Molecular phylogenetics reveals Leontodon (Asteraceae, Lactuceae) to be diphyletic

American Journal of Botany 93(8): 1193–1205. 2006.

MOLECULAR PHYLOGENETICS REVEALS LEONTODON (ASTERACEAE, LACTUCEAE) TO BE DIPHYLETIC1 ROSABELLE SAMUEL,2,8 WALTER GUTERMANN,3 TOD F. STUESSY,2 CLAUDETE F. RUAS,4 HANS-WALTER LACK,5 KARIN TREMETSBERGER,2 SALVADOR TALAVERA,6 BARBARA HERMANOWSKI,7 AND FRIEDRICH EHRENDORFER2 2

Department of Systematic and Evolutionary Botany, University of Vienna, Rennweg 14, A-1030 Austria; 3Department of Plant Biogeography, University of Vienna, Rennweg 14, A-1030 Austria; 4Departmento de Biologia General, Universidade Estadual de Londrina, Londrina, Parana´, Brazil; 5Botanic Garden and Botanical Museum Berlin-Dahlem, Free University Berlin, 141191 Berlin, Germany; 6Departamento de Biologı´a Vegetal y Ecologı´a (Bota´nica), Facultad de Biologı´a, Universidad de Sevilla, Avenida Reina Mercedes, E-41080 Sevilla, Spain; and 7University of Natural Resources and Applied Life Sciences, GregorMendel-Str., Vienna, A-1090 Austria The plastid matK gene, trnL/F spacer, and nuclear rDNA ITS were sequenced for 36 species of Leontodon and 29 taxa of related genera of tribe Lactuceae. Phylogenetic relationships inferred from the independent and combined data are largely congruent and reveal that Leontodon sensu lato (s.l.) as presently defined is diphyletic: L. subgenus Leontodon forms a clade with Helminthotheca, Picris and Hypochaeris as sister genera, whereas L. subgenus Oporinia appears as a separate clade with strong bootstrap support and is thus better treated as a separate genus. Previous sectional classifications of Leontodon s.l. are considered in the light of DNA and additional morphological and karyological data. Support is presented for a core group of Hypochaeridinae sensu stricto (s.s.) with the two clades of Leontodon s.l., Helminthotheca, Picris, and Hypochaeris, whereas Urospermum, Hyoseris, Aposeris, and Rhagadiolus appear to be positioned more distantly. Key words: Asteraceae; chromosome numbers; Hypochaeridinae; indumentum; ITS; Lactuceae; Leontodon; matK; Picris; phylogeny; trnL/F.

In the first edition of his Species Plantarum, Linnaeus (1753) recognized the genera Hypochaeris L., Leontodon L., and Picris L. These three taxa are widespread in western Eurasia, have a plumose pappus, and are members of subtribe Hypochaeridinae (Bremer, 1994). In 1754 the small genus Helminthotheca Vaill. was split off from Picris, but otherwise through the years the Linnaean generic concepts have been maintained based on overall vegetative characters, the presence or absence of receptacular bracts in the capitulum, and in recent decades the form of hairs and base chromosome numbers. Some synantherologists, such as Cassini (1829) or Schultz (Bipontinus) (1833, 1834), indulged in excessive splitting of these genera, resulting in an additional 11 genera as recognized, e.g., in Candolle’s Prodromus (1838). Bentham (1873), however, in his major synthesis of the family, returned to the original three Linnaean genera, which were structured internally into sections following some of the other generic distinctions. More recent studies have continued to support recognition of Hypochaeris, Helminthotheca, Picris, and Leontodon as distinct genera (Bremer, 1994; Lack, in press). Hypochaeris can be distinguished easily by its receptacular bracts. The distinction between Leontodon and Picris usually was thought to be clear, with the former having a scapose habit and the latter being typically branched. The two also differ in hair types 1

Manuscript received 14 January 2006; revision accepted 15 May 2006.

The authors wish to thank E. Grasserbauer and M. H. J. Barfuss for their help with laboratory work and to all those who kindly collected material during their field work, particularly Profs. M. Fischer and C. Zidorn, and Drs. E. Ho¨randl, M. Martı´nez Ortega, A. Tribsch, P. Scho¨nswetter, and G. Schneeweiss. This project was funded by grants P13055 and P15225 from the Austrian National Science Foundation (FWF) to T. F. S. 8 Author for correspondence (e-mail: mary.rosabella.samuel@ univie.ac.at)

and chromosome numbers (Lack, 1974), with Picris being x ¼ 5 (Holzapfel, 1994) and consistently possessing at least some anchor-shaped hairs and with Leontodon being x ¼ 4, 6, 7 (rarely 5, 11) (Pittoni, 1974; Izuzquiza, 1991) and possessing various other hair types if not glabrous (Pittoni, 1974). Helminthotheca is separated from Picris by its conspicuous outer involucral bracts. Considering that the presence/absence of receptacular bracts has been shown to be a weak character in some groups of Compositae and that some species of Picris have a scapiform habit (e.g., Picris olympica Boiss.) and some of Leontodon with a branched habit (e.g., Leontodon autumnalis L.), a new approach using molecular data was needed to test traditional limits among these genera of Hypochaeridinae. The correct placement of controversial species such as Hypochaeris robertia (‘‘Robertia taraxacoides’’), Picris (‘‘Leontodon’’) hispanica, or Leontodon (‘‘Picris,’’ ‘‘Microderis’’) rigens needs to be clarified, as well as the general relationship between Picris and Leontodon. Further, the infrageneric classification of Leontodon by Widder (1931, 1975), based on morphological characters, can now also be tested with new molecular data. He divided the genus into subg. Leontodon (comprising sects. Asterothrix, Leontodon, and Thrincia) and subg. Oporinia (comprising sects. Oporinia and Kalbfussia). According to Widder (1931), members of the former subgenus were suspected to be intermediate to Picris in some respects. Among molecular markers available for phylogenetic reconstruction, the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rDNA) has proven especially useful for elucidating relationships among congeneric species and closely related genera in Asteraceae (Baldwin, 1992; Baldwin et al., 1995; Kim et al., 1996). The efficacy of ITS for resolving the phylogeny of Hypochaeris and related genera such as Leontodon, Crepis, and Hieracium has already been demonstrated by Cerbah et al. (1998), Samuel et al. (2003), and

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Tremetsberger et al. (2005). Plastid noncoding regions are also suitable for phylogenetic investigations. They tend to evolve more rapidly than do coding sequences, by accumulation of insertions and deletions at a rate at least equal to that for nucleotide substitutions (Clegg et al., 1994; Kelchner, 2000). The plastid DNA sequences, trnL intron, and trnL/trnF intergenic spacer, have been used for phylogenetic analysis in Asteraceae at the tribal level (Bayer et al., 2000) and at generic and specific levels in Palmae (Baker et al., 2000). The matK gene is one of the most rapidly evolving plastid proteincoding regions (Wolfe, 1991). Recent studies have shown the usefulness of this gene for resolving intergeneric and interspecific relationships among flowering plants, e.g., in Nicotiana (Aoki and Ito, 2000), Orchidaceae (Salazar et al., 2003), and most recently across all angiosperms (Hilu et al., 2003; see also comparative review by Shaw et al., 2005). In the present phylogenetic investigations of Leontodon, Picris, and related genera, we have used nuclear and plastid sequences individually and in combination to evaluate previous generic, subgeneric, and sectional classifications that were based primarily on morphology and cytology. MATERIALS AND METHODS Collections sampled for DNA analyses—We used 102 accessions for the phylogenetic analyses, including 36 species of Leontodon sensu lato (s.l.) representing all traditional subgenera and sections, 17 species of Hypochaeris, 14 species of Picris, two of Helminthotheca, and 12 taxa from five outgroup genera. Previously collected herbarium specimens as well as field-collected material dried and stored in silica gel were used for DNA extraction. Relevant collection data are presented in Table 1. DNA extraction and amplification—Total DNA was extracted from material stored in silica gel as well as from herbarium specimens following the 23 cetyltrimethyl-ammonium bromide (CTAB) procedure of Doyle and Doyle (1987). The amplification of ITS (Taberlet et al., 1991; Baldwin et al., 1995) and the trnL intron and the trnL/F intergenic spacer and matK was done using universal primers. Polymerase chain reaction (PCR) amplification was carried out using PCR ready mix (AB-0619/LD; Abgene, Vienna, Austria), 45 lL PCR master mix each primer (total 4 lL of forward and reverse), and 2–8 ng (1 lL of 2–8 ng/lL) of template total DNA for a 50-lL reaction mixture. Amplified fragments were checked with 1% agarose gel and the amplified double-stranded DNA fragments were purified using Invisorb (Invitek, GmbH, D-13125 Berlin) gel purification kit. Sequencing—The purified fragments were directly sequenced on an ABI 377 automated sequencer (Applied Biosystems, Vienna, Austria) using dye terminator chemistry following manufacturer’s protocols. Two cycle sequence reactions were performed for each template using each of the two primers for PCR amplification. The programs Sequence Navigator and AutoAssembler (Perkin Elmer Applied Biosystems, Vienna, Austria) were used to edit and assemble the complementary sequences. Sequence alignment and phylogenetic analyses—Alignments were obtained using the program Clustal V (Higgins et al., 1992) and improved by visual refinement. Phylogenetic analysis was done using PAUP* (version 4.0b10; Swofford, 2003) for all four data sets, namely ITS, trnL intron, trnL/F spacer and partial matK, and the combined ITS and matK matrices. Heuristic searches were performed with equal weights, using 1000 random taxon addition replicates, and tree bisection–reconnection (TBR) branch swapping, and ‘‘keeping multiple trees’’ (MulTrees) in effect but holding 10 trees per replicate. Confidence limits for trees were assessed by performing 1000 replicates of bootstrapping (Felsenstein, 1985) using equal weighting, TBR swapping, MulTrees on, and holding only 10 trees per replicate. We also carried out a Bayesian analysis of the combined data set (ITS and matK sequences) using MrBayes version 3.0b4 (Ronquist and Huelsenbeck, 2003). The two data partitions (ITS and matK) were allowed to have different general

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time reversible (GTR) substitution models (Lanave et al., 1984; Rodriguez et al., 1990) with gamma-distributed rate variation among sites. The Monte Carlo Markov chain (MCMC) had 10 3 106 generations. The consensus trees from two independent runs were compared with one another and with the consensus tree from the parsimony analysis. The incongruence length difference (ILD; Farris et al., 1995) test was employed to detect incongruence among the data sets using the partition homogeneity test in PAUP*. We used 1000 replicates on parsimonyinformative characters using TBR branch swapping, with simple sequence addition and MulTrees option in effect. Siddal (1997) points out that the ILD test does not truly reveal the amount of incongruence and can be insensitive to small but significant topological differences suggested by the different data sets. Measures of incongruence like the incongruence length difference (ILD) test have been demonstrated recently not to be useful indicators of data partition combinability (Reeves et al., 2001; Yoder et al., 2001). Therefore visual inspection of the individual bootstrap consensus trees was used for determining combinability of the two data sets as done by Whitten et al. (2000). Bootstrap percentages (BP) are described as high (85–100%), moderate (75– 84%), or low (50–74%).

RESULTS Results from analyses of nuclear rITS, plastid and partial coding matK, and noncoding trnL/trnF sequences give generally congruent results. Analysis of ITS resulted in phylogenies with higher retention index (RI) and clades with high bootstrap percentage (BP) support. The plastid coding matK was less informative, but it showed better resolution than the noncoding trnL intron and trnL/F spacer. Not all accessions sampled were sequenced for ITS and matK due to problems with PCR amplification in some taxa. In the case of trnL/F, fewer species were analyzed because of overall poor resolution from initial samples. ITS—Results were obtained from 102 accessions including Hypochaeris, Leontodon, Helminthotheca, Picris, and 12 outgroup taxa (Fig. 1). Both spacer regions (ITS1, ITS2) and the 5.8S sequences were included in the analyses; no evidence for multiple rDNA repeat types was observed. The length of ITS1 ranged from 282 to 294 base pairs (bp) and that of ITS2 from about 201 to 241 bp. A total of 862 characters was included in the analysis, of which 466 (54%) were parsimony informative. The heuristic search generated 2650 equally parsimonious trees with 2340 steps (CI ¼ 0.47; RI ¼ 0.76); the strict consensus tree with bootstrap percentage (BP) greater than 50 is presented in Fig. 1. The ITS consensus tree shows that the genera Hypochaeris, Leontodon, Helminthotheca, and Picris form a monophyletic clade, supported by 100 BP. This clade corresponds to a core group of Hypochaeridinae. It consists of two subclades of which the first includes as sister groups (BP 72) Hypochaeris and Leontodon subg. Leontodon þ Helminthotheca þ Picris, of which the latter three are linked again (BP 82), whereas the second is equivalent to Leontodon subg. Oporinia. Within L. subg. Leontodon, three clades are equivalent to the three sections proposed by Widder (1975). The first two of these clades, L. sect. Asterothrix (with L. asperrimus, L. incanus, and others) and L. sect. Leontodon (with L. hispidus, L. kulczinskii, and L. rigens), are very strongly linked (BP 100) as sisters to one another and exhibit BP values of 66 and 98, respectively. The third clade stands separately, is well supported (BP 97), and corresponds to L. sect. Thrincia (with L. spec. A, L. saxatilis, L. tuberosus, and others). Leontodon subg. Oporinia (Widder, 1975) corresponds to a strongly supported clade (BP 100) that is widely separated

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Taxa of Asteraceae-Lactuceae (with localities, collectors, vouchers, and GenBank numbers) used for DNA analyses. Sequence(s) analyzed and GenBank accession number Taxon1

Aposeris foetida (L.) Less. Crepis aurea (L.) Cass. subsp. aurea C. alpestris (Jacq.) Tausch C. biennis L. C. mollis (Jacq.) Asch. Helminthotheca aculeata (Vahl) Lack H. echioides (L.) Holub Hieracium murorum L. Hyoseris radiata L. (1) H. radiata L (2) H. scabra L. Hypochaeris acaulis (Remy) Britton H. angustifolia (Litard. & Maire) Maire H. achyrophorus L. H. chillensis (Kunth) Britton H. cretensis (L.) Chaub. & Bory H. glabra L. H. illyrica K. Maly´ H. laevigata (L.) Ces. & al. H. maculata L. H. megapotamica Cabr. H. microcephala (Sch. Bip.) Cabr. H. oligocephala (Svent. & Bramw.) Lack H. pampasica Cabr. H. radicata L. H. robertia Fiori (1) H. robertia Fiori (2) H. robertia Fiori (3) H. sessiliflora Kunth H. uniflora Vill. Leontodon anomalus Ball L. asperrimus (Willd.) Ball L. [Op.] autumnalis L. L. berinii Reichb. L. boryi Boiss. L. [Op.] cantabricus Widder L. [Op.] carpetanus Lange L. [Op.] cichoriaceus Boiss. L. crispus Vill. (1) L. L. L. L.

crispus Vill. (2) [Op.] croceus Haenke [Op.] duboisii Sennen farinosus Merino & Pau

L. graecus Boiss. & Heldr. L. [Op.] helveticus Me´rat (1)

Locality

Austria (Ka¨rnten): Tro¨gerner Tal Austria (Steiermark): Loser Berg N Altaussee Austria (Niedero¨sterreich): Gahns N Payerbach Austria (Steiermark): Loser Berg N Altaussee Austria (Niedero¨sterreich): Gahns N Payerbach Italy, Sicily (Palermo): Chiusa Sclafani Greece, Ionian isl.: Kerkira, Akr. Asprokavos Austria (Steiermark): Loser Berg N Altaussee Italy (Fo´ggia): Gargano, Monte S. Angelo Spain (Malaga): Sierra del Torcal de Antequera Algeria: Akbou Chile: Laguana del Maule Morocco, Moyen Atlas, Mekne´s: near Timahdite Italy, Liguria Argentina, Prov. Buenos Aires Italy, Sicily Italy, Sicily Bosnia Italy, Sicily Austria (Niedero¨sterreich): Du¨rnstein Argentina, Prov. Buenos Aires Argentina, Prov. Buenos Aires cult. material of unspecified origin Argentina, Prov. La Pampa Switzerland (Zu¨rich): N of Greifensee Italy, Sicily Italy, Liguria (Genova): Monte Dente Italy (Lucca): Valle di Gramolazzo, Foce di Cardeto Ecuador, Pichincha France (Alpes Maritimes): Col de Tende Italy (Lucca): Alpi Apuane, Cresta di Capradosso Georgia (Samtskhe-Javakheti): around Azavreti Switzerland (Zu¨rich): N of Greifensee Italy (Pordenone): Cimoliana W San Floriano Spain (Granada): Sierra Nevada, Mulhacen Spain (Leo´n): La Ban˜a, Sierra de la Cabrera Spain (Salamanca): Laguna de los Lavajares Greece (Nom. Trika´lon): Pı´ndos, Ko´ziakas N Ela´ti Greece, Ionian isl.: Ke´rkira, SW La´kones Greece (Nom. Trika´lon) Pindos: Loupata Austria (Ka¨rnten): Koralpe France (Arie`ge): Querigut Spain (Leo´n): Montes Aquilianos (SE Ponferrada) Greece, Ionian isl.: Lefkaad, Ag. Nikitas Austria (Steiermark): Totes Gebirge, Elm

Collector(s) and number and voucher location2, or reference

ITS

trnL

matK

Go¨lles & Go¨lles GO657 (1) Stuessy 15536 (1)

DQ451822 AF528483

— AF528396

— —

Fischer s. n. (1)

DQ451817

—

DQ451749

Stuessy 15639 (1)

DQ451818

—

—

Fischer s. n. (1)

DQ451819

—

DQ451748

Certa & Ilardi 19759 (4)

DQ451797

—

DQ451731

Gutermann 23831 (2)

DQ451796

DQ449612

DQ451730

Stuessy 15535 (1)

AF528492

AF528400

—

Gutermann 23604 (2)

AF528494

AF528401

DQ451750

Gutermann 37277 (2)

DQ451824

—

—

Cerbah et al. (1998) Stuessy et al. 15571

Ref. text AF528433

— AF528360

— AF528403

Talavera et al. 676/03M (5)

AJ627260

—

DQ451689

Cerbah et Cerbah et Cerbah et Cerbah et Cerbah et Cerbah et Stuessy & Cerbah et Cerbah et Cerbah et

Ref. text Ref. text Ref. text Ref. text Ref. text Ref. text AF528454 Ref. text Ref. text Ref. text

AF528364 — — AF528369 AF528373 — AF528374 AF528375 AF528377 —

— — — AF528410 — — AF528413 AF528414 AF528416 AF528417

Cerbah et al (1998) Stuessy 15540 (1) Cerbah et al (1998) Bot. Institute ‘‘Hanbury,’’ Univ. of Genova Schratt & Gutermann 17105 (2)

Ref. text AF528457 Ref. text DQ451751

AF528378 AF528380 AF528382 —

AF528419 AF528420 — AF528422

DQ451752

—

DQ451690

Stuessy et al. 12332 (1) Gutermann et al. 32482 (1) Schratt & Gutermann 17067 (2)

AF528463 AF528481 DQ451753

AF528383 AF528390 —

AF528424 AF528428 DQ451691

Schneeweiß et al. 8182 (2)

DQ451754

—

DQ451692

Stuessy 15541 (1) Kuhns & Zidorn 970624h (1)

AF528486 DQ451756

AF528391 —

DQ451694 DQ451696

Rico s. n. (3)

DQ451757

DQ449600

DQ451695

Alamillo et al. s. n. (3)

DQ451758

—

DQ451720

Ladero & Gonzalez-Iglesias s. n. (3) DQ451759

DQ449602

DQ451699

Gutermann 35155 (2)

DQ451760

DQ449610

DQ451698

Ho¨randl 3308 (1)

DQ451761

DQ449601

—

Gutermann 35682 (2) Ho¨randl 2695 (1) Bosc s. n. (3) Alamillo et al. s. n. (3)

AF528488 DQ451762 DQ451763 DQ451764

AF528392 — — —

AF528430 DQ451697 DQ451700 DQ451701

Tremetsberger s. n. (1) Ho¨randl 1494 (1)

DQ451765 DQ451766

DQ449606 —

DQ451702 DQ451704

al. (1998) al. (1998) al. (1998) al (1998) al. (1998) al. (1998) Ehrendorfer s. n. (1) al (1998) al (1998) al. (1998)

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TABLE 1. Continued. Sequence(s) analyzed and GenBank accession number

Taxon1 L. [Op.] helveticus Me´rat (2)

Locality

Austria (Steiermark): Totes Gebirge, Rickmersscharte L. [Op.] helveticus Me´rat (3) Austria (Steiermark): Loser Berg N Altaussee L. [Op.] helveticus Me´rat (4) Austria (Ka¨rnten): Koralpe, Großes Kar L. hispidus L. (1) Austria (Steiermark): Loser Berg N Altaussee L. hispidus L. (2) Austria (Ka¨rnten): Reisskofel L. hispidus L. (3) Austria (Steiermark): Totes Gebirge, Gr. Priel L. hispidus L. (4) Austria (Niedero¨sterreich): Schneeberg L. incanus Schrank Austria (Niedero¨sterreich): Leobersdorf L. kulczinskii M. Popov Romania (Brasov): Pass Bratocea . Csukas L. [Op.] laciniatus (Bertol.) Widder Syria: between Palmyra and Damascus L. longirostris (Finch & Spain (Ma´laga): P.D. Sell) Talavera (1) Cortes de la Frontera L. longirostris (Finch & Spain (Ma´laga): P.D. Sell) Talavera (2) near Villanueva del Rosario L. longirostris (Finch & Spain (Almerı´a): P.D. Sell) Talavera (3) Urra ESE Sorbas L. maroccanus (Pers.) Ball (2) Spain (Malaga): near Villanueva del Rosario L. [Op.] microcephalus Boiss. Spain (Granada): Sierra Nevada, Laguna de la Caldera L. [Op.] montaniformis Widder Austria (Niedero¨sterreich): Schneeberg L. [Op.] montanus Lam. (1) Italy (Su¨dtirol, Bozen): Wolfendorn L. [Op.] montanus Lam. (2) Austria (Steiermark): Totes Gebirge, Gr. Priel L. [Op.] muelleri (Sch. Bip.) Spain, Andalucia (Almerı´a): Fiori (1) near Tabernas L. [Op.] muelleri (Sch.Bip.) Spain (Almeria): Alpujarras Fiori (2) L. [Op.] nevadensis (vel aff.) Spain (Granada): Sierra Nevada, Lange (2) Puerto de la Ragua L. [Op.] nevadensis Lange (1) Spain (Granada): Sierra Nevada, Puerto de la Ragua L. [Op.] palisiae Izuzq. Portugal (Alto Alentajo): Elvas L. [Op.] pyrenaicus Gouan Spain (Girona): Pirineos, Puigmal d’Err L. rigens (Ait.) Paiva & cult. material (achenes from Ormonde (1) ‘‘Jelitto Staudensamen’’) L. rigens (Ait.) Paiva & cult. material (achenes from Ormonde (2) Botanical Garden Basel) L. [Op.] rilaensis Hayek Romania (Sibiu): MtSii FaˇgaˇrasSului, NE of street pass L. rosani Ten. (1) Italy, Basilicata (Potenza): San Nicola L. rosani Ten. (2) Italy: Vaglia, Monte Morella L. saxatilis Lam. (1) Chile (Concepcio´n): Concepcio´n, Cerro Ponpo´n L. saxatilis Lam. (2) Austria (Niedero¨sterreich): Sierndorf L. tingitanus (Boiss. & Reut.) Spain (Ca´diz): Paloma Baja Ball N Punta Paloma L. tuberosus L. Greece, Ionian isl.: Lefkada, Nikiana L. [sect. Thrincia] spec. (A) Morocco, Atlas: Coll du Zad L. [Op.] spec. (B) Austria (Niedero¨sterreich): Schneeberg Picris abyssinica Schultz bip. Ethiopia: Debre Zeit, ILCA Experim. Field Station P. angustifolia subsp. merxmuelleri Australia, ACT: behind Bulls Lack & S. Holzapfel Head rest area P. coronopifolia (Desf.) DC. (1) Morocco, Anti Atlas: entre Agadir y Tafraoute

Collector(s) and number and voucher location2, or reference

ITS

trnL

matK

Ho¨randl 1493 (1)

DQ451767

—

DQ451705

Stuessy 15534 (1)

AF528484

DQ449605

DQ451703

Gutermann 26233 (2)

DQ451768

—

—

Stuessy 15537 (1)

DQ451769

—

DQ451706

Ho¨randl et al. 5389 (1) Ho¨randl 1495 (1)

DQ451770 DQ451771

— —

DQ451707 DQ451708

Stuessy 15546 (1)

AF528485

AF528393

AF528431

Gutermann 37630 (2)

DQ451772

DQ449603

DQ451709

Dobner & Zidorn 98-00113 (1)

DQ451773

—

DQ451721

Ehrendorfer s. n. (1) Gutermann 37343 (2)

DQ451774 DQ451775

— DQ449604

— —

Spitaler & Zidorn CZ-20030420A-1 (1) Gutermann 37111 (2)

DQ451776

—

—

DQ451777

—

DQ451710

Spitaler & Zidorn CZ-20030421B-1 (1) Rico s. n. (3)

DQ451778

DQ449608

DQ451715

—

—

DQ451711

Ho¨randl et al. 4615 (1)

DQ451780

—

DQ451714

Gutermann 26331 (2) Ho¨randl 1498 (1)

DQ451781 DQ451782

DQ449607 —

— DQ451713

Ortiz & Tremetsberger 7/04 (1)

DQ451783

—

DQ451716

Spitaler & Zidorn CZ-20020416A-1 (1) Gutermann 37221 (2)

DQ451786

—

DQ451717

DQ451779

DQ449609

DQ451712

Gutermann 37214 (2)

DQ451784

—

—

Guerra 1412 (3) Schneeweiß & Scho¨nswetter 8829 (1)

DQ451787 DQ451788

— —

DQ451718 DQ451719

Grass & Zidorn CZ-20040805A-1 (1)

DQ451789

—

DQ451723

Lack acc. no. 365 (4)

DQ451790

—

DQ451724

Dobner & Zidorn 98-00084 (1)

DQ451791

—

DQ451722

Spitaler & Zidorn CZ-20040413B-1 (1) Lack 64 (4) Stuessy et al. 15453B (1)

DQ451792

—

DQ451729

DQ451793 AF528489

— AF528394

— DQ451725

Ho¨randl & Hadacek 7076 (1) Gutermann 37432 (2)

DQ451794 DQ451795

DQ449599 DQ449611

DQ451726 DQ451727

Tremetsberger s.n. (1) Talavera et al. 281/03M (5) cult. at the Bot. Garden, Univ. of Vienna Boulos 9800 (4)

AF528487 DQ451755 DQ451785

AF528395 — —

DQ451728 DQ451693 —

DQ451798

—

DQ451745

Holzapfel, Thiele & Prober 012 (4)

DQ451799

—

DQ451734

Talavera et al. 148/03M (5)

DQ451800

—

—

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Continued. Sequence(s) analyzed and GenBank accession number

Taxon1

Locality

Collector(s) and number and voucher location2, or reference

ITS

trnL

matK

P. coronopifolia (Desf.) DC. (2) P. coronopifolia (Desf.) DC. (3) P. cupuligera (Durieu) Walp. (1)

as above as above DQ451802 — DQ451740 as above as above DQ451801 — DQ451741 Morocco, Gran Atlas: Carretera Talavera et al. 185/03M (5) DQ451803 — DQ451743 Taroudant—Marrakech P. cupuligera (Durieu) Walp. (2) as above as above DQ451804 — DQ451742 P. evae Lack Australia, Queensland: Lack acc. no. 110 (4) — — DQ451735 S of Oakey—cult in Berlin P. hieracioides L. Austria, Wien: Mauer, Stuessy & Swenson 15409 (1) AF528490 AF528398 AF528432 Wotruba church P. hispanica (Willd.) P. D. Sell (1) Morocco, Anti Atlas: Talavera et al. 161/03M (1) DQ451808 — DQ451733 Carretera Agadir—Tafraoute P. hispanica (Willd.) P. D. Sell (2) Spain (Granada): Sierra de Baza Valde´s et al. s. n. (4) DQ451809 — DQ451732 P. nuristanica Bornm. Pakistan, N. area: Nu¨sser 593 (4) DQ451810 — DQ451737 Rupal valley, Bayals P. pauciflora Willd. W. Greece (Etolia Akarnania): Willing 44043 (4) DQ451811 — — NNE Xiropigado P. rhagadioloides (L.) Desf. (1) Greece, Ionian isl.: Gutermann 23771 (2) DQ451815 — — Kerkira, S Afio´nas P. rhagadioloides (L.) Desf. (2) Greece, Etolia-Akarnania: Willing 32.478 (4) DQ451816 — DQ451739 SSW Maheras P. saharae (Coss. & Durieu) E. Algeria, Sahara: Wadi N Deb Deb Ehrendorfer s. n. (1) DQ451806 — — Hochr. (1) P. saharae (Coss. & Durieu) Morocco (Azrou): Vogt 14717 ¼ Oberprieler 8926 (4) DQ451807 — DQ451747 Hochr. (2) valley Oued Moulouya S Missour P. scabra Forssk. Yemen: Jebal Sabir Smalla 207b (4) DQ451812 — DQ451738 P. squarrosa Steetz S. Australia: Southern Lofty Holzapfel 14a & Cunningham (4) DQ451813 — DQ451736 Port Noarlunga dunes P. strigosa Marsch. Bieb. Iran (Yazd): Khormiz SW Mehriz Aryavand et al. 1468 (4) DQ451814 — DQ451746 P. willkommii (Sch.Bip) Nyman Spain (Huelva): Ayamonte Romero et al. s. n. (4) DQ451805 — DQ451744 Rhagadiolus edulis Gaertn. Greece, Ionian isl. (Nom. Lefka´dos): Gutermann 31695 (1) AF528495 AF528402 AF542066 Ka´lamos isl. DQ451823 — — R. stellatus (L.) Gaertn. Greece, Ionian isl. (Nom. Lefka´dos): Gutermann 31761 (1) Ka´lamos isl. Urospermum dalechampii (L.) Spain (Castello´n): Rico & Sa´nchez s. n. (3) DQ451820 — — F.W. Schmidt Desierto de las Palmas U. picroides (L.) F.W. Schmidt Spain (Badajoz): Puebla de Alcocer Rico s. n. (3) DQ451821 — — ___________________________________________________________________________________________________________________________________ 1

L. ¼ Leontodon subgen. Leontodon; L. [Op.] ¼ Leontodon subgen. Oporinia (1) ¼ University of Vienna, Herbarium (WU); (2) ¼ Herb. Gutermann, Vienna; (3) ¼ University of Salamaca, Herbarium (SALA); (4) ¼ Botanical Museum, Berlin-Dahlem (B); (5) ¼ University of Sevilla, Herbarium (SEV) 2

from the clades of L. subg. Leontodon discussed earlier. A differentiation into two subclades (I and II) is suggested, but these differ considerably from the sectional classification proposed by Widder (1975). Subclade I (with L. autumnalis, L. palisiae, and others) is strongly supported (BP 97), but subclade II (with L. cantabricus, L. helveticus, L. cichoriaceus, and others) is only weakly supported (BP 63). Within subclade I, L. autumnalis is sister to a well-supported species group with L. carpetanus, L. duboisii, L. microcephalus, and L. nevadensis (BP 95), and Leontodon palisiae is sister to L. muelleri (BP 86). Subclade II includes a poorly supported group with L. cantabricus, L. helveticus, and allies, the sister taxa L. montanus þ L. montaniformis (BP 86), and two more isolated species. The genus Helminthotheca with two accessions forms a separate and small, but 100-BP-supported clade, in an unresolved polytomy between Leontodon subg. Leontodon and Picris (Fig. 1). Picris itself is represented by 14 species and 20 accessions and forms a well-supported clade (BP 93). Two subclades within Picris can be recognized, the first (I, BP 98) with P. rhagadioloides, P. angustifolia, P. nuristanica, P. squarrosa, P. strigosa, P. hieracioides, and P. pauciflora, the

second (II, BP 91) with P. coronopifolia, P. cupuligera, P. hispanica, P. saharae, and P. abyssinica. Taxa of the latter group were for a long time uncertain with respect to their placement either in Leontodon (e.g., P. hispanica; Pittoni, 1974; Greuter, 2003), in Picris, or separated as Spitzelia (e.g., P. coronopifolia and allies; Ozenda, 1958), a problem that now appears settled. Analysis of Hypochaeris is based on 19 accessions from 17 species, representing all infrageneric groups. The genus is monophyletic although with weak support (BP 64). Four clades are recognizable and correspond with the infrageneric taxonomy of the genus. The first is well supported (BP 100), consists of a single species, H. robertia (three different accessions), which recently has again been classified as a separate genus (Robertia: Pignatti, 1982). The second clade (BP 99) includes members of H. sect. Seriola, i.e., H. achyrophorus and H. laevigata, and of H. sect. Hypyochaeris, i.e., H. radicata and H. glabra. The third and fourth clades are linked (BP 92). The third with BP 98 includes two subclades: one (BP 71) with H. cretensis and H. oligocephala (Lack, 1978; formerly Heywoodiella oligocephala) represents H. sect. Metabasis; the other (BP 100), with H. uniflora, H. maculata,

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Fig. 1. Nuclear rDNA ITS, strict consensus of 2650 equally parsimonious trees (length ¼ 2340, consistency index ¼ 0.47, retention index ¼ 0.76). Bootstrap percentages .50 are given above the branches.

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and H. illyrica, corresponds to H. sect. Achyrophorus. The fourth clade (BP 96) contains H. angustifolia (Morocco) and all the South American taxa, here represented with H. acaulis, H. chillensis, H. megapotamica, H. microcephala, H. pampasica, and H. sessiliflora; it is discussed below. matK—Seventy-three accessions from 56 taxa of Hypochaeris, Leontodon (both subgenera), Helminthotheca, and Picris were included together with four outgroup taxa (Fig. 2). Only a short fragment (800 bp) of the matK gene was included using the primers 880F and 1710R. A total of 938 bp characters was used for the analyses, of which 156 (16%) were parsimony informative. The heuristic search generated a total of 1620 most parsimonious trees with 603 steps with a CI ¼ 0.62 and RI ¼ 0.79. A strict consensus tree with BP . 50 is given in Fig 2. The tree obtained with matK is mostly congruent with that of ITS, showing close relationships among Leontodon subg. Leontodon, Helminthotheca, and Picris. These three genera form a well-supported clade (BP 93). Within Leontodon subg. Leontodon, the resolution between members of the traditional sections is not as clear as in ITS, and L. boryi þ L. rosani assemble with members of L. sect. Leontodon instead of L. sect. Asterothrix. Helminthotheca appears basal to Picris, which again is clearly differentiated into two clades (I, II). Leontodon subg. Oporinia forms a well-supported, and again quite separated, clade (BP 99), but within this subgenus, resolution is very limited. The genus Hypochaeris is monophyletic (BP 77). Hypochaeris robertia appears as sister to H. sect. Hypochaeris, i.e., H. radicata and H. glabra (BP 67). Hypochaeris angustifolia together with H. maculata (of sect. Achyrophorus) appear as sisters to the South American species of the genus. trnL/F—Included were 33 accessions representing eight species of Hypochaeris, 17 species of Leontodon, one each of Picris and Helminthotheca, and seven outgroup taxa. The total number of characters was 903, of which only 83 (9%) were parsimony informative. A heuristic search generated 3954 most parsimonious trees with 301 steps with CI ¼ 0.81 and RI ¼ 0.83. The resolution within Leontodon subg. Leontodon was poor compared to that of ITS, but the overall picture was the same with Picris and Helminthotheca sister to Leontodon subg. Leontodon and subg. Oporinia forming a basal clade (BP 80). Because the resolution was weaker in comparison with matK, we did not continue analysis of more samples, and no tree is shown here. Combination of ITS and matK data—Our combined analysis included 69 accessions, with 10 species of Hypochaeris, 32 of Leontodon, 11 of Picris, Helminthotheca echioides, and four outgroup taxa. A total of 1799 characters was included for the analysis, of which 535 (29.7%) were parsimony informative. Heuristic search generated 4805 equally parsimonious trees with 2050 steps; a strict consensus tree with bootstrap percentages (BP . 50) above and posterior probability values below each branch is shown in Fig. 3. This nrITS þ cp matK tree in its topology corresponds nearly completely with the nrITS tree alone. Hypochaeris, together with H. robertia, forms a monophyletic group with a moderate BP 70. Hypochaeris angustifolia is basal to the South American species (BP 85). Leontodon subg. Leontodon, together with Picris and Helminthotheca, forms a wellsupported clade with BP 94, which is sister to Hypochaeris.

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Leontodon subg. Oporinia constitutes a strongly supported separate clade sister to Hypochaeris þ Helminthotheca þ Picris (100 BP). These results clearly show that the genera Picris and Helminthotheca are embedded within the genus Leontodon s.l. Within subg. Leontodon, the sections Asterothrix (BP 74), Leontodon (BP 100), and Thrincia (BP 100) are clearly separated. In the parsimony analysis of the combined data matrix, the critical species L. rosani, together with L. boryi, group with Leontodon sect. Asterothrix. Picris forms a monophyletic clade (BP 100), again with two clear subclades. The first (BP 100) comprises both annual and perennial species from the eastern Mediterranean, Near East, Central Asia, and Australia, and the second (BP 92) contains both annual and perennial species from the western Mediterranean, and northern and tropical Africa. In Leontodon subg. Oporinia, L. autumnalis is sister (BP 97) to a strongly supported clade (BP 100) with L. carpetanus, L. duboisii, and L. nevadensis. Leontodon cichoriaceus and others connect to perennial mountain species, e.g., L. pyrenaicus þ L. cantabricus (BP 100) and to Mediterranean annuals, e.g., Leontodon muelleri þ L. palisiae (BP 100). Consensus trees from two independent runs of a Bayesian analysis (not shown) are completely congruent regarding major clades with those from the parsimony analysis. One minor exception is the position of Helminthotheca echioides, which is sister to Picris in Bayesian analysis (posterior probability 0.91). Another exception is the position of Leontodon boryi and L. rosani, which group with members of L. sect. Leontodon and not L. sect. Asterothrix in Bayesian analysis (posterior probability 0.95). Hypochaeris robertia is sister to all the other species of Hypochaeris, which have posterior probabilities of 0.92 and 0.93 in Bayesian analysis. DISCUSSION General—For taxa of Lactuceae in the present study, nuclear ITS gives a well-resolved tree with high bootstrap values (Fig. 1). Of the plastid markers, matK (Fig. 2) provides much better resolution than trnL/F (not shown). Visual inspection of the individual bootstrap trees of ITS and matK markers shows them to be largely congruent with each other, and therefore our discussion is based mainly on the ITS tree plus the combined data of ITS and matK sequences (Figs. 1, 3). All data confirm that Hypochaeris, the two clades of the traditional Leontodon s.l., Helminthotheca, and Picris form a well-supported core group in subtribe Hypochaeridinae. In recent treatments by Bremer (1994) and Lack (in press), this subtribe is circumscribed in a much wider sense, including in addition Urospermum, Hyoseris, Aposeris, Rhagadiolus, Gharadiolus, and Hedypnois. Our analysis (Fig. 1) indicates Urospermum as close to the Hypochaeridinae core group along with Hyoseris, Aposeris, Hieracium, and Rhagadiolus. These conclusions verify earlier reports (Samuel et al., 2003) and are supported by the independent molecular analyses of Whitton et al. (1995) and Gemeinholzer and Kilian (2005). Furthermore, the latter demonstrate that the annual Hedypnois (not available to us) does belong to the core group of Hypochaeridinae. We cannot make any conclusions regarding these outgroup taxa because sampling was very poor within the included genera.

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Fig. 2. Strict consensus tree (length ¼ 603, consistency index ¼ 0.62, retention index ¼ 0.79) of 1620 most parsimonious trees from plastid matK sequences. Bootstrap percentages .50 are shown above the branches.

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Fig. 3. Strict consensus tree of 4805 equally parsimonious trees from combined ITS and matK sequences (length 2050). Bootstrap percentages .50 are shown above and Bayesian posterior probability values below the branches.

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Leontodon s.l.—Available nr and cpDNA data (Figs. 1, 3) clearly show that the traditional genus Leontodon s.l. is diphyletic, Leontodon subg. Leontodon is sister to Picris and Helminthotheca, and the three are united with Hypochaeris in a more comprehensive clade. In contrast, L. subg. Oporinia forms a clade of its own basal to the other four genera with a very strong BP (100) support. Available chromosome data as well as indumentum types and phytochemical information support these molecular data. The occurrence of bifurcate or 3up to 11-fid leaf hairs (Pittoni, 1974) and hypocretenolids (Zidorn et al., 2001; Zidorn and Stuppner, 2001a, b; Zidorn, 2006) are characteristic of Leontodon subg. Leontodon, whereas L. subg. Oporinia is characterized by strictly simple hairs and guaianolides. Karyological data (Rousi, 1973; Pittoni, 1974; Izuzquiza, 1991; Mariotti Lippi and Garbari, 2004) demonstrate that L. subg. Leontodon has differentiated with respect to chromosome numbers (mostly x ¼ 4, 7, 11), which largely correspond with traditional infrageneric groups. Leontodon subg. Oporinia is rather homogeneous (x ¼ 6: Izuzquiza and Nieto Feliner, 1991; rarely x ¼ 5: Izuzquiza, 1991). All these findings strongly support the taxonomic treatment of the two subgenera of Leontodon as two separate genera. Leontodon subg. Leontodon would thus become Leontodon s.s. (based on L. hispidus L.). For Leontodon subg. Oporinia, the name Scorzoneroides Vaill. (based on Leontodon autumnalis L.) is apparently the earliest available (Greuter et al., 2005). Leontodon subg. Leontodon—The traditional, morphologically based classification of this monophyletic subgenus into three sections (cf. Widder, 1975) is well reflected in our consensus trees (Figs. 1, 3), even if their branches are relatively short as seen in a phylogram (not shown). Leontodon sect. Leontodon consists of the morphologically and genetically very diverse and ecogeographically differentiated European and Anatolian-Caucasean group of L. hispidus (Meusel and Ja¨ger, 1992: map 530a) with single capitula on simple stems without leaves. Leontodon kulczinskii from the eastern Carpathians also belongs here. With respect to chromosomes, L. hispidus s.l. is characterized by 2n ¼ 14 (Rousi, 1973), but rarely, triploids and dysploids (2n ¼ 18, 2n ¼ 16) have also been reported (cf. Izuzquiza and Nieto Feliner, 1991; Constantinidis et al., 2002). Our DNA data also contribute to the relationships of two aberrant species from the Azores that were thought earlier to belong to Picris, Crepis, or even a separate genus Microderis DC. In contrast to most other species of Leontodon, these taxa develop branched and leafy stems with numerous capitula. Already Paiva and Ormonde (1973, 1975) and Lack (1981) presented morphological, palynological, and karyological (2n ¼ 14) evidence that the two taxa involved should be included in Leontodon as L. rigens and L. filii and belong to (or are close to) L. sect. Leontodon and the L. hispidus group. For the first species, this inclusion in Leontodon is now clearly supported by molecular data (Figs. 1, 3). The taxa of Leontodon sect. Asterothrix, characterized by hairs that are more than two-fid or stellate and by a hairy pappus on all achenes, have a distribution throughout the Mediterranean, from the Iberian Peninsula to southwestern Asia (Meusel and Ja¨ger, 1992: maps 530b–d). A core group of species with 2n ¼ 8 is well supported as a monophyletic unit by congruent nrITS and cp matK data (Figs. 1, 2). It includes, i.e., L. incanus, L. berinii, and the L. crispus aggregate with L. asperrimus (type species of the section), L. anomalus, L. crispus, L. graecus, L. farinosus, etc., taxa often treated as

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subspecies only (e.g., Finch and Sell, 1976), but partly with quite divergent DNA sequences. In addition, Leontodon sect. Asterothrix includes taxa that deviate by their variable indumentum more or less approaching that of L. sect. Leontodon (Pittoni, 1974), by their chromosome number and by incongruent nr and cp sequences. Leontodon boryi (from the Sierra Nevada) and L. villarsii (from southwestern France to Spain) have 2n ¼ 14 (the 2n ¼ 8 report for ‘‘L. hirtus’’ in Finch and Sell [1976] is erroneous; it was originally published by Larsen [1956] for ‘‘L. crispus var. saxatilis’’ but refers to an annual species, presumably L. longirostris). In contrast, the morphologically very close L. rosani (Italy) has 2n ¼ 22 (Pittoni, 1974; Miceli and Garbari, 1977; Mariotti Lippi and Garbari, 2004). These three taxa form a subclade. In the present analysis collections of L. boryi as well as L. rosani were studied with respect to their nrITS and cp matK sequences. They associate with typical members of L. sect. Asterothrix in the nrITS tree (Fig. 1) and in the parsimony analysis of the combined data matrix (Fig. 3), but they group with taxa of L. sect. Leontodon in the matK tree BP 64 (Fig. 2) and in the Bayesian analysis of the combined data matrix (not shown). These DNA data, their aberrant karyotype, and more or less intermediate indumentum suggest hybrid origins of the three taxa in two steps: (1) a combination of an ancestral taxon with 2n ¼ 14, possibly from L. sect. Leontodon, and another from L. sect. Asterothrix, resulting in L. villarsii and L. boryi with x ¼ 7; and (2) production of L. rosani with x ¼ 11, from a combination of a L. villarsii-like ancestor with x ¼ 7 and another member of L. sect. Asterothrix with x ¼ 4. Pittoni (1974) has already speculated about such an allopolyploid origin, when she found 2n ¼ 22 in plants (then still called ‘‘L. villarsii’’) from Italy. Furthermore, population L. crispus 2 is incongruent for nrITS (corresponding to L. crispus 1 and other taxa of L. sect. Asterothrix with 2n ¼ 8) and for cp matK (corresponding to members of L. sect. Thrincia, also with 2n ¼ 8), which suggests that there may also have been hybridization between these two sections of Leontodon subg. Leontodon. The few (c. 5) species of Leontodon sect. Thrincia share an indumentum of hairs with 2–3 terminal, straight, or rarely more or less hooked branches, outer achenes with pappus reduced to scales or short hairs, and a chromosome complement of 2n ¼ 8 (Rousi, 1973; Pittoni, 1974). A new eudesmane-derived sesquiterpenoid occurs in L. tuberosus (Spitaler et al., 2004). Members of the section are centered in the western Mediterranean but extend considerably into northwestern Europe and southwestern Asia (Meusel and Ja¨ger, 1992: map 531a). Nuclear ribosomal ITS and cp matK data are available for all taxa including ‘‘Leontodon spec. A’’ (a possibly undescribed species from Morocco) (Figs. 1–3) and suggest monophyly of the section. Two well-supported clades can be recognized, one with the widespread perennial L. tuberosus and the southwestern Mediterranean annual L. maroccanus, the other including the perennial Leontodon species A, L. tingitanus, a local endemic of southern Spain and northern Morocco, and the very widespread polymorphic complex of L. saxatilis/L. longirostris with closely related perennials, biennials, and annuals. Changes from long- to short-lived growth forms thus have occurred more than once in L. sect. Thrincia. Leontodon subg. Oporinia—This subgenus is clearly monophyletic but not closely related to L. subg. Leontodon, and therefore the former should be classified as a separate

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genus. In contrast to the more strongly differentiated taxa within L. subg. Leontodon, the taxa within L. subg. Oporinia seem more closely related and are not well resolved by our plastid data (Fig. 2). Nevertheless, on the basis of their nrITS sequences (Fig. 1), they fall into two sufficiently supported subclades, designated I and II. However, these two clades and available phytochemical evidence correspond only partly to the traditional sections proposed by Widder (1975), i.e., L. sect. Oporinia and L. sect. Kalbfussia, based mainly on the presence or absence of an achenial beak, and designated in the following by O and K (in brackets). These conflicting findings also interfere with Widder’s earlier phytogeographical interpretation of L. sect. Kalbfussia (Widder, 1958). Subclade I includes the type species of L. subg. Oporinia, the perennial L. autumnalis [O], widespread from Europe to western Siberia (Meusel and Ja¨ger 1992: map 529d), which is sister to a well-supported clade of other perennials (L. carpetanus, L. duboisii, and L. nevadensis: all [K]), all from the mountains of the Iberian Peninsula. Together, these perennials are linked with a group of annual species (L. muelleri, L. palisiae, L. laciniatus: all [K]), which differ remarkably in their cp DNA (Fig. 2) and extend from the western to the eastern Mediterranean. Morphologically, all species of subclade I are united by the potential for branching flowering stems. Their chromosome number is nearly exclusively 2n ¼ 12 in the perennial taxa (Rousi, 1973; Vaarama, 1948: local tetraploid population in L. autumnalis). The finding of 2n ¼ 12 also has been recorded for the annuals L. muelleri (western Mediterannean; Izuzquiza, 1991, 1998; the report of 2n ¼ 8 by Gemeinholzer and Faustmann [2005] probably relates to an annual member of L. sect. Thrincia) and L. hispidulus (eastern Mediterranean; Brullo et al., 1990), and descending dysploidy (x ¼ 6–5) has occurred in L. palisiae (2n ¼ 10) centered in southern Spain (Izuzquiza, 1991). Subclade II of L. subg. Oporinia comprises exclusively perennial taxa with unbranched flowering stems, growing predominantly in the mountains of temperate Europe (Meusel and Ja¨ger, 1992: maps 529a–c) and mostly with 2n ¼ 12 (x ¼ 6; with one exception of x ¼ 5 in L. cichoriaceus). Taxa of the vicariant group of L. cantabricus, L. helveticus, and L. pyrenaicus (all [O]), were each treated as species by Widder (1937, 1967), but regarded as subspecies only by Finch and Sell (1976). Another group is formed by L. montanus and L. montaniformis (both [O]). In contrast, L. croceus [O] in the eastern Alps and Carpathians and L. rilaensis [O], a South Carpathian endemic, are more isolated species, and should certainly not be reduced to subspecies (as in Finch and Sell [1976]). Karyological data in the last two species are controversial. Tetraploidy with 2n ¼ 24 has been documented for a population of L. croceus from the eastern Alps (Favarger, 1959), whereas 2n ¼ 14 is known for plants from the eastern Carpathians (Pasˇuk, 1987). An earlier report for L. rilaensis was later corrected from 2n ¼ 14 to 2n ¼ 12 (Kuzmanov et al., 1993). All the montane species groups of clade II discussed earlier were placed by Widder (1975) into several corresponding series of L. sect. Oporinia together with L. autumnalis from our clade I. PCA analyses of phenolic compound profiles presented by Zidorn and Stuppner (2001b: fig. 1) are in line with these conclusions and also indicate that clade II taxa exhibit affinities with the base of clade I. Clade II of L. subg. Oporinia also includes an isolated Mediterranean mountain taxon, L. cichoriaceus [K], from Algeria and Italy to the Balkans and western Anatolia. It

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deviates by a specific sesquiterpene lactone (Zidorn et al., 2001) and, at least partly, by a dysploid chromosome number 2n ¼ 10 (x ¼ 5; Colombo and Trapani [1990] on plants from Sicily; but there is also a report of 2n ¼12 by Kuzmanov et al. [1987] for a collection from Bulgaria). Hypochaeris—Although the main focus of this paper is on Leontodon and relatives, the inclusion of a representative sampling of species of Hypochaeris, in conjunction with outgroup taxa, allows some observations to be made regarding relationships within the genus. Previous molecular phylogenetic studies by Cerbah et al. (1998) using ITS data and Samuel et al. (2003) with ITS, trnL intron, trnL-F spacer and matK data have emphasized several points that are, not surprisingly, again seen in analyses from the present data set. First, the genus is monophyletic. Here the genus barely holds together with a 64 BP in ITS (Fig. 1), a low value perhaps due to the attachment of H. robertia. Previously with other outgroup and ingroup taxa, this species fell outside Hypochaeris and among taxa of Leontodon, which resulted in a 98 BP in ITS for the former genus (Samuel et al., 2003). The status of H. robertia is obviously still not settled. Second, the new data set reflects the sectional structure established previously on morphological grounds for the Old World species (cf. Bentham, 1873), i.e., H. cretensis and H. oligocephala in sect. Metabasis; H. illyrica, H. maculata, and H. uniflora in sect. Achyrophorus; H. achyrophorus and H. laevigata in sect. Seriola; and H. glabra and H. radicata in sect. Hypochaeris. The remaining taxonomic challenge is how to classify the numerous South American species of the genus (c. 40 species, here in Fig. 1 represented by H. acaulis through H. sessiliflora), and also the northwest African H. angustifolia, which is now known to be the sister group (Tremetsberger et al., 2005). Although more work needs to be done on this issue, it might be appropriate to treat all these taxa in a new section, with perhaps series status for H. angustifolia. For this to be done carefully, however, a comprehensive view of molecular and morphological relationships within the large South American complex is required. Helminthotheca and Picris—These two genera of Hypochaeridinae are closely linked by their branched and leafy stems, 2- to 4-furcate and anchor-shaped hairs, lack of receptacular bracts, chromosome base number of x ¼ 5, and polyploidy (2x, only rarely 4x, 6x: Fernandes and Queiro´s, 1971; Oberprieler and Vogt, 1993; Holzapfel, 1994). Their only differences are the conspicuously enlarged and cordate outer phyllaries of the capitula (Lack, in press). Nevertheless, the combined tree (Fig. 3) shows them as two independent clades sister to Leontodon sect. Leontodon þ sect. Asterothrix and L. sect. Thrincia. Chloroplast matK (Fig. 2) combines them in a weakly supported tree, trnL/F (not shown) does not even separate them from Leontodon subg. Leontodon, and the combined tree (Fig. 3) links Helminthotheca to Leontodon subg. Leontodon but keeps Picris separate. Helminthotheca (four species) has a Mediterranean distribution, whereas Picris (about 50 species) extends to tropical Africa, throughout Eurasia to Australia and New Zealand (Meusel and Ja¨ger, 1992: maps 531b–d). Leontodon hispanicus was transferred to Picris by Sell (1976) but had been treated as a member of Leontodon by Pittoni (1974), and, with hesitation, by Greuter (2003). It is now obvious from the molecular data and its typical anchor-like trichomes that it belongs in Picris.

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