52 • August 2003: 463–476 Vargas • Alpine in Mediterranean Europe

Molecular evidence formultiple diversification patterns of alpine plants in Mediterranean Europe

Pablo Vargas

Real Jardín Botán ico, C.S.I.C., Plaza de Murillo 2, E-28014 Madrid, Spain. [email protected]

Apreliminary synthesis of diversification patterns of alpine plants in the Mediterranean region of Europe is presented based on seven groups displaying morphological differentiation and infraspecific taxa. Both previous and new phylogenetic results from ITS sequences and fingerprinting data suggest different coloniza- tion routes and modes of speciation in Androsace vitaliana (recent differentiation in the Iberian Peninsula), montana (west-to-east colonization and differentiation in Europe), Arenaria tetraquetra (colonization and differentiation from SE Iberian mountains to the Pyrenees; increasing number of chromosome comple- ments), Saxifraga oppositifolia (colonization from the arctic to the Iberian Peninsula), Saxifraga pentadactylis (differentiation in Mediterranean and Eurosiberian mountains by geographic isolation), and alpina (differentiation and colonization from northern Iberia to the Alps, and then to the Pyrenees and the Balkan Peninsula). Relative static diversification of Juniperus communis var. saxatilis in Europe, based on identity of chloroplast trnL-F sequences, is also described. Most morphological variation, expressed by number of sub- recognized in previous taxonomic treatments of the seven plant groups, appears to have occurred dur- ing the Pleistocene (< 1.75 Myr). Recurrent change of Quaternary climatic conditions in the Mediterranean Basin, coupled with geographic characteristics, life cycle, dispersal mechanisms, and pre-Holocene genetic structure are not convincing factors to account for all the observed diversification. Additionally, stoch astic processes are also considered for evaluating present-day distributions and processes of speciation.

KEYWORDS: Androsace, Anthyllis, Arenaria, colonization patterns, Juniperus, molecular d iversification, Saxifraga, Soldanella, subspecific differentiation.

Accordingly, floras of mountain ranges may have also INTRODUCTION evolved during the Pliocene and Pleistocene (Stebbins, Southern Europe encompasses elevated mountains 1984), including surviving floras in high altitudes encir- that harbour alpine plants in the Mediterranean region. cled by Mediterranean conditions. Glacial-interglacial The highest European mountains, which are still in an cycles in the Pleistocene promoted isolation of alpine uplifting process, are the result of the onset of the Alpine plants in high-altitude mountains of the Mediterranean orogeny in Mesozoic-Cenozoic times (c. 65 Myr). Plate Basin during warm (interglacial) periods. Conversely, tectonic activity of microplates generated main move- plant populations from isolated mountain ranges were ments during the latest uplifting periods in connected during long glacial stages. Most alpine plants Mediterranean Europe: Betic range (Jurassic-Miocene), that we find in southern European mountains are the Cantabrian range and the Pyrenees (Paleocene-Eocene), result of migrations from lower altitudes after glacial Jura (Miocene-Pliocene), the Alps (Oligocene-Miocene), periods or descendants of colonists from northern Europe Apennines (Paleocene-Miocene), and the Balkan (Stebbins, 1984). Peninsula (Paleocene-Pleistocene) (Ager, 1975). Molecular phylogenetics help infer historical bio- Important is that the present configuration of these geography and evolutionary patterns. Relationships mountains was established far earlier than differentiation among living populations of alpine plants in a phylogeo- of alpine species in Europe. graphic context seems to be the most appropriate Distribution and genetic structure of alpine plants approach to address diversification patterns of alpine have been ultimately molded by the geologic complexity plants in Europe (Avise & al., 1987; Comes & Kadereit, of the European continent, coupled with Tertiary and 1998; Avise, 2000). Intraspecific phylogeography is con- Quaternary climatic episodes. The occurrence of the cerned with the principles and processes governing the Mediterranean climate in the Pliocene (c. 3.2 Myr) geographic distributions of genealogical lineages, espe- caused seasonal rhythm and summer drought that cially those within and among closely related species became stable approximately 2.8 Myr ago (Suc, 1984). (Avise, 2000). This approach has been successfully

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implemented to describe postglacial colonization routes ies have not been included due to insufficient number of of trees in central Europe (Demesure & al., 1996; populations, low morphological variation or uninforma- Dumolin-Lapegue & al., 1997; King & Ferris, 1998; tive molecular results. Raspé & al., 2000; Petit & al., 2002) and to test the Molecular markers. — Molecular variation with- hypothesis of nunatak areas for in situ glacial survival in in the same species is the basic requirement to infer rela- arctic and alpine plants (Brochmann & al., 1996; tionships among populations. Fingerprinting techniques Gabrielsen & al., 1997; Stehlik & al., 2002; Schönswet- screening the nuclear genome, such as RAPDs, ISSRs, ter & al., 2002). Quaternary glaciations may not have AFLPs, and microsatellites, ensure a large number of dramatically affected the survival of plants in southern data to be analysed by phylogenetic methodologies Europe, in contrast with total extinction of angiosperms (Hillis & al., 1996a). In some cases, sequences of nuclear in northern Europe due to long-term establishment of ice ribosomal DNA(ITS, ETS) display a minimal number of sheets. In fact, three main refugia in the Iberian, Italian, phylogenetically informative characters among popula- and Balkan peninsulas have been proposed, from which tions. Recombination of biparental nuclear genomes may re-colonization occurred (Taberlet & al., 1998; Hewitt, obscure historical relationships based on hierarchical ge- 2000). Genetic structure and speciation patterns of alpine nealogies, particularly in cases of reticulation. Unipa- floras in southern Europe are more complex due to a con- rental haplotypes either from the mitochondria or chloro- tinuous presence of plants in Mediterranean mountains plast alleviate this problem because they are haploid and along fluctuating vegetation belts. non-recombinant macromolecules (Avise & al., 1987; This paper is a preliminary synthesis of results from Comes & Kadereit, 1998). However, low levels of genet- seven plant groups using molecular markers: Androsace ic variation in organelle genomes, together with phylo- vitaliana, Anthyllis montana , Arenaria tetraquetra , genetic reconstructions based on genes inherited by only Juniperus communis var. saxatilis, Saxifraga oppositifo- one parental organism (typically maternal inheritance in lia, Saxifraga pentadactylis , Soldanella alpina (Vargas, angiosperms; Mogensen, 1996), are disadvantages for 2002). Our working hypothesis is that geographic char- the use of these markers in phylogeography of plants and acteristics, recent climatic events (glaciations), life inference of morphological character evolution. cycles, dispersal mechanisms, and pre-Holocene genetic Phylogeographic reconstructions.— Tradi- structures are mostly responsible for present distribution tional biogeography considers two major possibilities to and differentiation within alpine plants. Subspecific taxa account for the origin of present distributions of popula- represent morphological differentiation of each species tions and species of alpine plants: dispersal and vicari- in an evolutionary context. The main objective is to test ance (Ronquist, 1997). The vicariance hypothesis argues this phylogeographic hypothesis based on variation of for isolation into island-like areas of populations on sun- molecular markers, phylogenetic reconstructions, and dered mountains from a continuous area separated during geographic arrangement of mountains that have led to interglacial periods. In contrast, under dispersal, distribu- present distributions and modes of speciation in Europe. tion of populations on elevated mountains is the result of colonization from one or few individuals. We abandon any inference based on area cladograms because our phylogenetic reconstructions are analysed at the popula- MATERIALS AND METHODS tional level, and distributions of alpine plants are pro- Species selection. — Alpine plants are typically foundly disparate. Genealogies of neutral genes are used defined as those obtaining optimal habitat conditions to evaluate colonization patterns and microevolutionary over 2300 m in the Mediterranean region (Körner, 1999). processes (Schaal & Olsen, 2000). The use of nuclear Unfortunately, we have found few examples in the liter- markers link morphological differentiation and phylo- ature of alpine vascular plants in southern Europe con- geography because most genes responsible for character taining subspecific taxa, which also have been analyzed evolution are contained in the nuclear genome. using phylogeographic data. Seven plant groups have Neighbor-joining and parsimony-based analyses of been considered in this paper thanks to previous publica- nuclear fingerprinting and ribosomal ITS sequences are tions from several authors on Anthyllis montana (Kropf implemented. & al., 2002a), Saxifraga oppositifolia (Abbott & al., The internal transcribed spacer (ITS) region of 2000; Holderegger & al., 2002), and Soldanella alpina nuclear 18S-26S ribosomal DNAhas proven to be (Zhang & al., 2001). Additionally, we have obtained and informative for both biogeographic studies and infer- analyzed new data from Androsace vitaliana , Arenaria ences of character evolution at inter- and intraspecific tetraquetra , Juniperus communis var. saxatilis, and levels (Baldwin & al., 1995). Availability of ITS Saxifraga oppositifolia , and extended the sample of sequences in GenBank is the result of ease of sequencing Saxifraga pentadactylis (Vargas, 2001). Some other stud- the ITS region coupled with an acceptable number of

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phylogenetically informative characters in some infra- most recent taxonomic treatment also includes subsp. specific studies (Baldwin 1993; Vargas & al., 1999a). flosjugorum Kress (Cantabrian range) described, as the Despite intrinsic problems in reconstruction of molecular other subspecies, under Androsace (Kress, 1997) (Table phylogenies by using multi-copy genes, such as the ITS 1). The ITS region is 604–606 bp in length: 222–223 bp region affected by concerted evolution (Fuertes & al., in ITS-1; 164 bp in 5.8S; 219–220 bp in ITS-2 (Álvarez, 1999), reliable phylogenetic signal at the infraspecific Luceño & Vargas, unpubl.). Eight variable and five par- level has been corroborated using different molecular simony-informative characters were obtained and markers (Kropf & al., 2002a; Zhang & al., 2001). analysed. When using Douglasia alaskana (Coville & Additionally, the body of ITS sequence analyses allow Standl. ex Hultén) S. Kelso, Douglasia beringensis S. comparison across angiosperms and the use of this mol- Kelso, Androsace ciliata DC., A. sempervivoides ecule to estimate maximum age of diversification Jaquem. ex Duby, and A. spinilifera as the outgroup, the through molecular clocks (Richardson & al., 2001). To accessions of A. vitalia form a monophyletic group. A obtain ITS and trnL-trnF sequences we used a sequenc- polytomy of three clades of A. vitaliana was retrieved ing strategy as described in Vargas & al. (1999a) and containing three samples from the Alps and Mount Taberlet & al. (1991). Ventoux, eight accessions from the Iberian Peninsula, Molecular clocks. — To include a time frame for and one from the Apennines (Fig. 1). Alarge polytomy is most molecular and morphological differentiation of the result of identity of ITS sequences from the entire each species, we searched for maximum ages of diversi- Iberian Peninsula, except for two samples from the fication. Diversification times were calculated from both Cantabrian range (subsp. flosjugorum ). Populations of previous estimates (Zhang & al., 2001; Kropf & al., subsp. flosjugorum have certain independence, as well as 2002a) and new ones based on the logic of Baldwin & those of subspp. cinerea and vitaliana from the Alps and Sanderson (1998) and Sanderson (2002). Calibration Mount Ventoux. Amaximum age of 0.7 Myr for the split points depend on the geological events and the plant of populations of Androsace vitaliana from Iberia is esti- group (e.g., origin of Madeira island and split of the mated from the highest K2P (Kimura-2-Parameter) pair- Strait of Gibraltar in Saxifraga; Vargas & al., 1999a) and wise distance between the populations from Mount the fossil record (Dorofeev, 1963, for ). A Ventoux and the Cantabrian range (1.16%). The popula- calibrated rate of Saxifraga in Richardson & al. (2001) is tions from the Iberian Peninsula may have diverged in also considered, even though it is clearly divergent in the last 0.2 Myr, as suggested by the highest K2P pair- comparison to other calibration points. Levels of diversi- wise distance (0.33%) between accessions from the fication are also compared using average molecular vari- Cantabrian range and the rest of the Iberian Peninsula. ation of ITS sequences in a group of angiosperms Anthyllis montana (Leguminosae) . — Morphol- (Richardson & al., 2001). ogical differentiation is reflected by four subspecies con- sidered in the last taxonomic treatments (Cullen, 1968; Greuter & al., 1989). They have two ploidal levels (2 n = 14, 28), fruits typically with one seed, and occur between RESULTS 200 and 2700 m. The ITS region is 605 bp in length: 231 Androsace vitaliana (Primulaceae). — The for- bp in ITS-1; 162 bp in 5.8S; 212 bp in ITS-2 (Kropf & mer genus Vitaliana has been lately considered as a sec- al., 2002a). The alignment of the nine accessions (Table tion (Vitaliana) of Androsace (Kress, 1997), a circum- 1) rendered six variable sites, of which three were parsi- scription in agreement with an ITS phylogeny where mony-informative. When using Hymenocarpos circinna- accessions of Androsace vitaliana form part of the line- tus (L.) Savi, Anthyllis vulneria L., and four species of A. age of Androsace sect. Aretia (L.) W .D.J. Koch (P. sect. Oreanthyllis Grisebach as the outgroup, the ITS tree Schönswetter, pers. comm.). This European endemic was resolved into four lineages of A. montana in a pecti- occurs in the Abruzzi, Alps, Pyrenees, and elevated nate topology: southern Spain, Algeria-French Alps, cen- mountains of the Iberian Peninsula, at an altitudinal tral Italy, and Austria-NE Italy-Slovenia-Croatia (Fig. 2). range between 1500 and 3300 m. It is characterized by Average K2P pairwise distance of sequences among the fruits in capsules with 2–3 seeds each and two ploidal four ITS lineages was 0.36 ± 0.16 %, which implies a levels (2n = 20, 40). The taxonomic treatment of maximum diversification time of 0.25–0.66 Myr (Kropf Ferguson (1972) includes five infraspecific taxa under & al., 2002a). The AFLPtree is congruent with that of Vitaliana primuliflora Bertol.: subspp. assoana M. Laínz ITS and provides further evidence of population discon- (S and E Iberia), canescens O. Schwarz (SW Alps and tinuity into western and eastern groups. Interestingly, Pyrenees), cinerea (Sünd.) I. K. Ferguson (C and SW populations from central and southern Italy belong to Alps and E Pyrenees), praetutiana (Buser ex Sünd.) I. K. two different groups, however weakly supported. Ferguson (Apennines), and primuliflora (SE Alps). A Arenaria tetraquetra (Caryophyllaceae). —

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Table 1. European subspecific taxa and accessions for the seven species investigated, including locality of wild popu- lations, vouchers, molecular markers, literature references, and GenBank accession numbers. MolecularGenBank Taxon Locality markeraccession no. Reference Androsace vitaliana (L.) Lapeyr. Álvarez & al., unpubl. ssp. assoana (M. Laínz) Kress Spain: Sistema Central (2 populations) ITS ssp. cinerea (Sünderm.) Kress France: Mount Ventoux. ITS ssp. flosjugorum Kress Spain: Cantabrian range; Montes Aquilianos.ITS ssp. nevadensis (Chiarugi) Kress Spain: Sierra Nevada. ITS ssp. praetutiana (Buser) Kress Italy: Apennines. ITS ssp. vitaliana Spain: Pyrenees (3 pops.). Switzerland: Alps ITS Anthyllis montana L. Kropf & al., 2002a ssp. atropurpurea (Vuk.) Pignatti Italy: Friuli. ITSAJ315504

ssp. hispanica (Degen & Hervier) CullenSpain: Sierra de Baza. ITSAJ315508

ssp. jacquini (A.Kerner) Hayek Austria: Geissberg Mt. Croatia: Velebit ITSAJ315503, AJ315501 Mts. Slovenia: Nanos Mt. AJ315502 ssp. montana Algeria: Massif du Djurdjura. France: Haute- ITSAJ315509, AJ315507 Savoie; Hautes-Alpes. Italy: Abruzzi AJ315506, AJ315505 Arenaria tetraquetra L. Valcárcel & al., un- publ. ssp. amabilis (Bory) H. Lindb. fil.Spain: Sierra Nevada (3 populations). ITS ssp. murcica (Font Quer) Favarger &Spain: Betic range (3 populations). ITS Nieto Fel. ssp. tetraquetra Spain: Sierra de la Pela; Pyrenees (2 pops.).ITS Juniperus communis L. ssp. communis var. communis Spain: Burgos trnL-trnF AY354286 ssp. communis var. saxatilis Pall. France: Hautes-Pyrenees. Iceland: Stögn. trnL-trnF AY354288, AY354289 Italy: Scanno. Spain: Ávila; Granada; Jaén; AY354290, AY354291 Madrid. Switzerland: Alps. Turkey: Caucasus Mts. AY354287, AY354292 AY354293, AY254294 AY354295 Saxifraga oppositifolia ssp. oppositifolia Austria: Salzburg. Iceland: Reykjavik. Italy:ITS A Y354303 Dolomites. Norway: Oppland. Spain: Cantabrian AY354304 + AY354306 range (Cantabria, Palencia), Sierra Nevada AY354396, AY354302 (Veleta Peak), Pyrenees (Huesca), Sierra de AY354298, AY354301 Urbión (La Rioja). AY354297, AY298906 ssp. paradoxa D. A. Webb Spain: S Pyrenees (Sierra del Cadí), N Pyrenees ITS A Y354299, AY354300 (Aran Valley). Saxifraga pentadactylis Lapyr. AY354307, AJ133031V argas, 2001 AY354308 ssp. almanzorii P. Vargas Spain: Sistema Central (Sierra de Gredos).ITS AJ133032 + AJ133026 ssp. pentadactylis Andorra: Pyrenees (El Serrat). ITSAJ233862 ssp. willkommiana (Boiss. Spain: Cantabrian range (Peña Prieta), Sistema ITS ex Willk.) M. Laínz Central (Sierra de Guadarrama), Sistema Ibérico (Sierra de Urbión). Soldanella alpina L. Zhang & al., 2001 ssp. alpina Austria: Carinthia. Spain: Basque Country. ITS AJ306323, AJ306322 Switzerland: Waadt. Yugoslavia: Montenegro. AJ306321, AJ306324 ssp. cantabrica Kress Spain: Cantabria. ITSAJ306325

Six ploidal levels have been found in this plant, which is drops (Goyder, 1987) seem to be mechanisms not favor- endemic to elevated mountains (between 1400 and 3400 able for long-distance dispersal. The ITS region is 633 bp m) of the Iberian Peninsula: subsp. amabilis (2x; Sierra in length: 258 bp in ITS-1; 159 bp in 5.8S; 216 bp in ITS- Nevada); subsp. murcica (3x, 4x, 5x; Betic range); and 2 (Valcárcel, Nieto & Vargas, unpubl.). We found a high subsp. tetraquetra (6x, Pyrenees; 7 x, Sierra de la Pela) number of variable sites (18) and parsimony-informative (López, 1990). Asynthetic treatment including only two characters (9) in nine populations sampled across the subspecies, tetraquetra and amabilis,is found in Chater range of the species. Phylogenetic analyses were per- & Halliday (1993). Acharacteristic cushion-like habit formed using three sequences of A. alfacarensis (endem- and capsules with seeds adapted to dispersal by rain ic to SE Iberian Peninsula) as the outgroup, which is the

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Fig. 1. Distribution (A) and phylogeogr aphic hypothesi s (B) for Androsace vitaliana (C, subsp. vitaliana; Spain, Pyrenees, Bonaigua pass) based on ITS sequences (Álvarez, Luceño & Vargas, unpubl.). Hypothetical barrier repre- sented by a hatched bar.

Fig. 2. Distribution (A) and phylogeographic hypothesis (B) for Anthyllis montana (C, subsp. montana; Spain, Pyrenees, Sallent de Gallego) based on ITS sequences and AFLPvariation. Hypothetical migration routes (arrows) inferred from Kropf & al. (2002a). most closely related species in the phylogeny of A. sect. not fully resolved, and all accessions of subsp. amabilis Plinthine (Reichenb.) Pau. As a result, a pectinate topol- form a monophyletic group. Considering molecular- ogy of the ITS tree was obtained (Fig. 3), where popula- clock evolution of ITS sequences and using variation tions from SE Iberia (Sierra Nevada and Betic range) are average within angiosperms (Richardson & al., 2001), basal, and northern accessions (Sierra de la Pela and ITS sequence variation (0–1.6 % K2P pairwise distance) Pyrenees) are at terminal nodes. Populations of subsp. is high enough to hypothesize a maximum age < 1.92 murcica are polyphyletic, accessions of tetraquetra are Myr for present distribution and increment of chromo-

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Fig. 3. Distribution (A) and phylogeographic hypothe sis (B) for Arenaria tetraquetra (C; subsp. amabilis, Spain, Sierra Nevada, Veleta Peak) based on ITS sequences (Valcárcel, Nieto & Vargas, unpubl.). Colonization and increment of chromosome complements in- dicated by an arrow.

some complements to 7 x. common chromosome number is 2 n = 26, but 2 n = 52 has Juniperus communis (Cupressaceae). — In also been reported from arctic areas. In Saxifraga, fruits Europe, two taxa have recently been recognized within are typically capsules with numerous seeds (Webb & this species: var. communis and var. saxatilis (Farjon, Gornall, 1989). We have sequenced nine populations of 2001). However, a different taxonomic treatment consid- subsp. oppositifolia and two of subsp. paradoxa (Table ered three subspecies (Franco, 1993): subspp. communis, 1). The ITS region is 659 bp in length: 277 bp in ITS-1; hemisphaerica (J. & C. Presl) Nyman, and alpina (Suter) 163 bp in 5.8S; 219 bp in ITS-2. Eleven nucleotide sub- Celak. This juniper occurs primarily between 1900 and stitutions and five parsimony-informative characters 2600 m in southern Europe, has a uniform chromosome were found. Chromatograms with nucleotide peaks over- number (2n = 22), and displays blue, fleshy cones. Six lapping at the same sites were observed in six popula- chlorotypes based on nucleotide substitutions and large tions after upstream and downstream sequencing reac- indels in trnL-trnF sequences have been found within a tions. IUPAC symbols were used for the analyses. The Mediterranean juniper ( J. oxycedrus L.; Martínez & semistrict consensus ITS tree of 140 minimum-length Vargas, 2002). This species also belongs to sect. Fitch parsimony trees yielded four major clades (Fig. 5) Juniperus and contains four subspecies. In contrast, trnL- when using S. spathularis and S. aizoides as the out- F sequences of nine European populations of J. commu- group. The first one supports monophyly of S. oppositi- nis var. saxatilis (Table 1) display no molecular varia- folia (95% bootstrap), a support value similar to that tion. Identical trnL-trnF sequences have also been obtained when including four more species from sect. obtained when sampling former infraspecific taxa con- Porphyrion (Conti & al., 1999; results not shown). A sidered within J. communis (subsp. communis var. com- basal polytomy includes four accessions of S. oppositifo- munis, subsp. hemisphaerica ). Distribution of Juniperus lia, three of samples from southern Norway, Iceland, and communis var. saxatilis in central and southern Europe Austrian Alps and one of the rest of the accessions (68% and the sample of nine populations (numbered from 1 to bootstrap; Fig. 5). This second clade is resolved, in part, 9) are shown in Figure 4. by a sequential branching pattern, where one accession Saxifraga oppositifolia (Saxifragaceae). — from the Central Pyrenees comes first, followed by a Synthetic taxonomic treatments of this species can be group (third clade, 72% bootstrap) of four accessions at found in Webb & Gornall (1989) and Webb (1993). a basal position [Dolomites, Cantabrian range (Palencia), Seven subspecies are considered, of which five occur in Sierra de Urbión (La Rioja), and Sierra Nevada] and a Europe (subspp. blepharophylla , oppositifolia , para- fourth clade (45% bootstrap) containing one sample of doxa, rudolphiana , speciosa). The species occurs subsp. oppositifolia from the Cantabrian range between 1700 and 3000 m in southern Europe. The most (Cantabria) and the two samples of subsp. paradoxa

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Fig. 4. Distributio n (A) of Juniperus communis var. saxatilis (B; Iceland, Skaf- tafel National Park) in cen- tral and southern Europe (A). The sequen cing of nine popula tions (num- bered from 1 to 9) rendered an identical sequence for the trnL-trnF spacer.

from the Pyrenees (Sierra del Cadí and Aran Valley). characters. Similar phylogenetic conditions were used as Four separated populations from the Alps (Dolomites), in Vargas (2001). The ITS consensus tree displays a Sierra de Urbión (La Rioja), Cantabrian range (Palencia), biphyletic topology (Fig. 6) with one clade weakly sup- and SE Iberia (Sierra Nevada) have the same ITS ported (52% bootstrap) of samples from northern Iberia sequence (Fig. 5). The highest K2P pairwise distance and a second one of two samples from central Iberia within Iberian accessions (0.31%) is found between sam- (70% bootstrap). Although a fully resolved tree was not ples from Sierra de Urbión and Aran Valley. Maximum obtained, this topology indicates that subsp. willkommi- age of diversification of S. oppositifolia in Iberia is esti- ana is not monophyletic. The average K2P pairwise dis- mated between 0.88 and 0.30 Myr using different cali- tance between ITS accessions was 0.58 %, being the bration events: unspecific calibration in Saxifraga (0.88 highest distance of 1.21 % (between Sierra de Urbión Myr, Richardson & al., 2001); origin of Madeira (0.60 and Sierra de Guadarrama). Separation times are sug- Myr, Vargas & al., 1999a); split of the Strait of Gibraltar gested at a maximum between 3.55 and 1.21 Myr by the (0.43 Myr, Vargas & al., 1999a); estimation average in use of different calibration events: unspecific calibration angiosperms (0.3 Myr, Richardson & al., 2001). in Saxifraga (3.55 Myr, Richardson & al., 2001); origin Saxifraga pentadactylis (Saxifragaceae). — of Madeira (2.42 Myr, Vargas & al., 1999); split of the This saxifrage is endemic to the northern half of the Strait of Gibraltar (1.72 Myr, Vargas & al., 1999a); esti- Iberian Peninsula and occurs between 1500 and 3000 m mation average in angiosperms (1.21 Myr, Richardson & (Vargas, 1997). Neighborhood diffusion (Shigesada & al., 2001). al., 1995) seems to be a predominant dispersal mechan- Soldanella alpina (Primulaceae). — Two sub- ism of plants with capsules containing over 100 seeds, as species are recognised (subspp. alpina and cantabrica) are populations of the three endemic taxa of S. pen- based on floral shapes and sizes (Kress, 1984; Zhang & tadactylis (Vargas & Nieto, 1996): subsp. pentadactylis al., 2001). Both of them are 2 n = 40. The ITS region is (Pyrenees); subsp. willkommiana (central part of north- 644–645 bp in length: 249 bp in ITS-1; 165 bp in 5.8S; ern half of Iberia); subsp. almanzorii (part of Sierra de 230–231 bp in ITS-2. Number of nucleotide substitutions Gredos) (Fig. 6). Asingle chromosome number has been within Soldanella was 30 (15 parsimony-informative found (2n = 32). An extended sample (Table 1) with two characters) and only one within S. alpina. Apolytomy more accessions from Sistema Central (Sierra de Urbión) included all accessions in the ITS tree. The AFLPrecon- and Cantabrian range (Peña Prieta) provided figures of struction, using Primula latifolia and P. bulleyana as the ITS sequence variation within the range described in a outgroup, depicted a pectinate tree in which subsp. previous publication (Vargas, 2001): 682–683 bp in cantabrica is the basal-most lineage followed by popula- length (281 bp in ITS-1; 168 bp in 5.8S; 233–234 bp in tions from the Alps. Populations from the Pyrenees and ITS-2); six variable sites; three parsimony-informative the Balkan Peninsula are intermingled among those from

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Fig. 5. Distribution (A) of Saxifraga oppositifol ia in southern Europe. Colored circles indicate the five sequences of nuclear ribosomal ITS sequences found. Semistrict consensus tree of 140 minimum-length Fitch parsimony trees (B) from analysis of ITS sequences (90 steps, CI: 0.99, RI: 0.96, including cladistically uninformative characters). Saxifraga spathularis and S. aizoides were used as the outgroup (not shown). Bootstrap values are above branches. Photograph of subsp. oppositifolia (C) from Spain (Pyrenees, Envalira pass). the Alps. Aclock-like evolution of ITS sequences of Central, and Sierra Nevada (Fig. 1). None of the two pre- Soldanella has been tested (Zhang & al., 2001). Asingle vious taxonomic treatments are in agreement with the nucleotide substitution between subsp. alpina and ITS tree, where populations of subspp. vitaliana from the cantabrica (~ 0.20 % K2P pairwise distance) indicates a Alps and Pyrenees are not monophyletic (Fig. 1). recent divergence time (< 0.13 Myr), using a conserva- West-to-east differentiation of Anthyllis mon- tive rate. tana. — Apectinate branching pattern of the ITS and AFLPphylogenies is described (Kropf & al., 2002a), in which the Iberian populations are basal, followed by those from the western, southern, central and eastern DISCUSSION Alps, and the Balkan Peninsula. These phylogenetic Recent migration an d differentiation of reconstructions suggest an eastward migration (Fig. 2). Androsace vitaliana in Iberia. — The branching pat- Tree resolution of European populations also revealed tern obtained from analyses of ITS sequences indicates fragmentation into western and eastern areas correspon- isolation between populations from the Iberian ding approximately with subspp. hispanica/montana and Peninsula, Alps, and Apennines. Two polytomies prevent jacquinii/atropurpurea , respectively. Character evolution inferring patterns of migration within Europe and within of floral traits based on morphometric and AFLPanaly- the Iberian Peninsula (Fig. 1). Afragmentation process ses leads to the conclusion that A. montana contains only separating populations from Iberia and the Alps is inter- two infraspecific taxa (subspp. montana and jacquinii) as preted from relatively significant DNAsequence diver- a result of vicariance in W-E Europe (Kropf & al., gence between them (1.16 % K2P pairwise distance), but 2002b). Evidence of a secondary contact was observed in similar levels occur within the two groups (0.33 and the Alps Maritimes/Liguria region. Estimation of a max- 0.16%, respectively). Similarly, Alpine populations are imum age of 700,000 years indicates infraspecific diver- isolated from that from the Apennines (0.66–0.83%). gence in Late Quaternary times (Kropf & al., 2002a). Separation times, based on the same logic as in Zhang & South-to-north colonization and increment al. (2001), suggest split of the three lineages in the late of polyploidy in Arenaria tetraquetra . — As part of Pleistocene (< 0.7 Myr), and then differentiation in the a wider investigation in Arenaria sect. Plinthine Iberian Peninsula in the last 0.24 Myr. Lack of variation (Valcárcel, Nieto & Vargas, unpubl.), preliminary data of in ITS sequences indicates recent isolation in four moun- ITS sequence evolution reveal that populations of subsp. tain ranges of Iberia: Pyrenees, Sistema Iberico, Sistema tetraquetra may have colonized the Pyrenees from SE

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Fig. 6. Distribution (A) of Saxifraga pentadactylis . Strict consensus tree (B) of two minimum-length Fitch parsimony trees from analysis of ITS sequences (122 steps, CI: 0.87, RI: 0.89). Saxifraga spathularis was used as the outgroup (not shown), as well as sequences and phylogenetic parameters from Vargas (2001). Bootstrap values over 50 % are shown above tree branches. Photograph of subsp. willkommiana (C) from Spain (Sierra de Guadarrama, Madrid).

Iberia (Fig. 3). Differentiation northwards from SE munis (Jordano, 1993; Martínez & Vargas, 2002), lead us Iberian populations of subsp. amabilis (2x) and subsp. to interpret that relatively recent colonization may have murcica (3x, 4x, 5x) is in agreement with a previous occurred across Europe. Amore variable molecular hypothesis of increment of chromosome complements marker is necessary to test this interpretation. (Nieto & Favarger, 1988). Assuming a clock-like evolu- Colonization of Saxifraga oppositifolia in the tion of the ITS region, this mode of speciation in a Iberian Peninsula from the arctic. — Survival of ploidal series is occurring since the onset of the an angiosperm ( Saxifraga oppositifolia ) in the arctic and Pleistocene (c. 1.75 Myr). The possibility of increment of further circumpolar migration from Beringia has been chromosome complements via hybridisation (allopoly- recently documented (Abbott & al., 2000). It has also ploiditation) should be cautiously contemplated (Nieto & been hypothesized that lack of geographical genetic pat- Favarger, 1988). Phylogenetic reconstructions based on terns in the Alps may be the result of both immigrations chloroplast markers (already in progress) may shed fur- after glaciations and in situ survival (Holderegger & al., ther light on modes of speciation and colonization of A. 2002). Phylogeographic studies including populations tetraquetra . from Mediterranean Europe have not been addressed for Static diversification in Juniperus communis . this species. Our results based on ITS sequences (Fig. 5) — Lack of molecular variation, not only within var. sax- are in agreement with those based on chloroplast varia- atilis from Iceland to the Caucasus but also with respect tion in the Eurosiberian region (Abbott & al., 2000; to var. communis, suggests a single origin of this species Holderegger & al., 2002). Terminal placement of ITS and a relative static diversification pattern in Europe accessions in a sequential branching pattern indicates (Fig. 4). Higher levels of variation have been obtained by that colonization in the Iberian Peninsula occurred from using AFLPdata in J. communis (van der Merwe & al., the north. Ancient arctic lineages may have been present 2000), suggesting multiple colonization patterns at a across Europe pre-dating Quaternary episodes (Abbott & local scale (the British Islands). Identity of trnL-F al., 2000). Maximum divergence times between 0.88 and sequences among populations separated over 3000 km, 0.30 Myr is herein estimated for the Iberian populations. coupled with active dispersal of fleshy cones of J. com- Interestingly, identity of ITS sequences in four separated

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Fig. 7. Distribution (A) and phylogeographic hypothe- sis (B) for Soldanella alpi- na (C, subsp. alpina; Spain, Pyrenees, Bonaigua Pass) based on ITS sequence s and AFLP variation. Hypothetical migrat ion routes (arrows ) inferre d from Zhang & al. (2001).

mountain ranges of Europe (Dolomites, Pyrenees, Gredos) from populations of subsp. willkommiana has Cantabrian range, and Sierra Nevada) indicates genetic already been described (Vargas, 2001). Differentiation of affinities (Fig. 5). Presence both of ancient and recent leaf and flower morphologies is estimated to occur at lineages depicts a complex genetic structure not only in most in the last 3.55 Myr, even though it is most plausi- the Eurosiberian region of Europe (Abbott & al., 2000), ble at the onset of the Pleistocene (c. 1.75 Myr). but also in the Mediterranean region. The finding of Differentiation from relict, Cantabrian pop- additive polymorphism in ITS sequences supports the ulations of Soldanella alpina . — Low levels of ITS likelihood of hybridization processes. Some genotypes sequence variation (one nucleotide substitution between surviving in Iberia after Quaternary climatic changes subspp. alpina and cantabrica) were found (Zhang & al., may have had secondary contacts, as in northern Europe 2001). The AFLPtree supports a basalmost placement of (Gabrielsen & al., 1997) and the Alps (Holderegger & subsp. cantabrica, followed by populations of subsp. al., 2002). Further investigations using markers unaffect- alpina in a sequential fashion, where populations from ed by concerted evolution (dissimilar to ITS sequences) the Pyrenees and the Balkan Peninsula are included in are needed to evaluate the impact of secondary contacts groups of populations from the Alps (Fig. 7). As the on the genetic structure of southern European popula- Pyrenees are situated between the Alps and the tions. Acquisition of alternate leaves in subsp. paradoxa Cantabrian range, two interpretations are considered for (Pyrenees) seems to be derived from opposite leaves, a the occurrence of S. alpina in the Pyrenees: (1) coloniza- character described for all the other subspecies (Fig. 5). tion to the Pyrenees from the Alps after long-distance Alarger sample of the entire subsection Oppositifoliae dispersal from the Cantabrian range to the Alps; and (2) (already in progress by R. Holderegger, pers. comm.) early colonization from the Cantabrian range, extinction, may shed light on character evolution within S. oppositi- and re-colonization from the Alps. Seed dispersal folia. inferred by using chloroplast markers would help to test Differentiation of Eurosiberian and these hypotheses, particularly in this case because evi- Mediterranean populations of Saxifraga pen- dence of hybridisation and introgression based on mor- tadactylis. — Our phylogenetic hypothesis (Fig. 6) is in phological, nuclear DNA, and distributional patterns has agreement with previous results of genetic drift in moun- been documented (Zhang & al., 2001). Using a conser- tains of the Iberian Peninsula (Vargas, 2001). vative estimated rate, divergence between subspp. alpina Populations of subsp. wilkommiana do not form a natu- and cantabrica may have taken place in the last 150,000 ral group because morphological differentiation, as years, coinciding with the Late Penultimate Glacial (van described in two taxonomic treatments (Webb & Gornall, Andel & Tzedakis, 1996). 1989; Vargas 1997), do not reflect isolation between To explore patterns of colonization and evolution of Eurosiberian (Cantabrian range, Pyrenees, Sierra de alpine plants, cladistic analyses based on DNAsequences Urbión) and Mediterranean (Sierra de Guadarrama, help infer hierarchical relationships of populations. Sierra de Gredos) mountains. In the same mountain Because of limitations in cytotaxonomic interpretations range (Sistema Central), genetic isolation through pre- from chromosome counts, Stebbins (1984) recommend- dominant autogamy in subsp. almanzorii (Sierra de ed use of cladistics and sequences of mitochondrial,

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chloroplast, and nuclear ribosomal DNAfor comparisons Sanicula; Vargas & al., 1999b). In contrast, speciation of between closely related species and races of the same a group of seven gentians ( Gentiana sect. Ciminalis) has species. This author warned that no absolute rules or been hypothesized to have occurred in the Pleistocene generalizations could be made on migration patterns of (Hungerer & Kadereit, 1998). Interestingly, the estimates arctic-alpine flora. The above-discussed plant groups of infraspecific variation herein presented based on occurring in southern Europe support this prediction also molecular data are mostly in consonance with previous for alpine species in Mediterranean mountains. Some estimates of speciation rates for herbs (1.15 Myr) based populations of the seven species studied in the present on chromosomal evolution (Levin & W ilson, 1976). paper cluster together into monophyletic groups of sub- Generalization of diversification times for angiosperms, species. Asearch for naturalness in the evolution of herbaceous plants, members of the same family, or even alpine plants reveals that populations of Androsace vital- species within the same genus should be cautiously con- iana subsp. flosjugorum (Fig. 1), Anthyllis montana templated because every plant group undergoes unique subsp. hispanica (Fig. 2), Arenaria tetraquetra subsp. differentiation histories (Hillis & al., 1996b). amabilis (Fig. 3), and Soldanella alpina subsp. alpina Diversification rates across taxa are the result of (Fig. 7) form natural groups in the phylogenetic recon- idiosyncratic characteristics of each species, extinction structions. Lack of resolution in some reconstructions is of their populations, and taxonomic artefacts (Barra- the result of identity of sequences ( Juniperus communis ), clough & Nee, 2001). Morphological differentiation of low number of characters ( Androsace vitaliana ) or char- the seven species has resulted in fluctuating taxonomic acter additivity ( Saxifraga oppositifolia ). In contrast, treatments at the infraspecific level. Infraspecific taxa some subspecies recognized in recent classifications are have been long discussed and subjected to historical not supported by molecular evidence for monophyletic interpretations (Stuessy, 1990). Irrespective of taxonom- populations: Anthyllis montana subspp. montana and ic subjectivity, present populations of alpine European jacquinii (Fig. 2), Arenaria tetraquetra subsp. tetraque- taxa display morphological differentiation as a result of tra and murcica (Fig. 3), and Saxifraga pentadactylis sorting and extinction primarily in the Pleistocene. subsp. willkommiana (Fig. 6). Island-like distribution of Further studies using taxonomic treatments of additional alpine plants in southern Europe suggests that allopatry genera, morphological differentiation, and molecular appears to be the main force for divergence and isolation divergence rates will serve to test the hypothesis that of subspecies. Infraspecific taxa are considered to be infraspecific variation does not predate the Quaternary intermediate stages in the speciation process in a geo- climatic changes in a significant number of cases. graphic context (Stuessy, 1990), and it is expected that Three basic hypotheses on glacial survival are pro- the phylogenetic status will vary over time to achieve posed: (1) total extinction through glaciations ( tabula monophyly via sorting and extinction of lineages in a rasa), as considered for thermophilous plants in the arc- sequence of polyphyly ® paraphyly ® monophyly tic (Brochmann & al., 1996) and trees in central and (Rieseberg & Brouillet, 1994). northern Europe (Demesure & al., 1996; Dumolin- Time of diversification and colonization was out- Lapegue & al., 1997; King & Ferris, 1998; Raspé & al., lined by applying a conservative rate of ITS sequence 2000; Petit & al., 2002); (2) in situ persistence of popu- evolution at the highest pairwise distances in all cases. lations within glaciated regions in ice-free mountains As a result, we found that maximum age of morphologi- (nunataks) (Stehlik, 2000; Gugerli & Holderegger, cal differentiation in southern Europe for six of the seven 2001); (3) glacial-induced migration to lowland and peri- study cases, expressed by subspecific taxa, is significant- pheral refugia, followed by recolonization of alpine habi- ly different for each plant group: Androsace vitaliana (< tats after ice-sheet retreat (altitudinal migration) 0.66 Myr); Anthyllis montana (< 0.88 Myr); Arenaria (Schönswetter & al., 2002; Gutiérrez & al., 2002). Un- tetraquetra (< 1.92 Myr); Saxifraga oppositifolia (< 1.37 like extinction of most trees and arctic plants in central Myr); Saxifraga pentadactylis (< 1.75 Myr); and and northern Europe under the tabula rasa hypothesis, Soldanella alpina (< 0.15 Myr). In any case, subspecific populations of alpine species in Mediterranean moun- taxa appear to have differentiated in the Pleistocene (< tains may have primarily survived by altitudinal migra- 1.75 Myr, www.iugs.org/iugs/pubs/intstratchart.htm) in tions due to absence of an ice sheet across lowlands in most of the estimates using different calibration points. southern Europe (Van Andel & Tzedakis, 1996; Hewitt, The above figures are higher than time of speciation cal- 2000). The importance of re-colonization from nunatak culated in oceanic islands (< 0.73 for silverswords areas should be analysed in future investigations. There- alliance, Baldwin & Sanderson, 1998; < 0.79 for fore, recurrent altitudinal re-colonizations of neighbour- Dendroseris , Sang & al., 1994), but similar to diversifi- ing populations, as found in Armeria from Sierra Nevada cation rates within herbaceous species from Medi- (Gutiérrez & al., 2002), and migrations from northern terranean floristic regions of America (< 1.5 Myr for regions, as inferred in Saxifraga oppositifolia from the

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arctic (Holderegger & al., 2002; this paper), may hend all factors responsible for present distributions and account, in part, for present distribution and genetic predominant modes of speciation in Mediterranean structure of alpine plants in Mediterranean Europe. Europe. Additionally, colonization patterns in the four geograph- ic directions (north-to-south, south-to-north, east-to- west, and west-to-east) are herein also documented. Multiple diversification patterns are indeed promot- ACKNOWLEDGEMENTS ed by disparate biological characteristics (population I thank the symposium group at Patras (Greece), our seminar structure, phylogenetic relationships, breeding system, participants, Rafael Álvarez, Rolf Holderegger, Modesto Luceño, dispersal syndromes, and ecological requirements), Gonzalo Nieto, Virginia Valcárcel, and an anonymous reviewer for together with geologic complexity of the European con- helpful comments on the manuscript; J. Muñoz for his help with tinent, recent climatic events (glaciations), and pre- map arrangements; and Tove Gabrielsen for leaf material of Holocene genetic structure (Taberlet & al., 1998; Comes Saxifraga oppositifolia from Norway. This research has been sup- & Kadereit, 1998; Hewitt, 2000). Lack of parallelism in ported by the Spanish Direcció n General de Investigació n diversification patterns among our seven plant groups, Científica y Té cnica (DGICYT) and Comunidad Autó noma de which share similar phylogenetic relationships Madrid through the projects PB98-0535 and 07M/0124/2000. (Primulaceae, Saxifraga spp.), dispersal syndromes (neighborhood diffusion through seeds in capsules for five cases), habitat requirements (rocky slopes), breeding systems (predominant allogamy), and existence in the LITERATURE CITED same continent during Quaternary episodes, indicates Abbott, R. J., Smith, L. C., Milne, R. I., Crawford, R. M. additional causes. Most alpine species are distributed M., Wolff, K. & Balfour, J. 2000. Molecular analysis of independently of one another along mountain ranges in plant migration and refugia in the arctic. Science 289: 1343–1346. southern Europe, which implies per se different colo- Ager, D. V. 1975. The geological evolution of Europe. Proc. nization and extinction histories. Recent studies by Geol. Ass. 86: 127–154. Taberlet (2002) and collaborators on phylogeography of Avise, J. C. 2000. Phylogeography. The History and six alpine species sharing similar distributions and habi- Formation of Species. Harvard Univ. Press, Cambridge, tats also illustrate that phylogeographic patterns are not Massachusetts. concordant (see also Gugerli & Holderegger, 2001). Avise, J. C., Arnold, J., Ball, R. M., Berminghan, E., Lamb, Additional biotic and abiotic causes may be responsible T., Neigal, J. E., Reeb, C. A. & Saunders, N. C. 1987. Intraspecific phylogeography: the mitochondrial DNA for disparate evolutionary patterns of alpine plants, bridge between population genetics and systematics. Ann. including random processes. Stochastic dispersal in Review Ecol. Syst. 18: 489–522. Saxifraga, with a non-specific long-distance syndrome, Baldwin, B. G. 1993. Molecular phylogenetics of Calycadenia appears to have been involved in colonization of Madeira (Compositae) based on ITS sequences of nuclear riboso- from the continent (c. 1000 km), but isolation of Medi- mal DNA: chromosomal and morphological evolution terranean populations of S. globulifera by the formation reexamined. Amer. J. Bot. 80: 222–238. of the Strait of Gibraltar (separated by only c. 14 km) Baldwin, B. G ., Sanderson, M. J., Porter, J. M., Wojciechowski, M. F ., Campbell, C. S. & Donoghue, appears to have been successful (Vargas & al., 1999a). M. J. 1995. The ITS region of nuclear ribosomal DNA: a This surprising result is in agreement with a considerable valuable source of evidence on angiosperm phylogeny. high number (c. 29 %) of colonizers of Macaronesia dis- Ann. Missouri Bot. Gard. 82: 247–277. playing non-specific long-distance syndromes (Vargas, Baldwin, B. G . & Sanderson, M. J. 1998. Age and rate of in press). Ecological facilitation (Callaway, 1995), in diversification of the Hawaiian silverswor d alliance which positive interactions among plants facilitate estab- (Compositae). Proc. Natl. Acad. Sci. U.S.A. 95: lishment and survival of species, has also played an 9402–9406. Barraclough, T. G. & Nee, S. 2001. Phylogenetics and speci- important role in stochastic processes for colonization. In ation. Trends Ecol. Evol. 16: 391– 399. this context, it has been recently demonstrated that inter- Brochmann, C., Gabrielsen, T. M., Hagen, A. & Tollefsrud, actions among alpine plants of physically harsh environ- M. M. 1996. Seed dispersal and molecular phylogeogra- ments in mountains of America and Eurasia are predom- phy: glacial survival, tabula rasa , or does it really matter? inantly positive (Callaway & al., 2002), including Norsk. Vidensk.-Akad. , Mat.-Naturvidensk. Kl. , Avh. 18: Juniperus communis , Saxifraga oppositifolia , and Solda- 54–68. nella alpina in the study. Comparative phylogeography Callaway, R. M. 1995. Positive interactions among plants. Bot. Rev. 61: 306–349. of alpine plants lead us to conclude that we are not only Callaway, R. M., Brooker , R. W., Choler, P., Kikvidze, Z., far from compiling data from a significant number of Lortie, C. J., Michalet, R., Paolini, L., Puignaire, F. I., species to infer common patterns, but also to compre- Newingham, B., Aschehoug, E. T., Armas, C., Kikodze,

474 52 • August 2003: 463–476 Vargas • Alpine plants in Mediterranean Europe

D. & Cook, B. J. 2002. Positive interactions among alpine Hewitt, G. M. 2000. The genetic legacy of the Quaternary ice plants increase with stress. Nature 417: 844–848. ages. Nature 405: 907–913. Chater, A. O. & Halladay, G. 1993. Arenaria L. Pp. 140–148 Hillis, D. M., Mable, B. K. & Moritz, C. 1996b. Applications in: Tutin, T. G., Burges, N. A., Chater, A. O., Edmondson, of molecular systematics: the state of the field and look to J. R., Heywood, V . H., Moore, D. M., V alentine, D. H., the future. Pp. 515– 543 in: Hillis, D. M., Moritz, C. & Walters, S. M. & Webb, D. A. (eds.), Flora Europaea , ed. Mable, B. K. (eds.), Molecular Systematics, Sinauer 2. vol. 1, Cambridge Univ. Press, Cambridge. Associates, Sunderland, Massachusetts. Comes, P . H. & Kadereit, J. W . 1998. The effect of Hillis, D. M., Moritz, C. & Mable, B. K. 1996a. Molecular Quaternary climatic changes on plant distribution and evo- Systematics. Sinauer Associates, Sunderland, Massa- lution. Trends Plant Sci. 3: 432–438. chusetts. Conti, E., Soltis, D. E., Hardig, T. M. & Schneider, J. 1999. Holderegger, R., Stehlik, I. & Abbott, R. J. 2002. Molecular Phylogenetic relations hips of the silver saxifrag es analysis of the Pleistocene history of Saxifraga oppositifo- (Saxifraga, Sect. Ligulatae Haworth): implications for the lia. Molec. Ecol. 11: 1409–1418. evolution of substrate specificity, life histories, and bio- Hungerer, K. B. & Kadereit, J. W. 1998. The phylogeny and geography. Molec. Phyl. Evol. 13: 536–555. biogeography of Gentiana L. sect. Ciminalis (Adans.) Cullen, J. 1968. Anthyllis L. Pp. 177– 182 in: Tutin, T . G ., Dumort.: a historical interpretation of distribution ranges Heywood, V. H., Burges, N. A., Moore, D. M., Valentine, in the European high mountains. Pers. Pl. Ecol. Evol. Syst. D. H., W alters, S. M. & W ebb, D. A. (eds.), Flora 1: 121–135. Europaea, vol. 2. Cambridge University Press, London. Jordano, P . 1993. Geographical ecology and variation of Demesure, B., Comps, B. & Petit, R. J. 1996. Chloroplast plant-seed disperse r interacti ons: southern Spanish DNA phylogeography of commom beech ( Fagus sylvatica junipers and frugivorous thrushes. Vegetatio 107/108: L.) in Europe. Evolution 50: 2515–2520. 85–104. Dorofeev, P. I. 1963. Primulaceae. Pp. 517–518 in: Takhtajan, King, R. & Ferris, C. 1998. Chloroplast DNA phylogeogra- A. L. (ed.), Oznovij Paleontologii . Akademia Nauka, phy of Alnus glutinosa L. Molec. Ecol. 7: 1157–1161. Moscow. Körner, C. 1999. Alpine Plant Life: Functional Plant Ecology Dumolin-Lapègue, S., Demesure, B., Fineschi, S., Le Corre, of High Mountain Ecosystems . Springer-V erlag, Heidel- V. & Petit, R. J. 1997. Phylogeographic structure of white berg. oaks throughout the European continent. Genetics 146: Kress, A. 1984. Primulaceen-Studien 7: Chromosomenzä h- 1475–1487. lungen an verschiedenen Primulaceen. Teil B, Soldanella. Farjon, A. 2001. World Checklist and Bibliograp hy of Botanical Garden, Munich. Conifers,ed. 2. Royal Botanic Gardens, Kew. Kress, A. 1997. Androsace. Pp. 22– 40 in: Castroviejo, S., Ferguson, I. K. 1972. Vitaliana Sesler. P. 20 in: Tutin, T. G., Aedo, C., Laínz, M., Morales, R., Muñoz Garmendia, F., Heywood, V. H., Burges, N. A., Moore, D. M., Valentine, Nieto Feliner, G. & Paiva, J. (eds.), Flora Iberica , vol. 5. D. H., W alters, S. M. & W ebb, D. A. (eds.), Flora C.S.I.C., Madrid. Europaea, vol. 3. Cambridge Univ. Press, Cambridge. Kropf, M., Kadereit, J. W . & Comes, P . 2002a. Late Franco, J. A. 1993. Juniperus L. Pp. 46– 48 in: Tutin, T. G., Quaternary distribution stasis in the submediterranean Burges, N. A., Chater, A. O., Edmondson, J. R., Heywood, mountain plant Anthyllis montana L. () inferred V. H., Moore, D. M., Valentine, D. H., Walters, S. M. & from ITS sequences and amplified fragment length poly- Webb, D. A. (eds.), Flora Europaea , ed. 2, vol. 1. morphism markers. Molec. Ecol. 11: 447–463. Cambridge Univ. Press, Cambridge. Kropf, M., Kadereit, J. W . & Comes, P . 2002b. Late Fuertes, J., Rosselló, J. A. & Nieto, G. 1999. Nuclear riboso- Pleistocene range fragment within the mountain plant mal DNA (nrDNA) concerted evolution in natural and arti- Anthyllis montana L. (Fabaceae) revealed by congruent ficial hybrids of Armeria (Plumbaginaceae). Molec. Ecol. patterns of morphometric and genetic variation. P. 301 in: 8: 1341–1346. Sixth Internati onal Congress of Systemati c and Gabrielsen, T . M., Bachmann K., Jacobson, K. S. & Evolutionary Biology. September 9– 16, Patras, Greece. Brochmann, C. 1997. Glacial survival does not matter: [Abstr.] RAPD phylogeography of Nordic Saxifraga oppositifolia . Levin, D. A. & Wilson, A. C. 1976. Rates of evolution in seed Molec. Ecol. 6: 831–842. plants: net increase in diversity of chromosome numbers Goyder, D. J. 1987. Observations on the geographical distri- and species numbers through time. Proc. Natl. Acad. Sci. bution, reproductive biology and ecology of Arenaria U.S.A. 73: 2086–2090. alfacarensis Pamp. Anales Jard. Bot. Madrid 44: 285–297. López, G. 1990. Arenaria L. Pp. 172–224 in: Castroviejo, S., Greuter, W., Burdet, H. M. & Long, G. 1989. Med-Checklist, Aedo, C., Laí nz, M., Ló pez, G ., Morales, R., Muñ oz vol. 4, Dicotyledones (Lauraceae-Rhamnaceae). Geneva, Garmendia, F., Nieto Feliner, G. & Paiva, J. (eds.), Flora Switzerland. Iberica, vol. 2.C.S.I.C., Madrid. Gugerli, F. & Holderegger, R. 2001. Nunatak survival, tabu- Martínez, J. & Vargas, P. , 2002. Chloroplast differentiation la rasa and the influence of the Pleistocene ice-areas on and active dispersal of Juniperus oxycedrus in the plant evolution in mountain areas. Trends Plant Sci. 6: Mediterranean basin based on trnL-trnF sequences. Pp. 26 397–398. in: Phylogeography in Southern European Refugia: Gutiérrez, B., Fuertes, J. & Nieto, G. 2002. Glacial-induced Evolutionary Perspect ives on the Origins and altitudinal migration s in Armeria (Plumbaginaceae) Conservation of European Biodiversity. International inferred from patterns of chloroplast DNAhaplotype shar- Symposium, Vairão (Portugal). March 11–15. Univ. Porto, ing. Molec. Ecol. 11: 1965–1974. Vairão. [Abstr.]

475 Vargas • Alpine plants in Mediterranean Europe 52 • August 2003: 463–476

Mogensen, H. L. 1996. The hows and whys of cytoplasmic Taberlet, P . 2002. Comparative phylogeography of alpine inheritance in seed plants. Amer. J. Bot. 83: 383–404. plants. P . 16 in: Phylogeography in Southern European Nieto, G. & Favarger, C. 1988. On the races of Arenaria tetra- Refugia: evolutionary perspectives on the origins and quetra L. (Caryophyllaceae). Bot. J. Linn. Soc. 97: 1–8. conservation of European biodiver sity. International Petit, R. J., Csaikl, U. M., Bordács, S., Burg, K., Coart, E., Symposium, Vairão (Portugal). March 11–15. Univ. Porto, Cotrell, J., V an Dam, B., Deans, J. D., Dumolin- Vairão. [Abstr.] Lapégue, S., Fineschi, S., Finkeldey, R., Gillies, A., Taberlet, P ., Fumagalli, L., Wust-Saucy, A.G., & Cossons, Glaz, I., Giocoechea, P. G., Jensen, J. S., König, A. O., J.-F. 1998. Comparative phylogeography and postglacial Lowe, A. J., Madsen, S. F., Mátyá s, G., Munro, R. C., colonization routes in Europe. Molec. Ecol. 7: 453–464. Olalde, M., Pemonge, M.-H., Popescu, F ., Slade, D., Taberlet, P ., Gielly, L., Pautou, G . & Bouvet, J. 1991. Tabbener, H., T aurchini, D., de Vries, S. G . M., Universal primers for amplification of three non-coding Ziegenhagen, B. & Kremer, A. 2002. Chloroplast DNA regions of chloropl ast DNA. Pl. Molec. Biol. 17: variation in European white oaks: phylogeography and 1105–1109. patterns of diversity based on data from over 2600 popu- Templeton, A. R. 1980. The theory of speciation via founder lations. Forest Ecol. Management 156: 5–26. principle. Genetics 94: 1011–1038. Raspé, O., Saumitou-Laprade, P ., Cuguen, J. & Jacque- Van Andel, T. H. & Tzedakis, P . C. 1996. Paleolithic land- mart, A.-L. 2000. Chloroplast DNA haplotype variation scapes of Europe and environs, 150,000–25,000 years ago: and population differentiation in Sorbus aucuparia L. an overview. Quat. Sci. Rev. 15: 481–500. (Rosaceae: Maloideae). Molec. Ecol. 9: 1113–1122. Van der Merwe, M., Winfield, M. O., Arnold, G . M. & Richardson, J. E., Pennington, R. T., Pennington, T. D. & Parker, J. S. 2000. Spatial and temporal aspects of the Hollingsworth, P . M. 2001. Rapid diversification of a genetic structure of Juniperus communis populations. species-rich genus of neotropical rain forest trees. Science Molec. Ecol. 9: 379–386. 293: 2242–2245. Vargas, P. 1997. Saxifraga L. Pp. 162–242 in: Castroviejo, S., Rieseberg, L. R. & Brouillet, L. 1994. Are many plant species Aedo, C., Laínz, M., Morales, R., Muñoz Garmendia, F., paraphyletic? Taxon 43: 21–32. Nieto Feliner, G. & Paiva, J. (eds.), Flora Iberica , vol. 5. Ronquist, F . 1997. Dispersal-vi cariance analysis: a new C.S.I.C., Madrid. approach to the quantification of historical biogeography. Vargas, P. 2001. Phylogenetic and evolutionary insights in the Syst. Biol. 46: 195–203. Saxifraga pentadactylis complex (Saxifragaceae): varia- Sanderson, M. J. 2002. Estimating absolute rates of molecular tion of nrITS sequences. Nordic J. Bot. 21: 75–82. evolution and divergence times: a penalized likelihood Vargas, P. 2002. Multiple diversification patterns of alpine approach. Molec. Biol. Evol. 19: 101–109. plants in the Mediterr anean Europe. P . 229 , Sixth Sang, T., Crawford, D. J., Kim, S.-C. & Stuessy, T. F. 1994. International Congress of Systematic and Evolutionary Radiation of the endemic genus Dendroseris (Asteraceae) Biology. September 9–16, Patras, Greece. on the Juan Fernández Islands: evidence from sequences Vargas, P. In press. Are Macaronesian islands refugia of relict of the ITS regions of nuclear ribosomal DNA. Amer. J. plant lineages?: a molecular survey. In: Weiss, S. J. & Bot. 81: 1494–1501. Ferrand, N. (eds.), Phylogeography in Southern European Schaal, B. A. & Olsen, K. M. 2000. Gene genealogies and Refugia: Evolutionary Perspectives on the Origins and population variation in plants. Proc. Natl. Acad. Sci. Conservation of European Biodiversity . Kluwer Academic U.S.A. 96: 302–306. Publishers, Dordrecht, The Netherlands . Schönswetter, P ., T ribsch, A., Barfuss, M. & Niklfeld, H. Vargas, P., Baldwin, B. G. & Constance, L. 1999b. Aphylo- 2002. Several Pleistocene refugia detected in the high genetic study of Sanicula sect. Sanicoria and S. sect. alpine plant Phyteuma globulariifolium Sternb. & Hoppe Sandwicenses (Apiaceae) based on nuclear rDNA and (Campanulaceae) in the European Alps. Molec. Ecol. 11: morphological data. Syst. Bot. 24: 228-248. 2637–2647. Vargas, P., Morton, C. & Jury, S. L. 1999a. Biogeographic Shigesada, N., Kawasaki, K. & Takeda, Y. 1995. Modeling patterns in Mediterranean and Macaronesian species of stratified diffusion in biological invasions. Amer. Nat.. Saxifraga (Saxifragaceae) inferred from phylogenetic 146: 229 251. analyses of ITS sequences. Amer. J. Bot. 86: 724–734. Stebbins, G . L. 1984. Polyploidy and the distribution of the Vargas, P. & Nieto Feliner, G. 1996. Artificial hybridization arctic-alpine flora: new evidence and a new approach. Bot. within Saxifraga pentadactylis (Saxifragaceae). Nord. J. Helv. 94: 1–13 Bot. 16: 257–266. Stehlik, I. 2000. Nunatak and peripheral refugia for alpine Webb, D. A. 1993. Saxifraga L. Pp. 437–458 in: Tutin, T. G., plants during Quaternary glaciation in the middle part of Burges, N. A., Chater, A. O., Edmondson, J. R., Heywood, the Alps. Bot. Helv. 110: 25–30. V. H., Moore, D. M., Valentine, D. H., Walters, S. M. & Stehlik, I., Blattner, F. R., Holderegger, R. & Bachmann, K. Webb, D. A. (eds.), Flora Europaea , vol. 1, ed. 2. 2002. Nunatak survival of the high alpine plant Cambridge Univ. Press, Cambridge. Eritrichium nanum (L.) Gaudin in the central Alps during Webb, D. A. & Gornall, R. J. 1989. Saxifrages of Europe. the ice ages. Molec. Ecol. 11: 2027–2036. Christopher Helm, London. Stuessy, T. F. 1990. Plant . The Systematic Zhang, L., Comes, H. P. & Kadereit, J. 2001. Phylogeny and Evaluation of Comparative Data. Columbia Univ. Press, Quaternary history of the European montane/alpine New York. endemic Soldanella (Primulaceae) based on ITS and Suc, J. P. 1984. Origin and evolution of the Mediterranean veg- AFLP variation. Amer. J. Bot. 88: 2331–2345. etation and climate in Europe. Nature 307: 429–432.

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