Molecular phylogeny and systematics ofGenista(Leguminosae) and ...

Plant Syst. Evol. 244: 93–119 (2004) DOI 10.1007/s00606-003-0091-1

Molecular phylogeny and systematics of Genista (Leguminosae) and related genera based on nucleotide sequences of nrDNA (ITS region) and cpDNA (trnL-trnF intergenic spacer) C. Pardo1, P. Cubas1, and H. Tahiri2 1 2

Departamento de Biologı´ a Vegetal II, Facultad de Farmacia, Universidad Complutense, Madrid, Spain De´partement de Biologie, Faculte´ des Sciences, Universite´ Mohammed V, Rabat, Morocco

Received April 9, 2003; accepted October 9, 2003 Published online: February 3, 2004 Springer-Verlag 2004

Abstract. Phylogenetic relationships of Genista and related genera (Teline, Chamaespartium, Pterospartum, Echinospartum, Ulex, Stauracanthus and Retama) were assessed by the analysis of sequences of the nrDNA internal transcribed spacer (ITS region), and the cpDNA trnL-trnF intergenic spacer. The tree obtained by combining both sets of data indicates the existence of three lines of diversification within Genista, that correspond to three subgenera: Genista, Phyllobotrys and Spartocarpus, however, each of these lineages encompass also species of the related genera Echinospartum, Teline, Retama, Chamaespartium, Pterospartum, Ulex, Stauracanthus. The molecular data do not support division of these subgenera into taxonomical units at the sectional level; only sections Genista and Spartocarpus are monophyletic groups. The sequences of both regions are also informative at the specific level, grouping morphologically related species (e.g. the G. cinerea aggregate). The molecular data have also helped to clarify the position of taxa whose relationships were not well established (e.g. G. valdes-bermejoi). The relationships of related genera that belong to the Genista lines of diversification have also been investigated. Echinospartum splits into two separate clades matching the separation of two ecological and caryological differentiated groups. Teline also forms two groups, both placed near to Genista

subgenus Genista, but that separated from the main core of the group. Retama, morphologically well differentiated from Genista, is close to Genista subgenus Spartocarpus. Chamaespartium and Pterospartum do not form a monophyletic group. Chamaespartium is closer to Genista subgenus Genista, whereas Pterospartum stands close to: 1) Genista subgenus Spartocarpus (particularly, sect. Cephalospartum); and 2) the Ulex-Stauracanthus clade (a terminal derivative of Genista subgenus Spartocarpus). Cases of incongruence (e.g. Echinospartum, Chamaespartium, Teline) between the trees obtained from the two molecular markers, may be indicating hybridisation and/or introgression between different lines of Genisteae. Key words: Genista, Genista-group, Genisteae, ITS region, trnL-trnF intergenic spacer, phylogeny, systematics.

Genista L. is a large genus of spiny and nonspiny shrubs centred in the Mediterranean region. The genus is also represented throughout most of western and central Europe, extending to the southeast of the former USSR, and to Turkey, Syria and North Africa. Gibbs (1966) recognised 72 species, ranging in distribution from virtually pan-European to restricted montane endemics such as G. teretifolia

94

C. Pardo et al.: Molecular phylogeny and systematics of Genista

and G. dorycnifolia, among others. Many more species have been added since Gibbs’ monograph (1966), and up to 100 entities have been recognised (e.g. Valsecchi 1993). Subgeneric and sectional units were first established by Spach (1844–1845). Later on Gibbs (1966) excluded from Genista several groups of species, transferring them to different genera (Echinospartum (Spach) Rothm., Teline Webb and Genistella Ortega), and reorganised the remaining ones in three subgenera: Genista, Spartocarpus, and Phyllobotrys. Taxonomical criteria were based on leaves (simple vs. trifoliate), branching pattern (alternate, subopposite and opposite), size and shape of petals, and legume characters. Gibbs (1966) indicated that the three subgenera are fairly distinctive, although examples of intermediate taxa can be found for almost all the differential characters. Other authors merged the two first subgenera, recognising only two: Genista and Voglera, e.g. Talavera and Salgueiro (1999). In the circumscription of Gibbs (1966), Genista subgenus Genista is distributed in the Mediterranean region, Balkan Peninsula and Turkey, with two species (G. tinctoria and G. pilosa) extending into NW Europe. Genista subgenus Phyllobotrys is mainly centred in the west of the Iberian Peninsula, but two species (G. anglica and G. germanica) extend into north and central Europe, and others can be found in North Africa, Yugoslavia, Sicily, Mallorca and W Turkey. Genista subgenus Spartocarpus is mainly centred in the Balkan Peninsula and the eastern Mediterranean, but has a secondary centre in the Balearic Islands, Sardinia, Sicily, North Africa, and southern Spain. Thus although there are some general trends, there is not a sharp distinctive geographic pattern of distribution that matches the morphological discontinuities in the present taxonomic scheme of the genus. Apart from nomenclatural discrepancies, at the sectional level there is a general agreement between different authors, except concerning subgenus Spartocarpus. This group, the most variable in vegetative characteristics, has been either regarded as a separate genus

(Cytisanthus O. Lang, Gams 1923) or has been included in Genista. As a subgenus of Genista, different taxonomic frameworks have been proposed which range from four (Gibbs 1966) to six sections (Valsecchi 1993, Talavera and Salgueiro 1999). There are several species close to Genista for which there is no general agreement on whether, or not, they should be included within the genus. Different taxonomic treatments have been proposed for these species, and for many authors they are considered as separate genera (i.e. Teline, Chamaespartium Adans. and Pterospartum Willk.). The mutual relationships and boundaries between these taxa have not been resolved yet, and consequently the rank deserved by some of them is still a matter of controversy. Other morphologically related genera that belong to the Genista line of diversification (Genista-group: Echinospartum, Ulex L., Stauracanthus Link and Retama Raf.; Bisby 1981) have also to be considered. Ulex and Stauracanthus include species diversified in the Iberian Peninsula and Morocco, with some of the Ulex species extending into western Europe. These genera are very close from the morphological point of view, and molecular data have shown that they form a monophyletic group (Aı¨ nouche et al. 2003). Their relationships with the other genera have not yet been established. Echinospartum comprises five species that form two groups, ecologically and caryologically differentiated: E. ibericum, E. barnadesii and E. algibicum (silicicolous, 2n ¼ 52 chromosomes), and E. boissieri and E. horridum (calcicolous, 2n ¼ 44). The genus is restricted to mountain areas of the Iberian Peninsula extending into southern France. Its systematic position and relationships within the tribe have been considered in different ways, depending on the features emphasised in classification. Retama is a small genus extending from the Iberian Peninsula to northern Africa, Israel and Syria. Morphologically, the genus is well characterised by its flowers in racemes and reduced legume, ovoid to globose.


Molecular data from DNA studies on Genista and related genera have been included in broader studies concerning the phylogeny of Papilionoideae (Ka¨ss and Wink 1995, 1997a), Genisteae (Crisp et al. 2000, Percy and Cronk 2002), Cytisus Desf. (Cubas et al. 2002), Lupinus L. (Ka¨ss and Wink 1997b, Aı¨ nouche and Bayer 1999), and Ulex (Aı¨ nouche et al. 2003), and recently, based on nuclear (ITS) and chloroplast (trnL intron) regions the molecular relationships in Genista sect. Spartocarpus have been studied by De Castro et al. (2002). However, specific molecular data on Genista and other related genera remain scarce. In this study we use molecular data from two different DNA regions, the nuclear ITS region and the chloroplast non-coding trnL-trnF intergenic spacer to elucidate taxonomic and phylogenetic relationships within the Genistagroup (Bisby 1981) and check the generic limits of the taxa included in this line (Echinospartum, Chamaespartium, Pterospartum, Ulex, Stauracanthus, Teline and Retama). Our goals were to: 1) compare the molecular phylogeny of Genista with the current taxonomic framework based on morphological, serological and other systematics analyses; and 2) address some of the controversies at the infrageneric and sectional ranks within Genista. Material and methods Plant material. Seventy eight taxa of Genista and related genera (Echinospartum, Chamaespartium, Pterospartum, Ulex, Stauracanthus, Teline and Retama) were analysed (Table 1). Multiple samples of one species were analysed to evaluate infraspecific variability. Plants were collected in the field by the authors or obtained through the courtesy of colleagues. Leaves and young twigs were either frozen or dried in silica gel, or seedlings were obtained from seeds of wild plants. Additionally several samples were obtained from herbarium specimens (up to 10 years old) or plants growing at different Botanic Gardens. We have concentrated the sampling in the western Mediterranean area, including representatives of all the sections recognised by Gibbs (1966), except Fasselospartum.

95

Sequences of Crotalaria L., Thermopsis Aiton & Aiton f. and Melolobium Ecklon & Zeyher obtained from GenBank were used as outgroups. Several sequences representing Genista subgenus Spartocarpus, already available in the GenBank, have also been included in the ITS analyses. In the separate analyses of both regions, selected sequences of the Cytisus group (Cytisus, Spartocytisus Webb & Berth., Chamaecytisus Link, Cytisophyllum O. Lang, Hesperolaburnum Maire, Laburnum Fabr., Calicotome Link) have also been used to obtain a more comprehensive picture of the tribe. Accession numbers and sources of these sequences are indicated in Table 2. DNA isolation and PCR amplification procedure. Total DNA was extracted from individual plants using DNeasy Plant Mini Kits from Qiagen (Courtaboeuf, France) following the manufacturer’s protocol. Double-stranded DNA amplifications were performed in 50 ll volumes containing 2 mM MgCl2, 200 lmol/l of each dNTP, 0.5 lmol/l of each primer, 1 U of DNA polymerase (Biotools), and approximately 50 ng of DNA. The entire ITS region, comprising ITS1, 5.8S gene and ITS2, was amplified with external ITS5 or ITS1, and ITS4 universal primers (White et al. 1990). The trnL-trnF intergenic spacer of the chloroplast genome was amplified with primers e and f (Taberlet et al. 1991). PCR conditions and purification of the amplicons follows the procedure of Cubas et al. (2002). Sequencing was performed on both strands using the ABI PRISMTM Dye Terminator Cycle Sequencing Ready Reaction Kit (PE Biosystems), with the amplification primer. Sequencing reactions were electrophoresed on a ABI PRISM 377 DNA Sequencer, or 3730 DNA analyzer (Applied Biosystems) at the Centro de Geno´mica y Proteo´mica (Universidad Complutense, Madrid). Phylogenetic analysis. Sequences of Leguminosae obtained from GenBank of Thermopsis (Thermopsidae), Crotalaria (Crotalarieae), and Melolobium (Genisteae) included in the genistoid alliance (Polhill 1994) were used as outgroups because they do not enter the core genistoid clade (Crisp et al. 2000). Multiple alignment of the sequences was obtained using the Clustal program (Higgins et al. 1992). Parsimony analyses were performed using PAUP version 4.0b4a for Macintosh (Swofford 2000) with unweighted characters and characters states. For the ITS region analysis, the very

439 382 438 424 478 500 530 299 451 245 449 205 263 335 296 536 400

Echinospartum boissieri (Spach) Rothm.

Echinospartum horridum (Vahl) Rothm.

Echinospartum ibericum Rivas Mart. et al.

Genista anglica L.

Genista aspalathoides Lam.

Genista benehoavensis (Bolle) Arco

Genista berberidea Lange Genista carpetana Lange

Genista cinerascens Lange

Genista cinerea (Vill.) DC. subsp. cinerea

Genista cinerea subsp. speciosa Rivas Mart. et al. Genista cinerea subsp. ausetana O. Bolo`s & Vigo 533

410 208

Echinospartum barnadesii (Graells) Rothm.

Genista aetnensis (Biv.) DC.

Sample

Taxon

Spain: Zaragoza, Los Pintanos, Collado Puig Darto, MAF162925

Spain, Avila, Puerto de Mijares, MAF160156 Spain: Avila, Plataforma Circo, Prado de las Pozas, MAF159291 Spain, Albacete, Puerto de las Crucetillas, MAF148144 Spain, Huesca, subida a Monrepo´s, MAF152961 Spain, Huesca, Vio, salida del Canõn del An˜isclo, MAF162314 Spain, Salamanca, Navasfrias, MAF160136 Spain, Zamora, Ribadelago Nuevo, MAF162105 Italy, Sicilia, Catania, MAF161132 Switzerland, Jardin Botanique de Gene`ve, MAF162677 Spain, Zamora, entre Ferreras de Abajo y Ferreras de Arriba, MAF148161 Italy, Sicile, Pizzo del Duce, Ile de Pantelleria, MAF160918 Spain, Canary Islands, La Palma, Cumbres de Puntallana Spain, Lugo, Vivero, LOU24707 Spain, Avila, Plataforma Circo, Prado de las Pozas, MAF159292 Spain, Zamora, Ribadelago Nuevo, MAF148173 Spain, Zamora, Ribadelago Nuevo, MAF148173 Spain, Avila, entre Ramacastanãs y Mombeltrań, MAF147898 France, Alpes de la Haute Provence, environs Gap, MAF139476 Spain, Cuenca, Casas de Haro, MAF160149

Origin, Voucher

AY263638

AY263637

AY263636

– – AY263635

AY263633 AY263634

AY263632

–

–

AY263628 AY263629 AY263630 AY263631

AY263626 AY263627

AY263625

AY263624 AF351091

ITS region

–

AY264006

–

AY264003 AY264004 AY264005

AY264001 AY264002

AY264000

AY263999

AY263998

AY263996 AY263997 – –

AY263994 AY263995

AY263993

AY263992 –

trnL-trn F IGS

Table 1. Accession data for the Genisteae taxa sampled for phylogenetic analyses for the ITS region of nuclear ribosomal DNA and trnL-trnF intergenic spacer of chloroplast DNA

96 C. Pardo et al.: Molecular phylogeny and systematics of Genista

Spain, Castelloń, Sierra Engarceran, Coll Bandereta, MAF159887 Spain, Salamanca, entre San Felices de los Gallegos y Lumbrales, MAF148697 Ibidem Spain, Palencia, Velilla del rı´ o Carrioń, MAF162926 Italy, Trieste, Draga Santa Elia, MAF144915 Spain, Jaen, El Yelmo, MAF148733 Spain, Jaen, El Yelmo, MAF148733 Spain, Mallorca, Coma de N’Arbona, Fornaluch, Jardı´ Bota`nic de So´ller 980603 261

Genista hystrix Lange subsp. hystrix

Genista majorica Canto´ & M.J. Sańchez

Genista hystrix subsp. legionensis (Pau) Gibbs Genista januensis Viv. Genista longipes Pau subsp. longipes 523 260 378 328

316 534

484 357

Genista haenseleri Boiss. Genista hirsuta Vahl subsp. hirsuta

Genista hirsuta subsp. lanuginosa (Spach) Nyman Genista hispanica L. subsp. hispanica

Spain, Alicante, Orihuela, Puerto de Rebate, MAF155287 Italy, Sicily, MAF159008 Spain, Eivissa, Port Roig, Jardı´ Bota`nic de So´ller 010059 Spain, Eivissa, Puig Falco´, Jardı´ Bota`nic de So´ller 010219 Spain, Avila, Pinar de Hoyocasero, MAF159295 Spain, Zamora, entre Ferreras de Abajo y Ferreras de Arriba, MAF149159 Spain, Avila, Puerto del Pico-Cuevas del Valle, MAF159294 Spain, Madrid, El Escorial, Silla de Felipe II, MAF159898 Spain, Caćeres, Herva´s, MAF159307 Spain, Leoń, Puerto de Tarna, MAF159896 Spain, Logronõ, Piqueras a Logronõ, MAF153725 Spain, Ma´laga, Ojeń, MAF153735 Spain, Caćeres, Montfraguë, MAF159913 Spain, Caćeres, Caćeres a Torrejoń el Rubio, desvı´ o a Moroy, MAF162129 Spain, Ma´laga, Marbella a Ojeń, MAF162117

233

250

209

213 339

331

455 330

562

249 292 448 322 462

Genista florida subsp. polygaliphylla (Brot.) Cout.

Genista florida L. subsp. florida

Genista cinerea subsp. murcica (Coss.) Canto´ & M.J. Sańchez Genista cupanii Guss. Genista dorycnifolia Font Quer subsp. dorycnifolia Genista dorycnifolia subsp. grosii (Font Quer) Font Quer & Rothm. Genista falcata Brot.

Table 1 (continued)

AY263650 AY263651 – AY263652

AY263648 AY263649

–

AY263647

AY263646

AF351093 – AY263643 AY263644 AY263645

AF351092

AY263642

–

AY263640 AY263641

AY263639

– –

–

AY264022 AY264023 AY264024 AY264025

AY264020 AY264021

AY264019

AY264018

AY264017

AF352189 AY264014 – AY264015 AY264016

AF352188

AY264013

AF352187

AY264011 AY264012

AY264010

AY264008 AY264009

AY264007

C. Pardo et al.: Molecular phylogeny and systematics of Genista 97

443 537 444 489 396

Genista pilosa L. Genista polyanthos Willk.

Genista pseudopilosa Coss.

quadriflora Munby radiata (L.) Scop. ramosissima (Desf.) Poir. sanabrensis Valde´s Berm. et al. scorpius (L.) DC.

Genista tinctoria L.

Genista spartioides Spach Genista sylvestris subsp. dalmatica (Bartl.) Lindb. Genista teretifolia Willk.

Genista sericea Wulfen subsp. sericea

Genista Genista Genista Genista Genista

445 230 234

535

252 452

262 313 454

510 521 256 482 228 380

470

264

440 287

Genista micrantha Gomez Ortega Genista obtusiramea Spach

Genista pumila (Hervier) Vierh. subsp. pumila Genista pumila subsp. rigidissima (Vierh) Talavera & Saéz

Sample

Taxon

Table 1 (continued)

Spain, Zaragoza, Los Pintanos, La Magdalena, MAF162924 Spain, Alava, Iruraiz-Gauna, Arrieta, MAF155263 Spain, Cantabria, Alonõs, MAF151253 Spain, Avila, Riofrı´ o y Gemiguel, Sierra Yerma, MAF159061

Spain, Zamora, Laguna de los Peces, MAF162137 Spain, Zamora, bajada de la Laguna de los Peces, MAF148171 Spain, Guadalajara, Chequilla, MAF148710 Spain, Ciudad Real, San Lorenzo, Calatrava MAF162928 Spain, Jaen, subida al Yelmo, MAF148151 Morocco, Jbel Rat, MAF162271 Spain, Cuenca, Fuentelespino de Haro, MAF160145 Spain, Soria, Medinaceli-Soria km 169, MAF153720 Spain, Guadalarara, Alcolea del Pinar, MAF162124 Morocco, Bad Berred a Ketama, MA571532 Spain, Jardı´ n Botańico Madrid, MAF162281 Spain, Almeria, Cala Raja, MAF159891 Spain, Zamora, Laguna de los Peces, MAF162113 Spain, Guadalajara, Ambite, MAF159298 Spain, Madrid, Arganda-Morata de Tajunã, km28, MAF160138 Spain, Jaen, Siles a Puerta de Segura, MAF148146 Spain, Jaen, Siles a Puerta de Segura, MAF148146 Italy, Trieste, entre Monrupino y Fernetti, MAF144911 Spain, Almeria, Majada Redonda, MAF159895 Italy, Foggia, Monte San Angelo, MAF 160925

Origin, Voucher

– AF351094 AF351095

AY263668

AY263666 AY263667

– AY263665 –

AY263660 AY263661 AY263662 AY263663 – AY263664

AY263659

–

AY263656 AY263657 AY263658

AY263655 –

AY263653 AY263654

ITS region

AY264045 AF352190 AF352191

–

AY264043 AY264044

AY264040 AY264041 AY264042

AY264035 – AY264036 AY264037 AY264038 AY264039

AY264034

AY264033

AY264030 AY264031 AY264032

AY264028 AY264029

AY264026 AY264027

trnL-trn F IGS

98 C. Pardo et al.: Molecular phylogeny and systematics of Genista

Spain, Valencia, Millares, hacia Dos Aguas, MAF160143 Spain, Granada, subida al Puerto de La Ragua desde Laroles, MAF158232 Morocco, km 108 N de Tafraoute, MA586956 Spain, Jardı´ n Botańico Madrid, MAF162279 Spain, Ca´diz, Los Barrios-Jerez, MAF162559

359 329

394 442 509 519 531

Genista versicolor Boiss.

Hesperolaburnum platycarpum (Maire) Maire Laburnum anagyroides Medikus Pterospartum tridentatum subsp. lasianthum (Spach) Talavera & Gibbs Retama monosperma (L.) Boiss.

379 465 468 487 458 343

Retama sphaerocarpa (L.) Boiss.

Stauracanthus boivinii (Webb) Samp. Stauracanthus genistoides (Brot.) Samp. Ulex europaeus L. subsp. europaeus Ulex micranthus Lange

Morocco, Kenitra, MAF162136 Spain, Toledo, carretera Madrid-Badajoz km 174, MAF162126 Spain, Madrid, Ciudad Universitaria, MAF160442 Spain, Caćeres, Alcańtara, junto al rı´ o Tajo, MAF162127 Spain, Ca´diz, Benalup-Casa Castanõs, MAF162132 Spain, Ca´diz, Chiclana a Vejer, MAF162120 Morocco, Kenitra, peaje, MAF162135 Spain, Pontevedra, Porrinõ, Gańdaras de Boudinõ, LOU24691

Spain, Mallorca, Coll des Coloms, Tosals Verds, Escorca, Jardı´ Bota`nic de So´ller 010058

295

Genista umbellata subsp. equisetiformis (Spach) Rivas Goday & Rivas Mart. Genista umbellata (L’He´r.) Poiret subsp. umbellata Genista valdes-bermejoi Talavera & L. Saéz (= G. acanthoclada var. fasciculata Knoche) Genista valentina (Sprengel) Steud.

461 464

Spain, Murcia, El Gorguel-Portman,, MAF159918

469

239 441 415 467

Genista tridens (Cav.) DC. subsp. tridens

Genista triacanthos Brot. subsp. triacanthos

Genista tournefortii Spach subsp. tournefortii

Spain, Avila, Aliseda de Tormes, MAF159296 Spain, Toledo, Los Ye´benes, MAF160762 Spain, Avila, cerca de la Angostura, MAF162343 Spain, Ca´diz, Benalup-Casa Castanõs, Tajo de Las Figuras, MAF162131 Spain, Ca´diz, Benalup-Casa Castanõs, Tajo de Las Figuras, MAF162133 Spain, Ma´laga, Coin-Mijas, MAF153712

Table 1 (continued)

AY263684 AY263685 AY263686 AY263687

AY263682 AY263683

– AY263681

AY263678 AY263679 AY263680

AY263677

AY263676

AY263675

AY263674

AY263673

AY263672

AF351096 AY263669 AY263670 AY263671

AY264060 AY264061 AY264062 AY264063

AY264058 AY264059

AY264056 AY264057

– – AY264055

AY264054

AY264053

AY264052

AY264051

AY264050

AY264049

AF352192 AY264046 AY264047 AY264048


100


Table 2. Accession data for the sequences obtained from the GenBank.(*) The sequence AF351087 was mislabeled as Echinospartum barnadesii in Cubas et al. (2002) Taxon

References

GenBank no. ITS region or ITS1 / ITS2

Calicotome intermedia C. Presl. Crotalaria podocarpa D.C. Chamaecytisus proliferus (Lf.) Link subsp. proliferus Chamaespartium sagittale (L.) P.E. Gibbs Cytisophyllum sessilifolium (L.) O. Lang Cytisus arboreus subsp. catalaunicus (Webb) Maire Cytisus commutatus (Willk.) Briq. Cytisus fontanesii subsp. plumosus (Boiss.) Nyman Cytisus grandiflorus subsp. haplophyllus (Maire & Sennen) Maire Cytisus heterochrous Colmeiro Cytisus multiflorus (L’He´r.) Sweet Cytisus oromediterraneus Rivas Mart. et al. Cytisus scoparius (L.) Link subsp. scoparius Cytisus striatus (Hill) Rothm. subsp. striatus Cytisus tribracteolatus Webb Cytisus valdesii Talavera & P. E. Gibbs Cytisus villosus Pourr. Echinospartum barnadesii (Graells) Rothm. Genista aetnensis (Biv.) DC. Genista anglica L. Genista aucheri Boiss Genista cinerea (Vill.) DC. Genista clavata Poiret Genista dorycnifolia Font Quer Genista ephedroides DC. Genista florida L. subsp. florida Genista florida L. Genista gasparrini (Guss.) Presl. Genista germanica L. Genista haenseleri Boiss. Genista januensis Viv. Genista nissana Petrovic Genista tinctoria L. (= G. elata Wender.)

Cubas et al. 2002 AF443634 Aı¨ nouche and Bayer 1999 AF007469 Cubas et al. 2002 AF351101

AF443659 AF385938 AF352197

Cubas et al. 2002

AF443630

AF443655

Cubas et al. 2002

AF351104

AF352200

Cubas et al. 2002

AF351124

AF352220

Cubas et al. 2002 Cubas et al. 2002

AF443649 AF351103

AF443674 AF352199

Cubas et al. 2002

AF351116

AF352212

Cubas et al. 2002 Cubas et al. 2002 Cubas et al. 2002

AF443636 AF351106 AF351109

AF443661 AF352202 AF352205

Cubas et al. 2002

AF351120

AF352216

Cubas et al. 2002

AF443642

AF443667


AF443638 AF443641

AF443663 AF443666


AF443639 –

AF443664 AF352183

Ka¨ss and Wink 1997a Cubas et al. 2002 De Castro et al. 2002 Ka¨ss and Wink 1997 Percy and Cronk 2002 De Castro et al. 2002 De Castro et al. 2002 Cubas et al. 2002 Ka¨ss and Wink 1997a De Castro el al. 2002 Ka¨ss and Wink 1997a De Castro et al. 2002 Ka¨ss and Wink 1997a De Castro et al. 2002 Ka¨ss 1995

Z72258/Z72259 AF351090 AJ294517/AJ294518 Z72260/Z72261 AF330658 AJ402864/AJ402865 AJ402866/AJ402867 AF351087 * Z72264/Z72265 AJ402872/AJ402873 Z72266/Z72267 AJ402876/AJ402877 Z72268/Z72269 AJ294519/AJ294520 Z72261/Z72262

– AF352186 – – – – – – – – – – – – –

trnL-trn F IGS


101

Table 2 (continued) Taxon

References

GenBank no. ITS region or ITS1 / ITS2

trnL-trn F IGS

Genista tricuspidata Desf. Genista umbellata (L’He´r.) Poiret Genista valsecchiae Brullo et De Marco Melolobium microphyllum (L.f.) Eckl. & Zeyh. Pterospartum tridentatum (L.) Willk. Pterospartum tridentatum subsp. tridentatum Spartocytisus supranubius (L.f.) Webb & Berth. Teline canariensis (L.) Webb & Berth. Teline canariensis (L.) Webb & Berth. Teline linifolia (L.) Webb Teline monspessulana (L.) K. Koch Teline monspessulana (L.) K. Koch Teline osyroides (Svent) Gibbs & Dingw. Teline pallida (Poir.) Kunkel Teline stenopetala (Webb & Berth.) Webb & Berth Thermopsis montana Torrey & A. Gray Ulex parviflorus Pourr. subsp. parviflorus

Ka¨ss and Wink 1997a Percy and Cronk 2002 De Castro et al. 2002

Z72272/Z72273 AF330665 AF402868/AF402869

– – –

Crisp et al. 2000

AF287683

–

Ka¨ss and Wink 1997a Cubas et al. 2002

Z72280/Z72281 AF443629

– AF443654

Cubas et al. 2002

AF351102

AF352198

Cubas et al. 2002 Ka¨ss and Wink 1997a Cubas et al. 2002 Cubas et al. 2002 Ka¨ss and Wink 1997a Ka¨ss and Wink 1997a

AF351098 [243] Z72282/Z72283 AF443631 AF443632 Z72284/Z2285 Z72286/Z72287

AF352194 – AF443656 AF443657 – –

Cubas et al. 2002 Ka¨ss and Wink 1997a

AF351097 Z72288/Z72289

AF352193 –

Aı¨ nouche et al. 2003

AF384336/AF384337

AF385937

Cubas et al. 2002

AF443626

AF443651

conserved 5.8S gene has been excluded from the data matrix in order to match the length of the sequences obtained from GenBank. In the trnLtrnF intergenic spacer (IGS), gaps were excluded in positions where the homology could not be ascertained, or mono- or dinucleotide repeats of different length occur. Indels of one or more base pairs were coded and considered as a single event, and the corresponding positions in the sequence alignment excluded from the analyses. For both regions we use starting trees obtained by simple stepwise addition sequences, with one tree held at each step and TBR branch swapping, MULTREES option in effect, accelerated transformation (ACCTRAN), branches of zero length collapsed and topological constraints not enforced. Several analyses were performed including or excluding the coded indels to check the degree of resolution provided by point substitutions alone

and by the indels. Separate and combined analyses of the two nuclear and chloroplast sequences data sets were performed, and strict and 50% majorityrule consensus trees were generated. A limit of 20000 trees was established due to the large number of taxa in the matrices. No differences in the topology of the trees were found in different runs using limits from 10000 to 20000 trees. In order to test the monophyly of Teline and Echinospartum, parsimony analyses with topological constraints were used to filter out trees discovered during the search, and the trees were compared using Templeton’s Wilcoxon signed-ranked test, as implemented in PAUP. The Bootstrap method (Felsenstein 1985) was used in order to estimate the robustness of the various clades revealed in the consensus tree. Bootstrap values (b.v.) were estimated from 1000 replicates of fast-heuristic searches using random addition sequence.

102


Results ITS region. The length of the ITS region (excluding the outgroups) varies from 582 to 606 bp. The aligned data matrix required the inclusions of gapped positions. The matrix used for the analyses includes 490 characters (172 constant and 227 potentially parsimonyinformative), corresponding to ITS1 and ITS2. Base frequencies for the complete ITS region are A ¼ 17.8%, C ¼ 28.6%, G ¼ 31.2%, and T ¼ 22.4%. Parsimony analysis of the ITS region recovered 20000 most parsimonious trees. The 50% majority rule consensus tree (tree length 1080, CI ¼ 0.47, RI ¼ 0.73, HI ¼ 0.53, RC ¼ 0.35) is shown in Fig. 1a and 1b. Similarly to the results found in Cubas et al. (2002), the analyses separate the species of the Cytisus group (Cytisus, Spartocytisus, Chamaecytisus, Cytisophyllum, Hesperolaburnum, Laburnum, and Calicotome) from all the species of the Genista line of divergence (Genista-group, Bisby 1981). Within the Genista line, two groups separate in the ITS tree. They are not supported by bootstrap values (b.v.) but form consistently in all analyses. Group 1 (Fig. 1a) includes all the species of Genista subgenus Genista. Group 2 (Fig. 1b) groups the species of Genista subgenera Phyllobotrys and Spartocarpus. The Teline species are nested in the first group, however they split into two separate clades. Similar results were found in more restricted analyses by Percy and Cronk (2002), and Cubas et al. (2002). Chamaespartium sagittale, Echinospartum boissieri and E. horridum are included in the first group whereas Pterospartum tridentatum, Ulex and Stauracanthus species, E. ibericum and E. barnadesii are in the second one. In Group 1 two clades with Echinospartum horridum and E. boissieri (92% b.v.), and

Teline linifolia and T. pallida (97% b.v.), separate from the rest of the species. The rest of species form two groups. In the first one (A, Fig. 1a) G. ramosissima, G. majorica, G. valentina and G. cinerea form a clade (88% b.v.) sister to a group where G. sanabrensis and G. pilosa separates from the T. canariensis clade (100% b.v.). The second group (B, Fig. 1a) splits in two groups: 1) G. carpetana as sister to two other clades formed by G. pseudopilosa and G. teretifolia (95% b.v.), and G. florida (56% b.v.), and 2) Chamaespartium sagittale and G. scorpius separated from a clade (52% b.v.) with G. januensis and G. tinctoria (67% b.v.); G. cinerascens, G. versicolor, G. benehoavensis and G. obtusiramea (55% b.v.); and a group with G. hystrix, G. longipes and G. pumila. In Group 2, a clade (E. ibericum and E. barnadesii, 100% b.v.) is sister to the rest of the taxa that split in two groups. The first one (C, Fig. 1b) includes two sister clades, the Retama (77% b.v.) and the G. aetnensis clades (79% b.v.; sect. Spartocarpus). G. valdes-bermejoi, which has been included either in subgenus Phyllobotrys (Gibbs 1966) or in subgenus Genista (Talavera and Salgueiro, 1999) appears in this clade. In the second group (D, Fig. 1b) Pterospartum and G. quadriflora form a clade (66% b.v.) sister to a group with G. clavata, G. umbellata, and the Ulex-Stauracanthus clade (58% b.v), and a clade with all the species of subgenus Phyllobotrys (83% b.v). Parsimony analyses with topological constraints were used to test the monophyly of the two genera (Teline and Echinospartum), which are split into two separate groups. Enforcing the monophyly to Teline species leads to an increase of 4 steps in tree length, but does not modify the topology of the tree. In the case of Echinospartum, the tree length increases 3 steps in the constrained analysis, and both groups

c

Fig. 1a. 50% majority rule consensus tree represented as phylogram from the ITS region data set. Numbers above branches represent branch length (ACCTRAN), and numbers below indicate bootstrap values above 50%. Branches present in more than 90% of the most parsimonious trees are indicated by bold lines


4

15 82

4

103

Melolobium microphyllum Crotalaria podocarpa // 31 12 36 // 74 Thermopsis montana 6 Cytisophyllum sessilifolium 1 Laburnum anagyroides 519 3 Cytisus fontanesii 6 Cytisus heterochrous 6 Hesperolaburnum platycarpum 509 8 Spartocytisus supranubius 1 3 16 Cytisus arboreus 8 4 Cytisus tribracteolatus 18 Calicotome intermedia 3 53 7 Cytisus villosus 5 Cytisus commutatus 2 3 6 Chamaecytisus proliferus 3 7 Cytisus valdesii 2 Cytisus striatus 4 Cytisus multiflorus 4 3 Cytisus oromediterraneus 3 71 Cytisus scoparius 2 Cytisus grandiflorus 10 Echinospartum boissieri 439 8 Echinospartum horridum 438 10 92 2 Echinospartum horridum 382 100 9 Teline pallida 6 5 Teline linifolia 97 13 Genista ramossisima 256 5 Genista cinerea 9 2 Genista majorica 328 Group 1 88 1 Genista cinerea subsp. cinerea 536 1 62 Genista8cinerea subsp. ausetana 533 2 Genista cinerea subsp. speciosa 400 3 1 Genista valentina 394 87 3 A 3 Genista sanabrensis 482 2 11 Genista pilosa 443 2 9 Teline canariensis 243 10 Teline stenophylla 1 100 3 Teline monspessulana 259 1 2 Teline monspessulana 1 1 3Teline canariensis 3 Teline osyrioides 21 Genista carpetana 205 Genista pseudopilosa 444 7 2 Genista pseudopilosa 489 1 95 2 Genista teretifolia 535 65 1 3 Genista florida subsp. polygaliphylla 249 5 1 Genista florida 82 2 Genista florida subsp. florida 250 56 1 1 Genista florida subsp. florida 209 4 5 59 Genista florida subsp. polygaliphylla 233 B 17 Chamaespartium sagittale 4 3 Genista scorpius 380 14 2 Genista scorpius 313 100 6 Genista januensis 523 8 Genista januensis 6 93 4 5 Genista tinctoria 8 67 Genista tinctoria 230 2 96 Genista tinctoria 234 4 61 6 Genista tinctoria 239 3 83 6 Genista cinerascens 296 52 3 4 Genista versicolor 442 1 2 Genista benehoavensis 245 55 3 Genista obtusiramea 287 1 8 Genista hystrix subsp. hystrix 316 4 Genista hystrix subsp. legionensis 534 1 9 Genista longipes 260 3 2 5 changes 65 2 1 Genista pumila subsp. rigidissima 470 Genista pumila subsp. pumila 396 90

4

Group 2 (Fig. 1b)

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Fig. 1a

11 100

3

C

4

Group 2

2

3

D

1 Echinospartum ibericum 478 Echinospartum ibericum 424 1 Echinospartum barnadesii 63 Echinospartum barnadesii 410 16 Retama monosperma 464 4 Retama sphaerocarpa 465 8 77 1 Retama sphaerocarpa 379 100 1 Genista aetnensis 8 Genista aetnensis 530 3 100 1 Genista aetnensis 500 94 8 10 Genista valdes bermejoi 329 1 Genista dorycnifolia 79 2 3 5 Genista dorycnifolia subsp. grosii 331 68 7 3 Genista gasparrini 70 100 1 Genista ephedroides 64 2 1 85 Genista valsecchiae 5 Genista radiata 521 6 Genista aucheri 3 9 Genista nissana 87 3 Genista spartioides 252 76 4 Genista haenseleri 3 98 Genista haenseleri 448 95 9 Genista quadriflora 510 4 1 Pterospartum tridentatum subsp. lasianthum 531 7 5 66 Pterospartum tridentatum subsp. tridentatum 2 100 1 Pterospartum tridentatum 67 13 Genista clavata 4 8 Genista umbellata subsp. equisetiformis 295 14 1 Genista umbellata subsp. umbellata 359 3 100 Genista umbellata 14 Stauracanthus genistoides subsp. genistoides 487 5 4 Stauracanthus boivinii 468 4 58 8 Ulex micranthus 343 3 1 79 Ulex parviflorus subsp. parviflorus 2 2 79 4 90 Ulex europaeus subsp. europaeus 458 15 Genista anglica 3 7 Genista hispanica subsp. hispanica 357 3 9 Genista berberidea 449 8 61 6 10 Genista micrantha 440 97 12 83 Genista sylvestris subsp. dalmatica 452 12 Genista triacanthos 467 1 16 Genista falcata 339 2 1 Genista falcata 213 100 1 2 10 Genista tournefortii 441 1 2 Genista tournefortii 415 100 13 Genista tricuspidata 11 3 Genista tridens 469 7 17 Genista germanica // 3 2 Genista hirsuta subsp. lanuginosa 484 13 Genista hirsuta subsp. hirsuta 462 100 1 5 changes Genista hirsuta subsp. hirsuta 322

Fig. 1b. (Continuation of Fig. 1a). 50% majority rule consensus tree represented as phylogram from the ITS region data set. Numbers above branches represent branch length (ACCTRAN), and numbers below indicate bootstrap values above 50%. Branches present in more than 90% of the most parsimonious trees are indicated by bold lines

(silicicolous and calcicolous) appear as sister to the rest of species of Group 2. The rest of the topology remains unchanged. The Templeton’s

Wilcoxon test indicates that there is no significant difference between the unconstrained and the constrained trees in both cases.


trnL-trnF IGS. The length of the trnLtrnF IGS region (excluding the outgroups) varies from 357 to 430 bp. Base frequencies are A ¼ 36.3%, C ¼ 14.5%, G ¼ 11.8%, T ¼ 37.7%. The aligned data matrix has 605 sites and required the inclusion of gapped positions. For the final analyses nineteen indels of one or more base pairs were coded and, where the homology could not be ascertained, gapped positions were excluded. Of the 324 included characters, 164 are constant and 90 potentially parsimony-informative. Most of the indels have high consistency indices, either at the specific level (e.g. G. florida subsp. florida and subsp. polygaliphylla; P. tridentatum subsp. tridentatum and subsp. lasianthum), for closely related species (e.g. E. horridum and E. boissieri), at the sectional level (e.g. G. januensis and G. tinctoria, sect. Genista), or even for genera (e.g. Ulex). Parsimony analysis recovered 20000 most parsimonious trees. The 50% majority rule consensus tree (tree length 269, CI ¼ 0.77, RI ¼ 0.85, HI ¼ 0.23, RC ¼ 0.65) is shown in Fig. 2. Due to the small number of parsimony informative character, the trnL-trnF IGS tree is less resolved than the ITS tree, a result similar to previous studies (Cubas et al. 2002, Aı¨ nouche et al. 2003). All the Genisteae species form a monophyletic clade (98% b.v.) that splits in two groups (Groups 1 and 2, Fig. 2) supported by one change each, and without bootstrap support. Group 1 includes all the species of Genista subgenus Genista (except G. pilosa and G. aspalathoides). In this group, a clade A formed by the species of section Genista (G. tinctoria and G. januensis, 99% b.v.) separates from the rest of the species that form an unresolved group B. Group 2 includes species of Genista subgenera Phyllobotrys and Spartocarpus, all the Genista-related genera and also the Cytisus-group. The topology of this group shows: 1) G. aspalathoides that separates from the rest of species; 2) G. pilosa, Teline species, Retama species, E. boissieri and E. horridum, all unresolved; 3) a group C with Chamaespartium sagittale, G. spartioides, and

105

G. dorycnifolia (section Spartocarpus) and G. valdes-bermejoi; 4) a set D which contains E. ibericum, E. barnadesii, G. quadriflora (sect. Cephalospartum) and P. tridentatum, and G. umbellata (sect. Cephalospartum), and Ulex and Stauracanthus species; 5) a group E formed by all the species of the Cytisus group; and 6) a group F including all the species of Genista subgenus Phyllobotrys, except G. valdes-bermejoi. Despite the small level of resolution at the internal branches, the terminal groups match most of the groupings found in the ITS tree. To compare the information provided by both molecular markers (nuclear and cpDNA), the trees were pruned to match both matrix sets. Comparison of the trees (Fig. 3) shows that both DNA regions recognise two large groups for the Genista line, despite the fact that the trnL-trnF IGS tree is less resolved. The topology of the trees is very similar although several differences are nevertheless apparent. The relationships among the terminal groups in Group 1 are resolved differently by the two DNA regions. Small rearrangements in the relative position of some taxa are indicated in Fig. 3. For example, the G. tinctoria clade is sister to the rest of the species in the trnL-trnF IGS tree, whereas the clade enters an internal group in the ITS region tree. The most conspicuous difference among trees is the position of the Teline species, Echinospartum boissieri and E. horridum, Chamaespartium sagittale and G. pilosa. These taxa stand in Group 1 of the ITS tree but enter in Group 2 in the chloroplast tree. As discussed later, this incongruence between the information provided by both genes could provide a helpful insight to the origin of these taxa. Combined analysis. The matrix of the combined data of the ITS region and trnLtrnF IGS includes 79 samples. Of the 814 characters included in the analysis, 388 are constant and 257 are potentially parsimonyinformative. The parsimony analysis recovered 3480 equally parsimonious trees. The 50% majority rule consensus tree is shown in Fig. 4

106


Crotalaria podocarpa Thermopsis montana Genista tinctoria 230 5 1 Genista januensis 523 99 A 64 Genista tinctoria 234 / 239 5 Genista carpetana 205 / 335 Genista carpetana 263 96 Genista scorpius 380 4 2 Genista scorpius 313 1 1 88 Group 1 Genista scorpius 228 / 262 2 Genista majorica 328 Genista sericea 454 1 Genista cinerea subp. speciosa 400 1 Genista cinerea subsp. murcica 562 1 Genista valentina 394 1 Genista ramosissima 256 Genista benehoavensis 245 2 B Genista sanabrensis 482 Genista obtusiramea 287 1 Genista cinerascens 296 Genista hystrix subsp. legionensis 534 Genista pumila subp. rigidissima 470 / 264 Genista pumila subsp. pumila 396 Genista polyanthos 537 2 2 Genista longipes 260 2 Genista longipes 378 84 Genista pseudopilosa 489 Genista teretifolia 445 versicolor 442 15 1 2 Genista Genista pseudopilosa 444 98 Genista hystrix 316 Genista hystrix 261 2 Genista florida subsp. polygaliphylla 292 1 2 Genista florida subsp. florida 209 / 250 Genista florida subsp. polygaliphylla 233 / 249 77 1 Genista aspalathoides 451 7 Genista pilosa 443 Teline pallida 1 Teline linifolia 4 Teline canariensis 6 Teline monspessulana 86 Echinospartum horridum 382 / 438 3 4 Echinospartum boissieri 439 86 3 Retama sphaerocarpa 379 / 465 1 89 1 Retama monosperma 464 3 90 8 Retama monosperma 461Chamaespartium sagittale 5 Genista spartioides 252 Group 2 1 2 1 Genista valdes bermejoi 329 2 Genista dorycnifolia subsp. grosii 331 C Genista dorycnifolia subsp. dorycnifolia 330 76 Echinospartum ibericum 478 Echinospartum ibericum 424 Echinospartum barnadesii Echinospartum barnadesii 410 3 Genista quadriflora 510 1 1 Pterospartum tridentatum subsp. tridentatum 3 2 Pterospartum tridentatum subsp. lasianthum 531 D 84 1 Genista umbellata subsp. equisetiformis 295 5 Genista umbellata subsp. umbellata 359 1 96 2 1 Stauracanthus boivinii 468 1 1 Stauracanthus genistoides 487 2 84 Ulex micranthus 343 4 81 Ulex parviflorus subsp. parviflorus 98 Ulex europaeus subsp. europaeus 458 4 Cytisus fontanesii 2 4 Cytisophyllum sessilifolium 2 Cytisus heterochrous 2 1 Cytisus tribracteolatus Calicotome intermedia 2 Spartocytisus supranubius E 2 Chamaecytisus proliferus Cytisus commutatus 1 3 Cytisus villosus Cytisus scoparius 1 Cytisus grandiflorus Cytisus arboreus 1 Cytisus striatus 61 1 Cytisus multiflorus Cytisus oromediterraneus 63 Cytisus valdesii Genista falcata 213 / 339 Genista radiata 469 Genista berberidea 449 3 Genista sylvestris subsp. dalmatica 452 2 Genista hispanica 357 1 1 Genista micrantha 440 2 Genista triacanthos 467 F 5 Genista cupanii 455 Genista tournefortii 441 6 Genista tournefortii 415 97 6 Genista anglica 1 Genista anglica 299 97 2 3 changes Genista hirsuta subsp. lanuginosa 484 Genista hirsuta subsp. hirsuta 322 / 462 86 Genista tridens 469 //

19

//


107

b Fig. 2. 50% majority rule consensus tree represented as phylogram from the trnL-trnF IGS data set. Numbers above branches represent branch length (ACCTRAN), and numbers below indicate bootstrap values above 50%. Branches present in more than 90% of the most parsimonious trees are indicated by bold lines

(TL ¼ 1012, CI ¼ 0.59, RI ¼ 0.72, RC ¼ 0.43, HI ¼ 0.41). The analysis shows: 1) a clade (87% b.v.) including all the species of Genista subgenus Phyllobotrys, except G. valdes-bermejoi, as sister to the rest; and 2) a group where a clade (E. ibericum and E. barnadesii, 99% b.v.) is sister to two separated groups. These groups lack bootstrap support but are found consistently in the analyses. The first one includes G. valdes-bermejoi, and all the species of Genista subgenus Spartocarpus, interspersed with Retama, Pterospartum, Stauracanthus and Ulex species. The relationships found in the ITS tree, and partially in the trnL-trnF IGS tree, have stronger support in the combined tree. Particularly, G. valdes-bermejoi forms a highly supported clade (98% b.v.) with G. spartioides and G. dorycnifolia, Pterospartum tridentatum and G. quadriflora forms a clade (81% b.v.), and G. umbellata forms a clade with Ulex and Stauracanthus species (57% b.v). The second group includes two separate clades with Teline pallida and T. linifolia (96% b.v.), Echinospartum boissieri and E. horridum (97% b.v.), and a main core formed by T. canariensis and T. monspessulana (100% b.v.), G. pilosa, Chamaespartium sagittale, and all the species of Genista subgenus Genista. Within the main core the relationships between the taxa are similar to those found in the ITS tree. Same as in the case of the ITS tree, we have enforced a monophyly constraint to Echinospartum and Teline species. Figure 5 shows the position of the tested clades in the constrained and unconstrained trees. In both cases, the Templeton’s Wilcoxon test indicates that there is no significant difference between the trees. Discussion Generic limits. One of the major goals of this work was finding the relationships between

several morphological related genera that are clearly part of the Genista line of diversification but that have been merged or excluded from Genista. The relationships between these genera (Teline, Echinospartum, Retama, Chamaespartium, Pterospartum, Ulex and Stauracanthus) and Genista are complex: some of these genera seem to have diverged from basal Genista ancestors, whereas others are nested inside Genista. Echinospartum. The molecular data indicate that despite the morphological similarities the genus Echinospartum is formed by two clades separately nested in the ITS tree. The calcicolous species (E. boissieri and E. horridum) separated from the line leading to Genista subgenus Genista (Group 1, Fig. 1a), whereas the silicicolous species (E. ibericum and E. barnadesii) diverged from the line which gave rise to Genista subgenera Spartocarpus and Phyllobotrys (Group 2, Fig. 1b). However, the information provided by the chloroplast region indicates that both groups of Echinospartum are related to this second line of Genista (Group 2, Fig. 2): the calcicolous species are unresolved, whereas the silicicolous species are more related to group D (species of sect. Cephalospartum, Pterospartum, Stauracanthus and Ulex) The combined tree positions: 1) E. ibericum and E. barnadesii as sister to the line that led to both Genista subgenera Spartocarpus and Genista, and 2) E. boissieri and E. horridum as a segregate of the Genista subgenus Genista. However, when monophyly of Echinospartum is enforced both Echinospartum clades (silicicolous and calcicolous) appear in the line of the subgenus Genista (Fig. 5A). From the systematic point of view, different taxonomic treatments have been applied to these taxa. Rothmaler (1941) defined a Genista group and an Ulex group, based on the relative depths of the lateral calyx-sinuses, and placed Echinospartum in the Ulex group. Polhill

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C. Pardo et al.: Molecular phylogeny and systematics of Genista trnL trnF IGS

ITS1+ ITS2

Group 1

Group 2

Crotalaria podocarpa Thermopsis montana G. carpetana 205 G. florida polygaliphylla 233 G. florida florida 209 G. florida florida 250 G. florida polygaliphylla 249 G. teretifolia 535 G. pseudopilosa 489 G. pseudopilosa 444 G. tinctoria 239 G. tinctoria 234 G. tinctoria 230 G. januensis 523 G. cinerascens 296 G. versicolor 442 G. benehoavensis 245 G. obtusiramea 287 G. hystrix hystrix 316 G. hystrix legionensis 534 G. longipes 260 G. pumila rigidissima 470 G. pumila pumila 396 C. sagittale G. scorpius 380 G. scorpius 313 G. valentina 394 G. cinerea speciosa 400 G. majorica 328 G. ramossisima 256 G. sanabrensis 482 G. pilosa 443 T. canariensis T. monspessulana T. pallida T. linifolia E. boissieri 439 E. horridum 438 E. horridum 382 R. monosperma 464 R. sphaerocarpa 465 R. sphaerocarpa 379 G. spartioides 252 G. valdes bermejoi 329 G. dorycnifolia 331 G. anglica G. hispanica 357 G. berberidea 449 G. micrantha 440 G. sylvestris 452 G. triacanthos 467 G. falcata 339 G. falcata 213 G. tournefortii 441 G. tournefortii 415 G. tridens 469 G. hirsuta lanuginosa 484 G. hirsuta hirsuta 462 G. hirsuta hirsuta 322 G. umbellata equisetiformis 295 G. umbellata umbellata 359 S. genistoides 487 S. boivinii 468 U. micranthus 343 U. parviflorus U. europaeus 458 G. quadriflora 510 P. tridentatum tridentatum P. tridentatum lasianthum 531

523 230

Group 1

Group 2

E. ibericum 478 E. ibericum 424 E. barnadesii E. barnadesii 410

Fig. 3. Comparison of the topology of the ITS region and the trnL-trnF trees of Figs. 1 and 2. The Cytisusgroup and the taxa where sequences of any of the regions are missing have been pruned


(1976) did not find any sharp demarcation in this character and includes Echinospartum within Genista, as a terminal group nearest Ulex. Gibbs (1966) separated Echinospartum from Genista because the species have an inflated-campanulate calyx, distinctive bracts and a different series of chromosome numbers. He noted similarities with Genista subgenus Spartocarpus in other respects as did Spach (1844–45). Based on serological studies Cristofolini and Feoli-Chiapella (1984) indicated that Echinospartum has connections with Retama, Cytisophyllum and to a lesser extent with Cytisanthus (Genista subgenus Spartocarpus), Spartium and Laburnum. The molecular data neither support a relationship between Echinospartum and Ulex nor a terminal position of Echinospartum within the Genista group. Our data clearly reflect the separation of the two ecological and caryological differentiated groups of Echinospartum, however, the constrained analysis does not reject the monophyly of Echinospartum. This result suggests that they are either phylogenetically unrelated (which conflicts with their morphological similarities) or separated so early from a common ancestor (that later diverged into the different lines of Genista) that the analyses of present species are not able to detect their relationships. On the other hand, our results indicate that at the molecular level E. ibericum and E. barnadesii are not very differentiated. Similar results were shown by Aparicio et al. (2002) using isozyme evidence for these two species and for the third taxa of this complex (E. algibicum, not included in our study). Teline. In the ITS region analysis Teline species split in two separate clades, both inside the line of Genista subgenus Genista (Group 1, Fig. 1a). The T. linifolia clade is sister to the rest of the species whereas the T. monspessulana clade is nested within the group. Similar results where found by Percy and Cronk (2002) and Cubas et al. (2002) with a smaller representation of species of Genista, which suggests that Teline is not a monophyletic genus but is associated with different groups of Genista

109

subgenus Genista. Similar to the results found in Echinospartum, the information provided by the chloroplast region places the Teline species more closely related to the line leading to Genista subgenera Spartocarpus and Phyllobotrys. The combined tree places both Teline clades near to Genista subgenus Genista. Although the infrageneric taxonomy of Genista subgenus Genista cannot yet be completely settled, the available results points that genus Teline should be included as part of this subgenus, bearing in mind that the information provided by the chloroplast region (Fig. 2) and the constrained analysis (Fig. 5C) does not rule out that other taxa related to Genista subgenera Spartocarpus, or even another genus of the tribe, could be involved in its origin. Retama. The ITS region indicates that R. sphaerocarpa and R. monosperma form a monophyletic group (77% b.v.), and in the trnL-trnF IGS region tree, both species also form a group, however, without bootstrap support. Although Retama is morphologically well differentiated from Genista, the molecular data provided by the combined analysis indicate that Retama is close to the species of Genista subgenus Spartocarpus, thus confirming the affinities suggested by Bisby (1981) and Cristofolini and Feoli Chiapella (1984). Chamaespartium and Pterospartum. C. sagittale and P. tridentatum have been included in a single genus (Vicioso 1953, Gibbs 1966, among others), or considered as independent genera (Verlaque 1992, Talavera 1999). However, other authors merge both into Genista (Polhill 1976, Greuter et al. 1989, ILDIS 2002). The molecular data suggest that C. sagittale and P. tridentatum do not form a monophyletic group. The combined tree shows that C. sagittale is closer to Genista subgenus Genista. On the contrary Pterospartum tridentatum stands close to G. quadriflora (within the group of Genista subgenus Spartocarpus sect. Cephalospartum), and to the Ulex-Stauracanthus clade.

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subgenus Phyllobotrys sect. Voglera

9

8 98

1 3

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11 98

subgenus Spartocarpus sect. Spartocarpus

38

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7 81

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sect. Cephalospartum

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0 1 7 3 10 8 2 6 17

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8 11 93

2 62

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2 58

5 4

0 1

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25 4

4 4

19 100

subgenus Genista

11

14 100

sect. Spartioides sect. Scorpioides sect. Erinacoides

4 7 94

6

sect. Genista

1 6 7 4 3 6

2

57 13

2 80 1

3 52 2 3 68

2

9 4 11 2 89

2 1

26

1

Genista valdes bermejoi 329 Genista spartioides 252 Genista dorycnifolia 331 Retama monosperma 464 Retama sphaerocarpa 465 Retama sphaerocarpa 379 Pterospartum tridentatum subsp. tridentatum 344 Pterospartum tridentatum subsp. lasianthum 531 Genista quadriflora 510 Genista umbellata subsp. equisetiformis 295 Genista umbellata subsp. umbellata 359 Stauracanthus boivinii 468 Stauracanthus genistoides 487 Ulex micranthus 343 Ulex parviflorus Ulex europaeus 458 Genista sanabrensis 482 Genista majorica 328 Genista ramossisima 256 Genista cinerea subsp. speciosa 400 Genista valentina 394 Genista pseudopilosa 444 Genista pseudopilosa 489 Genista teretifolia 535 / 445 Genista florida subsp. polygaliphylla 249 Genista florida subsp. polygaliphylla 233 Genista florida. subsp. florida 250 Genista florida subsp. florida 209 Genista carpetana 205 Genista scorpius 380 Genista scorpius 313 Genista januensis 523 Genista tinctoria 230 Genista tinctoria 234 Genista tinctoria 239 Genista cinerascens 296 Genista obtusiramea 287 Genista benehoavensis 245 Genista versicolor 442 Genista hystrix subsp. hystrix 316 Genista hystrix subsp. legionensis 534 Genista longipes 260 Genista pumila subsp. rigidissima 470 Genista pumila subsp. pumila 396 Chamaespartium sagittale

19

2 7 96 11 97

5 8 10 6

15 100 14 10 100

10

1

99

1 62

0 2 1 0 3

outgroups

Genista hispanica 357 Genista micrantha 440 Genista berberidea 449 Genista anglica Genista falcata 339 Genista falcata 213 Genista tournefortii 441 Genista tournefortii 415 Genista tridens 469 Genista triacanthos 467 Genista hirsuta subsp. lanuginosa 484 Genista hirsuta subsp. hirsuta 462 Genista hirsuta subsp. hirsuta 322 Genista sylvestris 452

2 2 2 2 16 12 2 1 1

16 100 16 100 3

2

87

sect. Phyllobotrys

9 7 12

Crotalaria podocarpa Thermopsis montana

Genista pilosa 443 Teline canariensis 243 Teline monspessulana 259 Teline pallida 241 Teline linifolia 258 Echinospartum Echinospartum Echinospartum Echinospartum Echinospartum Echinospartum Echinospartum

boissieri 439 horridum 438 horridum 382 ibericum 478 ibericum 424 barnadesii 208 barnadesii 410


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b Fig. 4. 50% majority rule consensus tree cladogram from the combined data for the Genista-group. Numbers above branches represent branch length (ACCTRAN), and numbers below indicate bootstrap values above 50%. Generic and subgeneric framework follows Gibbs (1966) with minor modifications

A

outgroups

B

outgroups

C

Phyllobotrys

Phyllobotrys

outgroups

Phyllobotrys

Retama Spartocarpus

Spartocarpus

Pterospartum

Stauracanthus Ulex

Retama

Retama

Spartocarpus

Pterospartum

Pterospartum

Stauracanthus

Stauracanthus

Ulex

Ulex E. boissieri E. horridum

Genista

Genista

Chamaespartium Teline Teline

0 changes

E. boissieri E. horridum

Genista

Chamaespartium Teline

Chamaespartium

Teline E. boissieri E. horridum

1 change

Teline Teline

E. ibericum

E. ibericum

E. ibericum

E. barnadesii

E. barnadesii

E. barnadesii

Fig. 5. Comparison of the consensus trees obtained with A) a monophyly constraint of Echinospartum, B) without any constraint, and C) a monophyly constraint of Teline

The question of how both taxa acquired their peculiar winged stem is still an unresolved issue. The morphological similarity between

Pterospartum and Chamaespartium, mainly the winged stem, could be only ‘superficial’, if they are phylogenetically unrelated. However, the

112


incongruence in the position of C. sagittale in the trees suggests clues for alternative explanations. In the ITS tree C. sagittale nests deeply inside Group 1, within Genista subgenus Genista. On the contrary in the chloroplast tree C. sagittale is part of Group 2, which includes the species of Genista subgenus Spartocarpus. Pterospartum tridentatum appears in both trees close to G. quadriflora, a taxon included in section Cephalospartum (part of the Genista subgenus Spartocarpus). These results suggest that the morphological similarity between Pterospartum and Chamaespartium could be the result of both taxa having evolved from the hybridisation of ‘Spartocarpus’ ancestors with different unrelated taxa, one from the Genista subgenus Genista (in the case of C. sagittale), and another from the Genista subgenus Spartocarpus (in the case of Pterospartum). Alternatively, C. sagittale could be the result of a cross between Pterospartum and a taxon of Genista subgenus Genista. Further studies will be required to offer a more definitive answer for this intriguing matter. Ulex and Stauracanthus. Ulex and Stauracanthus are morphologically related, sharing a calyx divided almost to the base in two lips, and a strong reduction of the leaves that in the mature plants become small phyllodes. Polhill (1976) and Vicioso (1962) merged Stauracanthus into Ulex. Reasons to maintain them as separate genera are provided by their chromosome numbers, multiple of n ¼ 12 in Stauracanthus and n ¼ 16 in Ulex (Cubas 1986). As indicated by Aı¨ nouche et al. (2003) and confirmed in our study, Ulex and Stauracanthus form a monophyletic group. However, despite the strong similarities of both genera, all the Ulex species studied so far present a characteristic insertion of 18 bp in the chloroplast sequence that is not shared by the Stauracanthus species. Regarding their relationships within the Genista-group, Gibbs (1966) indicated that Ulex and Stauracanthus share with Genista subgenus Phyllobotrys an important anatomical feature (the leaves with a single vascular trace). Furthermore, serological evidence

suggests that they are very similar and derived from this subgenus (Feoli-Chiapella and Cristofolini 1981). However, our data show that Ulex and Stauracanthus are closely related to G. umbellata, a species included in sect. Cephalospartum (Genista subgenus Spartocarpus). These results support their consideration as terminal derivatives of Genista subgenus Spartocarpus as proposed by Bisby (1981). Infrageneric and sectional boundaries. Another aim of this study was to examine the infrageneric framework of Genista at the subgeneric and sectional level with the molecular data (Table 3). Although not all the species of Genista have been included in this study, the major groups are fairly well represented, which allows to extract some conclusions in this matter. As indicated above, based on morphological characters Gibbs (1966) reorganised the species of Genista into three subgenera: Genista, Spartocarpus, and Phyllobotrys. However, other authors merge the two first subgenera, recognising only Genista (including Spartocarpus) and Voglera (e.g. Talavera and Salgueiro 1999). The ITS and trnL-trnF IGS trees support the separation of two lines of diversification within Genista (Group 1 and Group 2). Group 1 leads to all the species included in Genista subgenus Genista whereas Group 2 includes both Genista subgenera Phyllobotrys and Spartocarpus. The combined tree, however, shows Genista subgenus Phyllobotrys as sister to the other two subgenera. Some incongruence in the relationships between the three subgenera have been found in the analyses, depending on the number of the genera of the tribe that are included. Moreover, some important taxa of sect. Cephalospartum (Genista subgenus Spartocarpus, G. microcephala, G. capitellata and G. cephalantha) are still missing from the analyses. As shown by the combined analysis, the molecular data indicate the existence of three lineages of diversification that correspond to the subgenera of Gibbs (1966), however, two of them encompass not only Genista species, but also species of the related genera


113

Table 3. Position of the studied taxa of Genista in previous classifications. The asterisk (*) indicates taxa not included in this work After Gibbs (1966)

After Talavera and Salgueiro (1999) Subgenus/sectio

Subgenus/sectio Genista/Genista Genista/Spartioides Spach

Genista/Scorpioides Spach Genista/Erinacoides Spach

Spartocarpus Spach / Cephalospartum Spach

Spartocarpus Spach / Spartocarpus

Spartocarpus Spach/ Acanthospartum Spach Phyllobotrys Spach/ Voglera Spach

G. tinctoria L. G. januensis Viv. G. florida L. G. benehoavensis (Svent.) Arco G. obtusiramea Spach G. ramosissima (Desf.) Poiret G. cinerascens Lange G. cinerea (Vill.) DC. G. valentina (Spreng.) Steud G. majorica Canto´ & M.J. Sańchez G. sericea Wulfen G. teretifolia Willk. G. pilosa L. G. pseudopilosa Coss. G. scorpius (L.) DC. G. carpetana Lange G. pumila (Hervier) Vierh. G. aspalathoides Lam. G. hystrix Lange G. polyanthos Willk. G. longipes Pau G. versicolor Boiss (= G. baetica Spach) G. sanabrensis Valde´s Berm. et al. G. quadriflora Mumby G. clavata Poiret G. umbellata (L’Her.) Poiret [G. microcephalla, G. capitellata, G. cephalantha] * G. radiata (L.) Scop. G. aucheri Boiss. G. aetnensis (Biv.) DC. G. ephedroides DC. G. gasparrini (Guss.) Presl. G. valsecchiae Brullo & De Marco G. nissana Petrovic G. dorycnifolia Font Quer G. haenseleri Boiss. G. spartioides Spach [G. acanthoclada DC.] * G. valdes-bermejoi Talavera & L. Saéz (= G. acanthoclada var. fasciculata Knoche)

Genista/Genista Genista/Chamaesparton Griseb

Genista/Scorpioides Spach Genista/Erinacoides Spach

Genista/Phyllodioides Talavera & Gibbs Genista/Lasiospartum Spach Genista/Cephalospartum Spach Genista/Asterospartum Spach

Genista/Retamospartum Spach ex Coss. Genista/Acanthospartum Spach

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Table 3 (continued) After Gibbs (1966)

After Talavera and Salgueiro (1999) Subgenus/sectio

Subgenus/sectio

Phyllobotrys Spach / Phyllobotrys (Spach) Gibbs

G. G. G. G. G. G. G. G. G. G. G. G. G.

micrantha Go´mez Ortega hispanica L sylvestris germanica L. hirsuta Vahl tournefortii Spach triacanthos (Brot.) tridens (Cav.) DC tricuspidata Desf. cupanii Guss. anglica L. falcata Brot. berberidea Lange

Echinospartum, Teline, Retama, Chamaespartium, Pterospartum, Ulex, and Stauracanthus. The three lineages are: 1) ‘Phyllobotrys’, that forms a very consistent clade (80% b.v.), and is the most distinctive morphologically, with sterile axillary spines, leaves and branching pattern alternate, and leaves usually simple (3 exceptions) taking a single vascular trace (Gibbs 1966). Only Genista species enter in this clade; 2) ‘Genista’, which includes spiny and non spiny species, forms consistently a group. Although a more extensive study of this group must include more representatives of Genista of the eastern Mediterranean area, the subgenus Genista may represent a phylogenetically related group. Teline species are closely related to this group. However, the information provided by the chloroplast marker could indicate that another taxon has played a part in the origin of Teline species. This could explain the complex combination of morphological features, and accompanying taxonomic instability, i.e. merged in Genista, in Cytisus, or considered as a separate genus; and 3) ‘Spartocarpus’ includes a set of morphologically heterogeneous taxa. In our analyses, only Genista sect. Spartocarpus forms a consistent clade. The other taxa separate in several groups related to Pterospartum (G. quadriflora), and Ulex and Stauracanthus (G. umbel-

Voglera (P. Gaert. et al.) Rchb./ Voglera (P. Gaert. et al.) Spach

Voglera (P. Gaert. et al.) Rchb. / Phyllospartum Willk.

lata). On the other hand, Retama may have segregated from this group. Further study is needed, including a more comprehensive representation of sections Cephalospartum and Acanthospartum, to better explain the internal and external (to other genera) relationships within this subgenus. Thus, although the division of Genista into three subgenera (Gibbs 1966) is well supported by the molecular data, a taxonomic revision that reflects the relationships to the other related genera still remains to be done. On the contrary, the current framework of the genus at the sectional level is not supported by the molecular analyses. Most of the boundaries between sections are not reflected in the tree, and although there are still unsampled species, it does not seem that this conclusion will change with the inclusions of more taxa. In Genista subgenus Genista, only sect. Genista, represented by G. tinctoria and G. januensis, forms a constant and supported clade in all the analyses. The species of all the other sections do not form monophyletic groups. G. scorpius and G. carpetana (sect. Scorpioides) are not related. The species of sect. Spartioides are included in separated groups intermingled with species of other sections: 1) Genista cinerascens, G. benehoavensis and G. obtusiramea form a clade with


G. versicolor (sect. Erinacoides) sister to the rest of species of sect. Erinacoides (G. hystrix, G. longipes, and G. pumila); 2) G. ramosissima, G. majorica, G. cinerea and G. valentina form a separate clade; and 3) G. florida, G. pseudopilosa and G. teretifolia are grouped with G. carpetana (sect. Scorpioides). Additional information provided by the alkaloid data available do not support the actual concept of sect. Spartioides (Greinwald et al. 1995). Moreover, several intersectional hybrids have been described involving taxa of sect. Scorpioides, sect. Erinacoides and sect. Spartioides, e.g. G. ·segurae (= G. pumila · G. scorpius, Uribe-Echevarrı´ a and Urrutia 1992). On the other hand, our data do not match the groups of species based on isoflavone patterns (van Rensen et al. 1999). Within the clade of Genista subgenus Phyllobotrys neither the species of sect. Phyllobotrys form a separate group in the analyses nor the species of sect. Voglera do. Thus the separation of two sections (Phyllobotrys and Voglera; Gibbs 1966, Talavera and Salgueiro 1999) is not supported by the molecular data. Within Genista subgenus Spartocarpus, section Spartocarpus has been thoroughly studied by De Castro et al. (2002). The new samples of G. radiata, G. aetnensis, G. dorycnifolia, G. spartioides and G. haenseleri added here do not change the topology and confirm that sect. Spartocarpus forms a monophyletic clade. In our analyses this clade has bootstrap support, in contrast to those of De Castro et al. (2002). The difference may be related to the choice of the outgroups. De Castro et al. (2002) included Retama species as one of the outgroups but in our trees Retama is very close to sect. Spartocarpus. Genista valdes-bermejoi also joins the sect. Spartocarpus clade. The position of this species was not well established previously. It was included under the name of G. lucida in sect. Voglera (Gibbs 1966), and later transferred to sect. Acanthospartum of subgenus Spartocarpus (Talavera 1999). Other proposed rearrangements of subgenus Spartocarpus such as the splitting of sect. Retamospartum (G. haenseleri and G. spartioides) are

115

not reflected in our analyses. These species are nested well inside sect. Spartocarpus. Moreover, this section is very uniform serologically (Cristofolini and Feoli Chiapella 1984) and palynologically (Rizzi Longo and Feoli Chiapella 1994). Sect. Cephalospartum as defined by Gibbs (1966) splits into two groups. Genista clavata and G. umbellata (in the ITS tree) form a group sister to an Ulex-Stauracanthus clade, whereas G. quadriflora is close to Pterospartum tridentatum. Gibbs (1966, p.94) indicated that section Cephalospartum is a perplexing and possibly heterogeneous grouping. The species are united principally by the characters of capitate inflorescences and partly opposite branching. The results found in our analyses indicate that G. quadriflora separates from the rest of sect. Cephalospartum. However, the proposal to join G. quadriflora with G. sanabrensis (sect. Phyllodioides; Talavera and Gibbs in Talavera and Salgueiro 1999) is not supported by the molecular data. Genista sanabrensis, the type of this new section, is closer to species of Teline and Genista subgenus Genista. The molecular data provide evidence that G. sanabrensis belongs to Genista subgenus Genista, but are not conclusive regarding its most related taxa. Species and species aggregates. The final aim of this work was to evaluate the taxonomic level at which the ITS region allows recognition of the different taxa. The trnL-trnF gene lacks resolution to resolve the relationships between close species. The main conclusion is that within the Genista group, the sequences of the ITS region are conserved within species. In most of the taxa, where two or more sequences from different subspecies or populations have been studied, the sequences are very similar, forming clades with high levels of bootstrap support (e.g. G. falcata 100% b.v., G. tournefortii 100% b.v., G. hirsuta subsp. hirsuta and subsp. lanuginosa 100% b.v., G. pumila subsp. pumila and subsp. rigidissima 90% b.v., G. umbellata subsp. umbellata and subsp. equisetiformis 100% b.v., Pterospartum tridentatum subsp. tridentatum and subsp. lasianthum 100% b.v.). More

116


infraspecific variation in the ITS sequences have been detected in other cases (e.g. G. florida subsp. florida and subsp. polygaliphylla, or G. tinctoria). However, all the samples of the species form monophyletic groups. The ITS region also groups morphologically related taxa that form consistent clades and have very similar ITS sequences. For example, G. ramosissima groups with the G. cinerea aggregate (G. cinerea subsp. cinerea, subsp. ausetana, and subsp. speciosa, G. valentina, and G. majorica). On the contrary, G. cinerascens is not related to this aggregate although it is currently included here (Canto´ and Sańchez 1988, Greuter et al. 1989). The distribution of major alkaloids also reveals that G. cinerascens is clearly distinct from G. majorica, G. valentina and G. cinerea (Greinwald et al. 1992). The analyses also provide some preliminary clues for the positioning of some species. In this sense, G. aspalathoides (subgenus Genista sect. Erinacoides) in the chloroplast tree does not group with other species of this section. Gibbs (1966) indicated that the trifoliate leaves of this species are an anomalous character for the section, and also that G. aspalathoides shows a striking resemblance with some specimens of G. ephedroides (a taxa of subgenus Spartocarpus). At present, no information of the sequence of the ITS region of this taxon is available, thus a clear conclusion cannot be drawn regarding the affinities of this species. In the case of G. pilosa more information is needed to explain its position near to the Teline canariensis clade in the ITS tree, and the incongruent result in the chloroplast tree. Conclusions The molecular data indicate the existence of three lineages of diversification within the Genista-group that correspond to the subgenera of Gibbs (1966), however, two of these lineages encompass not only Genista species, but also species of the related genera Echinospartum, Teline, Retama, Chamaespartium, Pterospartum, Ulex, Stauracanthus. The relationships of these genera to the main core of

Genista vary: some genera are nested well inside a line, whereas others are later segregates. These results point the need for a taxonomic revision that reflects the relationships inferred here. The three lineages of diversification of the Genista-group are 1) ‘Phyllobotrys’, a monophyletic group, corresponding to the Genista subgenus Phyllobotrys, distinctive both morphologically and from the molecular point of view; 2) ‘Spartocarpus’, the most heterogeneous group, including a monophyletic clade (Genista section Spartocarpus) but also a conglomerate of species of Genista sectio Cephalospartum, closely related to several terminal segregates: G. umbellata joins the Ulex-Stauracanthus clade, and G. quadriflora is sister to Pterospartum. Retama, morphologically well differentiated from Genista, separates from this line; and 3) ‘Genista’, including all the species of Genista subgenus Genista and other related genera: Chamaespartium sagittale (with still unresolved relationships), and the Teline species that split in two clades (separately placed near Genista subgenus Genista). Contrary to what is suggested by the present taxonomy, the molecular data do not support division of these subgenera at the sectional level. A feature shown by the molecular analyses of Genisteae, and in particular by the Genistagroup, is the short branches that appear near to the base of the group. This results in clades that tend to be weakly supported, often decaying in trees only one to several steps longer than the most parsimonious trees. One plausible explanation for this phenomenon is that the group, as a whole, has undergone a rapid organismal diversification compared to the rate of molecular evolution (e.g. Wendel and Doyle 1998). At the terminal branches, the ITS region provides parsimony informative characters that: 1) group closely related species (e.g. the G. cinerea aggregate); 2) separate those unrelated (e.g G. cinerascens); and 3) characterise others (G. florida subsp. florida and subsp. polygaliphylla). The trnL-trnF IGS region resolves less than the ITS region, but shows some indels that characterise species,


groups of species, and even genera (e.g. Ulex ). In conclusion, the molecular data provide a powerful tool to better support the systematics of this group, where processes such as hybridisation, introgression, aneuploidy and polyploidy have played an important role in its diversification (San˜udo 1979, Bisby and Nicholls 1977, Bisby 1981). The incongruence detected in some cases between the information provided by organellar and nuclear markers, rather than representing a problem, may be reflecting that two different components have played a part in the origin of a taxon. This information may help to better explain the origin and complex combination of morphological features that contribute to the taxonomic instability of some taxa (e.g. Chamaespartium and Teline). The study was financially supported by the projects DGICYT PB98-0774 from the Ministerio de Educacioń y Ciencia, and PR52/00-8849, Universidad Complutense of Madrid, Spain. The authors thank Prof. A. Crespo (Madrid) for encouraging this research and providing constructive suggestions. Valuable aid with collection of material was received from V. J. Arań (Madrid), A. Barra (Jardı´ n Botańico de Madrid, Spain), J. Fernańdez Bobadilla (Ca´diz), A. Guerra (Canary Islands), F. J. Silva Pando (LourizańPontevedra), M. Vicens (Jardı´ Bota`nic de So´ller, Balearic Islands, Spain), and friends and colleagues of our Departments. Prof. A. Liston and an anonymous referee are thanked for helpful comments and suggestions during the reviewing of the manuscript.

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Address of the authors: Cristina Pardo ([email protected]) and Paloma Cubas ([email protected]), Departamento de Biologı´ a Vegetal II, Facultad de Farmacia, Universidad Complutense,E-28040 Madrid, Spain. Hikmat Tahiri ([email protected]), De´partement de Biologie, Faculte´ des Sciences, Universite´ Mohammed V, BP 1014 Rabat, Morocco.