and therefore they will not be further discussed here. Samples from continental H. valentina formed a discrete group, whereas H. balearica samples clustered with western Balearic samples (H. grosii). Within H. balearica, the UPGMA dendrogram showed clusters each containing samples from Mallorca (plus the Cabrera and Dragonera islets) and Minorca. The first three axes of the PCOA accounted for 74·62 % of the variance. The 17 samples formed three discrete groups in the multivariate space, in agreement with those depicted by the clustering method (Fig. 3). A minimum spanning tree imposed on the PCOA representation revealed that samples from the western and eastern Balearics were the most closely related in the ordination analysis (not shown).
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DISCUSSION |
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All authors dealing with the H. balearica complex have stated that plants from the eastern Iberian peninsula and the Balearic islands are closely related (Boissier, 1841; Burnat and Barbey, 1882; Knoche, 1922; Bellot, 1946; Ball, 1968; Garrido and Escarré, 1973). However, based on non-explicit taxonomic methods, the treatments of the complex have ranged from recognition of one taxon (Rouy, 1888; Chodat and Lendner, 1905) to three species (Talavera and Domínguez, 2000). Llorens et al. (1995) supported the presence of two species within the complex and, for the first time, provided an objective framework which could be tested using independent markers. However, it should be noted that the morphometric results obtained by these authors were contradictory. Thus, principal component analysis showed three non-overlapping groups in the multivariate space, but results from the clustering analyses failed to discriminate between H. balearica and H. valentina samples. Both numerical methods agree that H. balearica and H. valentina samples are more closely related to each other than to H. grosii.
The fact that continental (H. valentina) and eastern insular populations (H. balearica) of the complex could not be fully discriminated using numerical methods could be explained by several biogeographic and evolutionary hypotheses: (1) mainland and insular populations have diverged from a common ancestor on a recent evolutionary time scale; (2) long diverged populations have experienced secondary contacts through migration, so that gene flow has partially blurred morphological boundaries; or (3) morphological similarities could be due to convergence, since plants from the western Balearics and the continent share the same rupicolous habitat. Under hypotheses (1) and (2) we would expect RAPD markers to show either an incomplete differentiation between continental and eastern insular samples or, alternatively, to be geographically structured but closely related. In contrast, under hypothesis (3) we would expect low similarity and a lack of intermediates between continental and insular samples.
Our analyses of RAPD profiles from 17 populations belonging to the H. balearica complex revealed a highly structured geographic pattern, not only among continental–insular territories but also within the eastern Balearic islands. In sharp contrast to the results obtained by Llorens et al. (1995), a clear separation of continental and insular samples belonging to the H. balearica complex was found, and no intermediates linking H. balearica and H. valentina were detected in the phenetic analyses of the RAPD data. In fact, molecular data suggested a rather different biogeographic picture: western and eastern Balearic populations of the complex (H. grosii and H. balearica) were more closely related to each other than either of them was to the continental populations (H. valentina), as already suggested by Mus et al. (1990). Evidence gained from the multivariate analyses of RAPD data suggests that the similarities between continental H. valentina and insular H. balearica taxa recovered by numerical or by other non-explicit methods are not due either to a recent speciation event or to gene flow between them. If we assume that RAPD fingerprinting provides a fair representation of genomic relatedness, parallel evolution or, alternatively, retention of plesiomorphic features is the most likely explanation for the high phenetic similarity detected by Llorens et al. (1995). A taxonomic corollary is that the circumscription of H. balearica, as proposed by Llorens et al. (1995), is artefactual and should be re-evaluated. The type of markers analysed (RAPD), the experimental approach (pooled DNA samples) and the sampling procedure used in this study are not adequate for suggesting a sound taxonomy for the complex, which would ideally involve inclusion of all perennial taxa of the western Mediterranean.
Overall, RAPD data obtained for the H. balearica complex are strongly concordant with the regional biogeographic relationships based on diverse sources of biological evidence (animal endemic taxa, Palmer et al., 1999; flora and landscape, Font Quer, 1927; vertebrate fossils, Alcover et al., 1999) and those inferred from the palaeogeographical history of the western Mediterranean basin (Cardona, 1979). Thus, the facts that (1) the Balearic samples of the H. balearica complex are more closely related to each other than to continental ones; (2) a clear molecular distinction between samples belonging to western and eastern Balearic islands has been found; and (3) the clustering of samples within the eastern Balearics is in agreement with insular boundaries, are correlated to three palaeogeographical events. First, the Balearic archipelago was linked to Iberian territories by land bridges during the Messinian transgression at the end of the Miocene. During this period, the western and eastern islands were also connected. The splitting of these territories after the Messinian salinity crisis allowed the separation of the western and eastern Balearic islands and their isolation from the continent. Secondly, both Balearic subarchipelagos have remained isolated since the opening of the Gibraltar strait (approx. 5·3 mya; Gautier et al., 1994). Lastly, in the Upper Pliocene all the eastern Balearic islands were once again connected by a land bridge as a consequence of the glaciation episodes (the first northern glaciation occurred approx. 2·36 mya; Shackleton and Opdyke, 1977).
The identification of the closest relatives of many Balearic endemic taxa is highly uncertain since most of the available knowledge is based on intuitive comparisons of morphological characters (Knoche, 1922) or chromosome number data (Cardona and Contandriopoulos, 1979; Contandriopoulos and Cardona, 1984). That this information alone may be misleading has recently been exemplified by the case of Naufraga balearica Constance and Cannon, a monotypic genus of Apiaceae now restricted to the Balearic islands (extinct in the wild from Corsica). Since its description, Naufraga has been placed in the subfamily Hydrocotyloideae Link, with presumed relatives from the southern hemisphere. However, recent analyses using nuclear ribosomal DNA sequences (ITS) (Downie et al., 2000) have shown that this taxon falls in another subfamily, Apioideae Seem., as sister to Apium L., a mainly Mediterranean genus. The data reported in this study show that caution should be taken when inferring biogeographic relationships or speciation patterns on Mediterranean islands using single or few organisms. If these explicit models are not tested using objective and contrastable techniques, then most of the debate involving the testing of biogeographic hypotheses will be useless. RAPD markers have contributed greatly to the testing of some alternative biogeographical hypotheses concerning Scandinavian and alpine plants (Gabrielsen et al., 1997; Bauert et al., 1998; Brochmann and Gabrielsen, 1998; Tollefsrud et al., 1998). While not being an appropiate choice for population-based research, the DNA pooling approach has been used to search for genetic relationships (Furman et al., 1997) and to gain insights into hybridization processes (Díaz Lifante and Aguinagalde, 1996) in several plant groups. Our study suggests that both the type of markers used and the approach followed could also be promising in revealing phytogeographic patterns in a complex area such as the western Mediterranean basin.
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ACKNOWLEDGEMENTS |
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We are indebted to G. Nieto and M. Mus for helpful comments on the manuscript. M. B. Crespo made useful criticisms that improved the paper. J. A. Alcover helped with palaeontological literature, and improved the paper through stimulating discussions on island biogeography.
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