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Department of Botany, University of Reading, Whiteknights, P.O. Box 221, Reading RG6 2AS, UK
Received for publication April 9, 1998. Accepted for publication October 15, 1998.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: biogeography • ITS data • Macaronesian species • Mediterranean species • plant evolution • Saxifragaceae • Saxifraga • Saxifraga globulifera.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Despite the separation of northern Africa from southern Europe by the Mediterranean Sea, there is a weaker floristic relationship between the eastern and western Mediterranean than between the northern (European) and southern (African) Mediterranean. The connection of the continents in the east through Asia Minor and the Middle East and the proximity of the Iberian Peninsula to Africa in the west account for two exclusive groups of species in the east-west extremes of the Mediterranean basin.
The union of the Iberian Peninsula to Africa 
5–6 million years (mya) ago was probably an important factor in forming the flora of the Ibero-Mauritanian zone. According to Quézel (1978)
more than 500 species are endemic to this Euro-African zone (Fig. 1). The proximity of the continents (minimum separation of 14 km today) and extensive bird migration across the Strait of Gibraltar may have promoted dispersal of a large number of species between Europe and Africa. These factors, along with similar ecological conditions, may help to explain why 75% of the plant species are shared between the small area of Andalusia and the Moroccan Rif (Valdés, 1991
).
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). Recent treatments of Saxifraga in the Iberian Peninsula (Vargas, 1997
) and northern Morocco (Vargas, unpublished data) reveal that further taxonomic studies within S. globulifera are needed.
Two species also belonging to section Saxifraga [series Ceratophyllae (Haw.) Pawlowska] have been described from the Madeiran archipelago. These endemics are the only representatives of Saxifragaceae in the flora of Macaronesia: S. maderensis D. Don (Madeira) and S. portosanctana Boiss. (Porto Santo). Macaronesia consists of five major groups of islands (the Azores, Madeira, the Salvagems, the Canary Islands, and the Cape Verde Islands) situated close to the Mediterranean zones of Africa (Fig. 1). The origin of the Macaronesian flora has been primarily explained by dispersal from the Mediterranean flora that occurred before the massive glacial extinctions in the late Miocene and Pliocene (Bramwell, 1972
). Molecular evidence for a Mediterranean origin of several Macaronesian genera has already been obtained (Böhle, Hilger, and Martin, 1996
; Francisco-Ortega et al., 1997
; Francisco-Ortega et al., unpublished data), but the origin of the Macaronesian Saxifraga is uncertain (Webb and Press, 1987
).
This paper is part of a larger molecular phylogenetic study of section Saxifraga and is primarily focused on the series Ceratophyllae and Gemmiferae. Assessment of previous taxonomic classifications and biosystematic studies led us to conclude that these series encompass the best candidate lineages for determining the origin of both Macaronesian and Mediterranean species (Webb and Gornall, 1989
; Webb, 1993
; Vargas, 1994
, 1997
). The two series are defined by morphological characters that are approximately unique in the genus: axillary flowering stems arising from rosettes (Ceratophyllae) and presence of axillary and terminal summer-dormant buds (Gemmiferae).
The specific objectives of this study were to: (1) examine correlations between morphological and genetic variation in S. globulifera; (2) assess the placement of S. globulifera within series Gemmiferae; (3) infer historical biogeographic patterns of a Mediterranean plant group shared by the Iberian and North African floras using molecular markers; (4) provide molecular evidence on the origin of Saxifraga in Macaronesia; and (5) test the monophyly of series Ceratophyllae.
In order to achieve the above aims we have sequenced the internal transcribed spacer (ITS) region of nuclear 18S–26S rDNA. The ITS region has become widely used by systematists (Baldwin et al., 1995
) and has proven to be informative for biogeographic studies in some angiosperm genera, even at infraspecific levels (Baldwin, 1993
; Vargas, Baldwin, and Constance, 1998
). Chloroplast DNA regions have been used in systematic studies of Saxifraga, providing reliable information at generic and frequently at specific levels: rbcL and matK (Soltis et al., 1993
; Soltis et al., 1996
). Sequencing of the ITS region seems to be promising for detecting variation in Saxifraga at the populational level (Brochmann et al., 1998
), and it has been used successfully in S. tridactylites L. and S. osloensis Knaben, which also belong to section Saxifraga (Brochmann, Nilsson, and Gabrielsen, 1996
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Vargas, 1991
, for taxonomy of the series). Voucher information is listed in Table 1.
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, as modified by Loockerman and Jansen, 1996
). Organic compounds were removed with SEVAG (24:1 chloroform: isoamyl alcohol), followed by DNA precipitation with isopropanol at -20°C. Fresh material typically provided much higher DNA yields than silica-gel dried material or herbarium material. PCR (polymerase chain reaction) amplifications were performed using the external oligonucleotide primers 17SE and 26SE described by Sun et al. (1994)
. Slight modifications of the reaction conditions (e.g., the annealing temperatures, MgCl2 concentration) were sometimes necessary. Amplified products were cleaned using QIAGEN QIAquickTM PCR Purification kit (QIAGEN Inc., Chatsworth, California, USA) following the protocols provided by the manufacturer. Cleaned products were then directly sequenced using either the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase, FS (Perkin-Elmer Corp., Foster City, California, USA) or the Thermo Sequenase dye terminator cycle sequencing pre-mix kit (Amersham LIFE SCIENCE Corp., Little Chalfont, Buckinghamshire, UK). For cycle sequencing the primers ITS4 and ITS5 were used and, if necessary, primers ITS2 and ITS3 (White et al., 1990
). Unincorporated dye terminators were removed using a 3 mol/L sodium acetate and absolute alcohol precipitation, as recommended by the manufacturer. Samples were then loaded into a 5.75% polyacrylamide gel on an ABI 373A DNA Sequencer (Perkin-Elmer Corp., Foster City, California, USA), and data were collected on a Macintosh platform. Sequencing data were edited and assembled using the program "LaserGene 2" (DNASTAR, Madison, Wisconsin, USA).
Analysis
This analysis is a subset of a more complete analysis for section Saxifraga taken from a larger systematic project (in preparation). Initially, using an unpublished matrix of 75 ITS sequences for 47 Saxifraga species, a heuristic search and bootstrap analysis with 100 replicates were performed to identify general positions for the species of series Gemmiferae and Ceratophyllae. Based on the results of this analysis, we selected a more restricted set of taxa (Table 1) representing the major clades and the two series. Thirty-one sequences from the series Arachnoideae, Cespitosae, Ceratophyllae, Gemmiferae, Pentadactyles, and Saxifraga were compared and aligned by visual inspection. We used S. adscendens L., S. osloensis, S. spathularis Brot., and S. tridactylites as outgroup taxa based on the results from the larger analysis. The boundaries of the ITS and rDNA coding regions were identified by comparison to Daucus carota L. and Vicia faba L. (Yokota et al., 1989
), and to S. spathularis, S. rotundifolia L., and S. cernua L. (unpublished sequences from D. Soltis). Five equivocal sites of five ITS sequences (S. fragosoi, S. globulifera 4, S. globulifera 6, S. pentadactylis, and S. rigoi) were coded as polymorphic by using International Union Pure and Applied Chemistry ambiguity symbols. Three more imprecise sites in the sequences of S. pentadactylis and S. trifurcata were coded as missing data. The resulting matrix was analyzed with PAUP version 3.1.1 (Swofford, 1993
) and PAUP version 4d61 (test version) using heuristic searches. One hundred replicates of random taxon entries were performed using TREE BISECTION RECONNECTION (TBR), MULPARS, and STEEPEST DESCENT, with all characters and character states weighted equally and unordered (Fitch parsimony; Fitch, 1971
). Branch lengths for trees were calculated using the accelerated transformation optimization (ACCTRAN). Relative support for clades identified by parsimony analysis was assessed by using bootstrap (BS) analysis (100 replicates, branch swapping, and steepest descent in PAUP 3.1.1) and by "autodecay" analysis (Bremer, 1988
). Two heuristic analyses were performed with the ITS data. The first one examined the data set without recoding of indels, while in the second analysis, 15 synapomorphic indels were recoded [13 of one base pair (bp), one of 2 bp, and one of 3 bp].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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).
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Phylogenetic analysis of ITS sequences
The first analysis (without recoding of indels) of 696 ITS characters produced a single minimum-length Fitch tree of 360 steps, with a consistency index (CI) of 0.80 (including autapomorphies) and a retention index (RI) of 0.84 (Fig. 2). Analyses of the second data set (with recoded indels) of 712 characters produced four minimum-length trees of 385 steps with a CI of 0.79 (including autapomorphies) and a RI of 0.83 (Fig. 3). Analysis of the two data sets yielded congruent topologies except for the relative position of S. babiana T. E. Díaz & Fern. Prieto and S. camposii Boiss. & Reuter. Because the second analysis yielded more trees, slightly more homoplasy, and slightly less support, we focused on the results of the first analysis.
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Within clade 2, the five populations of S. globulifera from North Africa composed a well-supported clade (BS 98) that was sister to a clade containing two populations of S. globulifera (BS 61) and two of S. reuteriana Boiss. (BS 100) from the Iberian Peninsula (BS 95). Saxifraga globulifera and S. reuteriana constituted a robust clade (BS 100) that was (with weak support) sister to a clade consisting of S. erioblasta Boiss. & Reut. and S. rigoi Porta (BS 100). Clade 1 formed a weakly supported sister clade to clade 2 (BS 53). Saxifraga exarata Vill. (Cespitosae (Reichenb.) Pawlowska) and S. pentadactylis (Pentadactyles) formed a strongly supported (BS 87) clade that was sister (BS 91) to clade 1 and clade 2. Two populations of S. conifera Coss. & Durieu (Gemmiferae) formed a strongly supported clade (BS 100), which was sister to the group containing S. exarata, S. pentadactylis, clade 1, and clade 2 (BS 99). Saxifraga arachnoidea Sternb. (Arachnoideae (Engl. & Irmsch.) Gornall) was strongly positioned (BS 100), as sister to the rest of section Saxifraga.
Sequence divergence
DNA divergence values (uncorrected Kimura's two-parameter distances) between the ITS sequences are shown in Table 3. The average sequence divergence between members of the ingroup was 4.43%. The highest divergence (17.64%) within the data set was observed between S. spathularis and S. adscendens. The highest divergence (9.41%) within the ingroup was observed between S. granulata and S. arachnoidea. The lowest divergence (0.0%) was found between two populations of S. globulifera and S. reuteriana. The average sequence divergence between the populations of S. globulifera was 1.57%.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and other angiosperms (Baldwin et al., 1995
). Saxifraga is the largest genus in the Saxifragaceae (450 species) and it shows considerable morphological diversity. The sequence divergence observed in this small sample of the genus Saxifraga is far higher than within other genera of Saxifragaceae. In fact, the maximum divergence found between two species of Saxifraga (17.6% between S. spathularis and S. adscendens) is similar to the divergence observed among members of Heuchera, Darmera, and Rodgersia (15.1–19.9% for ITS1 and 14.6–18.1% for ITS2; Soltis and Kuzoff, 1995
). Similarly, the highest sequence divergence found between populations of a single species of Saxifraga (3.5% in S. globulifera) is in the range of the highest pairwise divergences within genera of Saxifragaceae: Mitella (8.4%), Heuchera (3.8%), Tellima (2.0%), and Tiarella (1.1%; Soltis and Kuzoff, 1995
). In some cases, the nucleotide substitutions provided phylogenetic information at the infraspecific level within species of the above genera. Similar divergences have been found within Saxifraga sect. Mesogyne Sternb. (4.9% between two populations of S. cernua L.; Brochmann et al., 1998
). The ITS region in Saxifragaceae is therefore a useful source of molecular phylogenetic data to infer relationships at the populational level, whereas in other families such sequence divergence seems to be scarce (Soltis and Kuzoff, 1993
).
The rapidity of adaptive radiation in most island genera of angiosperms is reflected by low levels of phylogenetic information in ITS sequences (Sang et al., 1994
; Francisco-Ortega, Jansen, and Santos-Guerra, 1996
; Kim et al., 1996
). The average ITS sequence divergence at the populational level between European and African S. globulifera (2.4%) is similar to the interspecific average divergences in some insular genera: 2.7% in Dendroseris (Sang et al., 1994
); 1.1% in Argyranthemum (Francisco-Ortega, Jansen, and Santos-Guerra, 1996
); 1.0% in Taeckholmia (Kim et al., 1996
). The low species diversity of Saxifraga in Macaronesia contrasts with the ITS sequence divergence between S. maderensis and S. portosanctana (1.2%) that is equivalent to the average divergences found in the above-noted, species-rich island genera.
Taxonomic implications
Morphologically, species assigned to series Ceratophyllae share only one feature: axillary growth of flowering stems (Vargas, 1991
, 1994
). However, the ITS data suggest that series Ceratophyllae is not monophyletic; two species belonging to series Gemmiferae (S. fragosoi and S. hypnoides) are nested within the clade corresponding to series Ceratophyllae (Fig. 2). The summer-dormant buds shared exclusively by species of series Gemmiferae allow survival of the plants during the dry season. However, our molecular tree suggests also that series Gemmiferae is nonmonophyletic. The three clades containing species of series Gemmiferae (S. conifera, S. fragosoi-hypnoides, and S. erioblasta-rigoi-globulifera-reuteriana) occur scattered in the tree (Fig. 2). In addition, S. conifera (series Gemmiferae) is sister to the clade comprising the remaining species of Gemmiferae and the other series of section Saxifraga. Hence, the molecular evidence does not support a single origin of summer-dormant buds, which have been considered a shared, derived characteristic of Gemmiferae species (Webb and Gornall, 1989
; Webb, 1993
; Vargas, 1997
). However, a single morphological character unites the species included in clade 2. They have rounded summer-dormant buds, in contrast to the acute buds of the species in the other two clades. Additional studies are needed to reassess the taxonomic relationships of series Ceratophyllae and Gemmiferae.
At least two geographically distant populations have been sequenced for any species that showed taxonomic conflicts in our ITS tree with previous classifications. Monophyletic groups of ITS sequences confirmed their position in the tree. Our results indicate that S. globulifera forms a monophyletic group only if S. reuteriana is included (Fig. 2, clade 2), and these two species form a very well-supported clade (BS 100). The relationships among the populations from the Iberian Peninsula and North Africa suggest that S. reuteriana arose from an Iberian ancestor. Our molecular results for S. globulifera and S. reuteriana are supported by previous morphological data, suggesting that the endemic S. reuteriana evolved from Iberian populations of S. globulifera without reticulation. Although hybridization is common in section Saxifraga, only a few cases of introgression have been reported based on field observations and artificial hybridization results (Vargas and Nieto Feliner, 1996
). On the other hand, several cases of chloroplast capture and a number of allopolyploid species have been identified within genera of Saxifragaceae (Soltis and Kuzoff, 1995
; Soltis, Johnson, and Looney, 1996
) and species of Saxifraga (Brochmann, Nilsson, and Gabrielsen, 1996
; Brochmann et al., 1998
) by use of molecular markers. Based on morphology, we conclude that hybridization between S. reuteriana and S. globulifera is unlikely to account for the nesting of S. reuteriana within S. globulifera in the ITS tree. Hybrids and species of hybrid origin in Saxifraga usually display intermediate morphological features relative to their progenitors (Vargas and Nieto Feliner, 1996
; Brochmann, Nilsson, and Gabrielsen, 1996
), but S. reuteriana does not show intermediate characters relative to other species in section Saxifraga. In addition, S. reuteriana has floral features that are unique in the genus, i. e., yellowish petals with margins reflected towards the tip, producing a star-shape flower, and all this leads us to suggest a nonhybridogeneous origin of S. reuteriana (but see McDade, 1990
, 1992
).
The sampling of S. globulifera was intended to elucidate the historical context of the geographic and morphological variation in this complex species. The lack of correlation between morphological variation and geographic origin of S. globulifera populations from North Africa is also seen with molecular evidence, which does not support grouping by geographic proximity or morphological similarity (Figs. 1, 2). For instances, the distant population 7 (Fig. 1) is unresolved in the ITS clade containing also the morphologically dissimilar populations 3 and 4, whereas population 5 displays no characteristic morphological feature but ten nucleotide autapomorphies and a distinctive position in the ITS tree (Fig. 2). The only correlated molecular phylogenetic and distributional patterns are seen between the Iberian populations of S. globulifera (clade supported by BS 61, but BS 95 in the Iberian clade including S. reuteriana), although these populations are morphologically most similar to some North African populations, in particular population 7. The nested position of S. reuteriana within the paraphyletic S. globulifera in the ITS tree fits the local-speciation model considered by some authors (Levin, 1993
; Rieseberg and Brouillet, 1994
) to be a common evolutionary pattern, with paraphyly being a temporary stage that will evolve into monophyly over time, following sorting and lineage extinction.
Biogeographic implications
Although ancient archipelagos close to continents, such as the Madeiran islands, could be subjected to multiple colonization events by similar species, molecular evidence from several genera does not support this expectation (Böhle, Hilger, and Martin, 1996
; Kim et al., 1996
; Mes and 't Hart, 1996
). The strong support for monophyly of the Madeiran taxa of Saxifraga (BS 99) suggests that this northern Macaronesian archipelago has been colonized a single time by a member of this genus (Fig. 2). Previous studies based on morphological characters and chromosome counts (S. maderensis, 2n = 100, 124; S. portosanctana, n = 27) were unable to shed light on the origin of these morphologically dissimilar Madeiran species (Vargas and Nieto Feliner, 1995
). The north-to-south arrangement of the Macaronesian islands, along with the prevailing southward direction of the trade winds, seem to have played an important role in the formation of the Macaronesian flora. Despite the lack of special fruit adaptations to dispersal in Saxifraga, the seeds may be sufficiently light or attachable to have been wind or bird dispersed to the islands (Ridley, 1930
). Clade 1 comprises the Madeiran lineage plus taxa characterizing two different floristic regions (Fig. 2): the Mediterranean region (S. camposii, S. cuneata, and S. fragosoi), and the Eurosiberian region (S. trifurcata, S. babiana, and S. hypnoides). Interestingly, colonization of Macaronesia by a Eurosiberian ancestor is indicated by the clade (BS 83) containing the Macaronesian lineage and its sister species, S. trifurcata, which is endemic to the extreme north of the Iberian Peninsula (Fig. 1). The limited radiation of Saxifraga in Macaronesia may be explained in part by its northern Iberian ancestry. The genomic constitution of a likely Eurosiberian ancestor may have forced the Macaronesian lineage to occupy high altitudes where new populations would have found habitat conditions similar to those of extreme northern Iberia. A similar colonization of Macaronesia has been preliminary assessed for Silene in the Canary Islands (Clement et al., 1997
). Our molecular evidence for another Eurosiberian-Macaronesian relationship argues for careful consideration of Eurosiberian taxa in biogeographic studies of Macaronesian groups.
The analyses of the ITS sequences of S. globulifera and S. reuteriana suggest that the North African and Iberian populations represent two divergent lineages (Fig. 2, clade 2). The first lineage contains the North African populations (S. globulifera), and the second lineage consists of the Iberian populations of S. globulifera and S. reuteriana. The two lineages within S. globulifera are probably isolated by the geographic barrier of the Strait of Gibraltar. Conversely, the poor geographic structuring of the morphological and molecular variation among the North African populations of S. globulifera may have been caused by massive migrations during the Pleistocene (Quézel, 1978
). Recent connections with Iberian populations are, however, unlikely. Therefore, the Mediterranean Sea has been an effective isolating barrier for some plant groups that occur between Europe and North Africa.
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FOOTNOTES |
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2 Author for correspondence, permanent address: Real Jardín Botánico, c/ Claudio Mollano, s. n., 28014-Madrid, Spain.
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  M. Escudero, P. Vargas, V. Valcarcel, and M. Luceno Strait of Gibraltar: an effective gene-flow barrier for wind-pollinated Carex helodes (Cyperaceae) as revealed by DNA sequences, AFLP, and cytogenetic variation Am. J. Botany, June 1, 2008; 95(6): 745 - 755. [Abstract] [Full Text] [PDF]
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V. Malecot, T. Marcussen, J. Munzinger, R. Yockteng, and M. Henry On the origin of the sweet-smelling Parma violet cultivars (Violaceae): wide intraspecific hybridization, sterility, and sexual reproduction Am. J. Botany, January 1, 2007; 94(1): 29 - 41. [Abstract] [Full Text] [PDF]
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K. M. Kay, P. A. Reeves, R. G. Olmstead, and D. W. Schemske Rapid speciation and the evolution of hummingbird pollination in neotropical Costus subgenus Costus (Costaceae): evidence from nrDNA ITS and ETS sequences Am. J. Botany, November 1, 2005; 92(11): 1899 - 1910. [Abstract] [Full Text] [PDF]
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M. A. Carine, S. J. Russell, A. Santos-Guerra, and J. Francisco-Ortega Relationships of the Macaronesian and Mediterranean floras: molecular evidence for multiple colonizations into Macaronesia and back-colonization of the continent in Convolvulus (Convolvulaceae) Am. J. Botany, July 1, 2004; 91(7): 1070 - 1085. [Abstract] [Full Text] [PDF]
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G. D. Hoggard, P. J. Kores, M. Molvray, and R. K. Hoggard The phylogeny of Gaura (Onagraceae) based on ITS, ETS, and trnL-F sequence data Am. J. Botany, January 1, 2004; 91(1): 139 - 148. [Abstract] [Full Text] [PDF]
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R. Holderegger and R. J. Abbott Phylogeography of the Arctic-Alpine Saxifraga oppositifolia (Saxifragaceae) and some related taxa based on cpDNA and ITS sequence variation Am. J. Botany, June 1, 2003; 90(6): 931 - 936. [Abstract] [Full Text] [PDF]
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R. K. Hoggard, P. J. Kores, M. Molvray, G. D. Hoggard, and D. A. Broughton Molecular systematics and biogeography of the amphibious genus Littorella (Plantaginaceae) Am. J. Botany, March 1, 2003; 90(3): 429 - 435. [Abstract] [Full Text] [PDF]
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D. M. Percy and Q. C. B. Cronk Different fates of island brooms: contrasting evolution in Adenocarpus, Genista, and Teline (Genisteae, Fabaceae) in the Canary Islands and Madeira Am. J. Botany, May 1, 2002; 89(5): 854 - 864. [Abstract] [Full Text] [PDF]
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 J. E. Richardson, R. T. Pennington, T. D. Pennington, and P. M. Hollingsworth Rapid Diversification of a Species-Rich Genus of Neotropical Rain Forest Trees Science, September 21, 2001; 293(5538): 2242 - 2245. [Abstract] [Full Text] [PDF]
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series Arachnoidea, 
series Ceratophyllae, 
series Cespitosae, • series Gemmiferae, 
series Granulatae, and + series Pentadactyles. Numbers after species names refer to populations in 