Annals of Botany 98: 741–753, 2006 doi:10.1093/aob/mcl157, available online at www.aob.oxfordjournals.org

Centaurea Revisited: A Molecular Survey of the Jacea Group

N. GARCIA-JACAS1,*, T. UYSAL 2, K. ROMASHCHENKO3,V.N.SUA´ REZ-SANTIAGO4, K. ERTUG˘ RUL2 and A. SUSANNA1 1Botanic Institute of Barcelona (CSIC-ICUB), Passeig del Migdia s.n., E-08038 Barcelona, Spain, 2Department of Biology, Faculty of Science and Art, Selcuk University, Konya, Turkey, 3Institute of Botany M. G. Kholodny, Tereshchenkovska 2, 01601 Kiev, Ukraine and 4Department of Botany, Science Faculty, University of Granada, 18071 Granada, Spain

Received: 9 December 2005 Returned for revision: 11 April 2006 Accepted: 6 June 2006 Published electronically: 27 July 2006

Background and Aims The genus Centaurea has traditionally been considered to be a complicated taxon. No attempt at phylogenetic reconstruction has been made since recent revisions in circumscription, and previous reconstructions did not include a good representation of species. A new molecular survey is thus needed. Methods Phylogenetic analyses were conducted using sequences of the internal transcribed spacers (ITS) 1 and 2 and the 5.8S gene. Parsimony and Bayesian approaches were used. Key Results A close correlation between geography and the phylogenetic tree based on ITS sequences was found in all the analyses, with three main groups being resolved: (1) comprising the most widely distributed circum- Mediterranean/Eurosiberian sections; (2) the western Mediterranean sections; and (3) the eastern Mediterranean and Irano-Turanian sections. The results show that the sectional classification in current use needs major revision, with many old sections being merged into larger ones. A large polytomy in the eastern Mediterranean clade suggests a rapid and recent speciation in this group. Some inconsistencies between morphology and molecular phylogeny may indicate that hybridization has played a major role in the evolution of the genus. Conclusions Phylogenetic analysis of ITS has been useful in identifying the major lineages in the group, and unraveling many inconsistencies in the sectional classification. However, most recent groups in the eastern Mediterranean clade are not resolved and reticulation in the western Mediterranean group of sections makes phylogenetic relationships within these two groups somewhat obscure.

Key words: Cardueae, Centaureinae, Centaurea, Centaurea jacea group, hybridization, ITS sequences, phylogeny, systematics.

INTRODUCTION Centaurea, not for the numerous species in the core group. In line with this suggestions, Greuter et al. (2001) proposed The genus Centaurea L., with nearly 300 species (Bremer, conserving the name Centaurea with a new type species 1994; Wagenitz and Hellwig, 1996), has traditionally been for the genus, Centaurea paniculata L., from the group considered problematic and none of the early attempts to Jacea. Under his proposal, the old section Centaurea subdivide it (Cassini, 1829; Boissier, 1875; Lo¨ve and Lo¨ve, would acquire the rank of genus, namely Rhaponticoides 1961; Dosta´l, 1969, 1973; Holub, 1973, 1974) was widely Vaillant (Greuter, 2003; Greuter et al., 2005). The old accepted. However, more recent molecular analyses of the sections Acrocentron and Cyanus are likewise considered genus and of subtribe Centaureinae, together with studies by Greuter et al. (2001) to be separate genera, namely of morphology, pollen type and karyology, have enabled Colymbada Hill and Cyanus Mill., respectively. In the the natural limits of Centaurea to be established with authors’ opinion, however, this separation of Acrocentron greater confidence (Susanna et al., 1995; Wagenitz and and Cyanus is not fully supported by the available Hellwig, 1996; Garcia-Jacas et al., 2000, 2001). morphological, molecular and karyological evidence The nomenclatural consequences of establishing the (Garcia-Jacas et al., 2001). In view of the evident connec- natural limits of Centaurea were significant. The type tions between Acrocentron/Cyanus and the sections of the species of the genus, Centaurea centaurium L. (section group Jacea, it is suggested (Susanna and Garcia-Jacas, Centaurea), occupied an isolated position in the internal 2006) that all these sections should be maintained within transcribed spacer (ITS) and matK phylogenetic trees, the genus Centaurea, at least until there is firm evidence to distant from the monophyletic group that includes the great the contrary. Partly coinciding with this circumscription, majority of species in the genus (Garcia-Jacas et al., 2000, Greuter (2003) re-included Acrocentron in Centaurea at 2001). sectional rank, leaving Cyanus as a distinct genus. In previous studies (Garcia-Jacas et al., 2000, 2001) it The name ‘Jacea group’ denotes those taxa with the was suggested a new type species for the genus Centaurea, Jacea pollen type (Garcia-Jacas et al., 2000). Within this selected from the monophyletic core group, not from the group there is marked among-section diversity in habit current section Centaurea. This solution makes it necessary and in the morphology of bract appendages and cypselas, to change the name of only the 17 species of the section but the species of this group have various characters in * For correspondence. E-mail [email protected] common, including (a) the Jacea pollen type, (b) a lateral The Author 2006. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected] 742 Garcia-Jacas et al. — Molecular Analysis of the Centaurea jacea Group hilum (considered a derived character by Dittrich, 1968), with its current assignment to the section Acrocentron, and (c) a peculiar floral morphology with showy sterile whereas it is the commonest number in the Jacea group. peripheral florets lacking staminodes (Wagenitz and There is also a group of eastern species the sectional Hellwig, 1996). These latter two characters are shared affiliations of which are unclear (C. antitauri Hayek, with the sections Acrocentron and Cyanus. The Jacea C. ensiformis Davis, C. isaurica Hub.-Mor.) or need group shows descending dysploidy, with chromosome confirmation, including C. cankiriensis A. Duran & numbers ranging from x = 12 to x = 7 (Garcia-Jacas et al., H. Duman. 1996). Following a period of intensive collecting in the Iberian Most species of the Jacea group are distributed in the Peninsula and the eastern Mediterranean, a study based on eastern Mediterranean region and the Irano-Turanian analysis of ITS region sequences, like previous ones, was regions, although some species are exclusive to the western carried out which had the following main aims: Mediterranean region and others have a wider distribution. (1) To verify whether the basically biogeographical group- The widely distributed species are mostly associated with ings proposed in a previous study (Garcia-Jacas et al., crop cultivation and livestock farming, and in some areas 2000) are confirmed when species from a wider geo- they are economically significant weeds (C. diffusa Lam., graphical range are considered, notably including the C. maculosa Lam. and C. nigra L.). most representative species of the eastern Mediterran- In a preliminary study of Garcia-Jacas et al. (2000), the ean and the circum-Mediterranean/Eurosiberian clades. Jacea group was found to be monophyletic, in contradic- (2) To verify the existing sectional classification, and to tion with other authors who considered some sections to investigate relationships between sections. be separate genera (Chartolepis Cass., Cheirolepis Boiss., (3) To clarify the position of certain species the sectional Cnicus L., Grossheimia Sosn. & Takht., Tomanthea DC.; affiliation of which is unclear. cf. Klokov et al., 1963). Molecular phylogeny also allowed the possibility to be ruled out that Oligochaeta (DC.) C. Koch and Zoegea L. form part of this group, as already indicated by chromosome numbers (Garcia-Jacas et al., MATERIALS AND METHODS 1998a, b) and pollen types (Martı´n and Garcia-Jacas, 2000). Plant material Studies have divided the Jacea group into three major Sampling focused on the species of Centaurea with the clades, largely corresponding to geographical distributions: Jacea pollen type which constitute the Jacea group one clade with widely distributed species (circum- (Wagenitz and Hellwig, 1996; Garcia-Jacas et al., 2000). Mediterranean/Eurosiberian), a second clade of species Included are 98 taxa of the Jacea group, representing from the western Mediterranean, and a third clade of 27 sections out of the 29 accepted by Wagenitz and species from the eastern Mediterranean and adjacent areas Hellwig (1996); only the monotypic sect. Gymnocyanus of the Irano-Turanian region. In addition, these studies from Morocco and the bitypic sect. Corethropsis from the indicate that the sectional classification requires a deep Middle East are missing. The five outgroup species were review. Some sections cannot be segregated from the chosen from Centaurea sect. Cyanus, which is sister to the others on the basis of the authors’ analyses (specifically the Jacea group (Susanna et al., 1995; Garcia-Jacas et al., sections Melanoloma and Seridia of the western clade 2001). Voucher data, source and GenBank accession and the sections Cynaroides and Paraphysis of the eastern numbers for the ITS sequences are given in Table 1. clade). However, despite these previous findings (Garcia- All the sequences that had been used in previous studies Jacas et al., 2000), sampling limitations meant that several (Susanna et al., 1995; Garcia-Jacas et al., 2000) have questions remained to be resolved. been redone, with the addition of the 5.8S gene and The first question is the division into three major the elucidation of ambiguities that characterized the old clades (circum-Mediterranean/Eurosiberian, western Medi- manually generated sequences. terranean and eastern Mediterranean). Previous sampling of the circum-Mediterranean/Eurosiberian and eastern Mediterranean clades was limited, and it would be of DNA extraction, amplification and sequencing interest to confirm that the three-clade classification is Total genomic DNA was extracted following the maintained when a wider range of species is sampled. 2xCTAB method of Doyle and Doyle (1987) as modified The second question to be resolved is that of the by Soltis et al. (1991) and Cullings (1992) from silica sectional classification, notably of the eastern Mediterran- gel-dried leaves collected in the field or fresh leaves of ean group. We have noted a number of specific problems plants cultivated in the Botanic Institute of Barcelona. (e.g. the intercalation of sections Cynaroides–Paraphysis In some cases, herbarium material was used. and Cheirolepis–Plumosipappus) in a previous report Double-stranded DNA of the ITS region was mostly (Garcia-Jacas et al., 2001), and it remains unclear whether amplified using 17SE as the forward primer and 26SE as the current sections are natural entities. the reverse primer (Sun et al., 1994). The profile used for The third unresolved question relates to the taxonomic amplification included a hot start at 94 C for 2 min, placement of certain species. A clear case is that of followed by 80 C for 5 min, during which the polymerase C. chrysantha Wagenitz, the chromosome number of which (Ecotaq, Ecogen S. R. L., Barcelona, Spain) was added. (2n = 18; Romashchenko et al., 2004) is incompatible Then 30 cycles of amplification were carried out under the Garcia-Jacas et al. — Molecular Analysis of the Centaurea jacea Group 743

T ABLE 1. Origin of the materials, herbaria where the vouchers are deposited and GenBank accession numbers

Species Range Voucher ITS accession

Centaurea aggregata Fisch. & Caucasus, Iran, Turkey, Adana: Ala Dag˘ above Dag˘dibi, 2000 m, Ertug˘rul, Garcia-Jacas, DQ319077 C. A. Mey. ex DC. Turkey (weed) Susanna 2305 & Uysal, 3.8.2002 (BC) Centaurea aladaghensis Turkey endemic Turkey, Adana: Ala Dag˘ above Dag˘dibi, 2000 m, Ertug˘rul, Garcia-Jacas, DQ319078 Wagenitz Susanna 2304 & Uysal, 3.8.2002 (BC) Centaurea alba L. subsp. Iberian Peninsula Spain, Huesca: Pen˜a de Oroel, Ferna´ndez-Galiano & Rivas Goday 23733, AM114325 costae (Willk.) Dosta´l endemic 15.7.1947 (GDA) Centaurea alba L. subsp. Iberian Peninsula Spain, A´ vila: La Adrada, Sa´nchez-Mata & Canto´ 24946, 27.7.1982 AM114326 latronum (Pau) Dosta´l endemic (GDAC) Centaurea albonitens Turrill Caucasus, Iran, Turkey Garcia-Jacas et al. (2000) DQ319081 Centaurea alexandrina Delile Egypt, Libia, Egypt, Alexandria: 20 km W Alexandria on the coast road, Susanna 1854 DQ319082 Middle East & Vilatersana, 8.6.1998 (BC) Centaurea amadanensis Iran endemic Garcia-Jacas et al. (2000) DQ319083 Schultz-Bip. Centaurea antitauri Hayek Turkey endemic Turkey, Adana: Ala Dag˘ above Dag˘dibi, 2000 m, Ertug˘rul, DQ319084 Garcia-Jacas, Susanna 2306 & Uysal, 3.8.2002 (BC) Centaurea armena Boiss. Caucasus, Turkey Turkey, Van: Van, between Van and Gu¨rpinar, Kurubas¸ pass, DQ319085 2237 m, Ertug˘rul 2940, 5.7.2003 (KNYA) Centaurea aspera L. W Mediterranean Susanna et al. (1995) DQ319086 Centaurea aucheri (DC.) Caucasus, Iran, Turkey Garcia-Jacas et al. (2000) DQ319087 Wagenitz Centaurea avilae Pau Iberian Peninsula Spain, A´ vila: Sierra de Gredos, Blanca 6087, 30.7.1979 (GDAC) AM114309 endemic Centaurea aziziana Rech. f Turkey endemic Iran, Azarbayjan-e-Sharghi: between Tatar and Golfa, DQ319089 85 km from Golfa, Garcia-Jacas, Mozaffarian, Susanna 1680 & Valle`s, 7.8.1996 (BC) Centaurea behen L. Caucasus, Iran, Iraq, Susanna et al. (2006) AY826250 Middle East, Turkey Centaurea behen L. Caucasus, Iran, Iraq, Armenia, Abovian: between Gehart and Garni, Fajvush, Gabrielyan, DQ319090 Middle East, Turkey Garcia-Jacas, Guara, Hovannisyan, Susanna 1568, Tamanian & Valle`s, 23.8.1995 (BC) Centaurea benedicta L. Widespread Nancy Botanical Garden (1993) DQ319091 Centaurea boissieri DC. Iberian Peninsula Spain, Granada: Sierra de Ca´zulas, Blanca 6597, 8.6.1979 (GDAC) AM114278 subsp. boissieri endemic Centaurea bruguierana (DC.) Caucasus, Iran, Turkey Garcia-Jacas et al. (2000) DQ319093 Hand.-Mazz. Centaurea cadmea Boiss. Turkey endemic Turkey, Burdur: 4 km from Burdur on the road to Sparta, mountains above DQ319094 Burdur, 1200 m, Ertug˘rul, Garcia-Jacas, Susanna 2249 & Uysal, 28.7.2002 (BC) Centaurea calcitrapa L. Circum-Mediterranean Egypt, Alexandria: Alexandria road to Wadi Natrum, 130 km from Cairo, AY826252 Susanna 1866 & Vilatersana, 9.6.1998 (BC) Centaurea calolepis Boiss. Turkey endemic Turkey, Burdur-Mug˘la: Dirimli mountain pass, 1600 m, Ertug˘rul, DQ319095 Garcia-Jacas, Susanna 2254 & Uysal, 29.7.2002 (BC) Centaurea cankiriense A. Turkey endemic Turkey, Cankiri:¸ Kalfat–Atkaracalar road, 1455 m, 4041049N and DQ319096 Duran & H. Duman 3305027E, Uysal 524, 26.7.2003 (KNYA) Centaurea cariensis Boiss. Turkey endemic Turkey, Antalya: 40 km from Elmali on the road to Korkuteli, N slopes of DQ319097 the Karamanbeli mountain pass, 1400 m, Ertug˘rul, Garcia-Jacas, Susanna 2258B & Uysal, 30.7.2002 (BC) Centaurea carratracensis Iberian Peninsula Spain, Ma´laga: Carratraca, Sierra de Aguas, Blanca 42802, AM114302 Lange endemic 4.7.1998 (GDAC). Centaurea cataonica Turkey endemic Turkey, Gaziantep: Gaziantep, at the entry of the town, 800 m, Ertug˘rul, DQ319099 Boiss. & Hausskn. Garcia-Jacas, Susanna 2319 & Uysal, 4.8.2002 (BC) Centaurea cheirolepidoides Turkey endemic Turkey, Antalya: Elmali, from Tekke to Ciglikara,¸ 1500 m, Uysal 1000, DQ319100 Wagenitz 28.6.2004 (KNYA). Centaurea cheirolopha Lebanon, Turkey Turkey, Maras:¸ 40 km from Gaziantep on the Maras¸ road, DQ319101 (Fenzl) Wagenitz 2 km from Narh, 500 m, Ertug˘rul, Garcia-Jacas, Susanna 2324 & Uysal, 4.8.2002 (BC) Centaurea chrysantha Turkey endemic Turkey, Nig˘de: Ala Dag˘lar, track above the village of Demirkazik, DQ319102 Wagenitz 2000 m, Ertug˘rul, Garcia-Jacas, Susanna 2298 & Uysal, 3.8.2002 (BC) Centaurea corymbosa Pourr. S France endemic France, Narbonne: La Clappe, M. Riba, 1995 (BC) DQ319103 Centaurea debdouensis Morocco endemic Morocco, Debdou: Gaada de Debdou, Pasquier & Ch. Rungs, 18.6.1954 AM114317 Breitw. & Podlech (MPU) Centaurea deflexa Wagenitz Turkey endemic Turkey, Konya: between Cukuryurt¸ pass and Gevne valley, 25 km from DQ319105 Taskent,¸ 1700 m, Ertug˘rul, Garcia-Jacas, Susanna 2274 & Uysal, 1.8.2002 (BC) Centaurea derderiifolia Turkey endemic Turkey, Sivas: between Gu¨ru¨n and Divrigi, 18 km from Divrigi, DQ319106 Wagenitz 1500–1600 m, Uysal 530,39160530N and 3759027E 28.6.2003 (KNYA) Centaurea deusta Ten. Italy endemic Italy, Calabria: Crotone, Torrente Matassa near Caccuri, 360 m, DQ319107 Vogt 15531, Berlin Botanical Garden, Index Seminum 1997 744 Garcia-Jacas et al. — Molecular Analysis of the Centaurea jacea Group

T ABLE 1. Continued

Species Range Voucher ITS accession

Centaurea diffusa Lam. Widespread (weed) Armenia, Talin : between vil. Pokr Arthik and Bagravan, Fajvush, DQ319108 Gabrielyan, Garcia-Jacas, Guara, Hovhannisyan, Susanna 1589, Tamanyan & Valle`s, 26.8.1995 (BC) Centaurea diluta Aiton Iberian Peninsula, Morocco, Ksar es Souk: 10 km N of Midelt to Meknes, 1700 m, Garnatje, DQ319109 Morocco (weed) Susanna 1793 & Vilatersana, 17.6.1997 (BC) Centaurea donetzica Klokov Ukraine endemic Ukraine, Donetzkaya: Krasny Liman, Romashchenko, DQ319110 12.8.2002 (BC) Centaurea drabifolia Sm. Irano-Turanian Garcia-Jacas et al. (2000) DQ319111 Centaurea ensiformis Turkey endemic Turkey, Mug˘la: Ko¨yceg˘iz district, Sandras Dag˘ range 13 km from Ag˘la, DQ319112 P. H. Davis 1700 m, Ertug˘rul, Garcia-Jacas, Susanna 2251 & Uysal, 29.7.2002 (BC) Centaurea exarata Iberian Peninsula Spain, Huelva: road A-983, Almonte to Matalascan˜as km 25, DQ319113 Boiss. ex Coss. endemic Roche´ & Susanna 1909, 9.7.1999 (BC) Centaurea fenzlii Reichardt Turkey endemic Turkey, Erzurum: between Erzurum and Varto, 1 km from Varto, 1650 m, DQ319114 Uysal 895, 31.7.2004 (KNYA). Centaurea gadorensis Blanca Iberian Peninsula Spain, Almerı´a: Sierra de Ga´dor, Pico La Estrella, 1730 m, Martı´nez AM114298 endemic Lirola & Salinas 44171, 29.7.1996 (GDAC) Centaurea glastifolia L. Caucasus, Garcia-Jacas et al. (2000) DQ319116 Middle East, Turkey Centaurea glomerata Vahl Egypt, Middle East Egypt, Alexandria: Alexandria to Burg-el-Arab, Badr, Guara, DQ319117 Kamel & Valle`s, 10.4.1995 (BC) Centaurea hermanni F. Herm. Turkey endemic Turkey: Saray to Istambul, 35 km from Saray, Baytop, 17.6.1972 (E) DQ319118 Centaurea hyssopifolia Vahl Iberian Peninsula Spain, Toledo: near Ontı´gola, 500 m, Garcia-Jacas, Susanna 1600 & DQ319119 endemic Vilatersana, 22.6.1996 (BC) Centaurea iberica Trev. ex Caucasus, Iran, Turkey, Ankara: 20 km from Ankara to Eskisehir, 900 m, DQ319120 Sprengel Turkey (weed) R. Ilarslan 4308 & H. Ilarslan, 5.8.1994 (ANK) Centaurea imperialis Iran endemic Garcia-Jacas et al. (2000) DQ319121 Hausskn. ex Bornm. Centaurea inexpectata Turkey endemic Turkey, Antalya: Gevne valley, above village Ku¨cu¸ ¨klu¨, 1750 m, DQ319122 Wagenitz Uysal 598, 30.6.2004 (KNYA). Centaurea involucrata Desf. Algeria, Morocco Susanna et al. (1995) DQ319123 Centaurea isaurica Turkey endemic Turkey, Konya: Bozki, Sorkun town, 2000 m, Uysal 509, 13.7.2003 (BC) DQ319124 Hub.–Mor. Centaurea jacea L. Eurosiberian Spain, Barcelona: La Garrotxa, Pass of Bracons, 1100 m, Garcia-Jacas & DQ319125 Susanna 1593 (BC) Centaurea jaennensis Iberian Peninsula Spain, Jae´n: Pozo Alco´n, La Bolera dam, Blanca & Varo 6724, 19.6.1978 AM114287 Degen & Debeaux endemic (GDAC) Centaurea kotschyi (Boiss. & Caucasus, Iran, Turkey Garcia-Jacas et al. (2000) DQ319127 Heldr.) Hayek Centaurea kurdica Reichardt Turkey endemic Turkey, Elaˆzig˘: road Elaˆzig˘ to Bingo¨l, 1 km from the cross to Alacakaya, DQ319128 700 m, Ertug˘rul, Garcia-Jacas, Susanna 2360 & Uysal, 6.8.2002 (BC) Centaurea linifolia L. Eurosiberian Garcia-Jacas et al. (2000) DQ319129 Centaurea lycopifolia Boiss. Turkey endemic Turkey, Adana: Osmaniye–Yarpuz road, 800 m, Uysal 534, 30.6.2003 DQ319130 & Kotschy (KNYA) Centaurea macrocephala Caucasus, Turkey Armenia, Idjevan: N side of Sevan Pass, 1900 m, Fajvush, Gabrielyan, DQ319131 Muss. Puschk. ex Willd. Garcia-Jacas, Guara, Hovhannisyan, Susanna 1519, Tamanyan & Valle`s, 17.8.1995 (BC) Centaurea melitensis L. Widespread (weed) France, Montpellier: in front of museum ‘Agropolis’, Roche´ & Susanna DQ319132 2013, 2.7.1999 (BC) Centaurea monticola Boiss. Iberian Peninsula Spain, Granada: Pantano del Cubillas, Blanca 6750, 6.6.1977 (GDAC) AM114313 ex DC. endemic Centaurea napifolia L. Italy, Sardinia, Sicilia Italy, Calabria: Crotone, La Castella, 80 m, Vogt 15542, Berlin Botanical DQ319135 Garden, Index Seminum 1997 Centaurea nemecii Na´b. Iran, Turkey Garcia-Jacas et al. (2000) DQ319137 Centaurea nigra L. Eurosiberian Garcia-Jacas et al. (2000) DQ319138 Centaurea nivea (Bornm.) Turkey endemic Turkey, Eskisehir:¸ Mihalıcık–Alpu¸ road 19–20 km, 900–950 m, DQ319139 Wagenitz Uysal 519, 15.7.2003 (KNYA) Centaurea odyssei Wagenitz Greece, Turkey Turkey, Balikesir: Kaz mountains, Sarikiz hill, 1650–1675 m, DQ319140 Uysal 520,16.7.2003 (KNYA) Centaurea pallescens Delile Egypt, Libya Egypt, Alexandria: Alexandria between Amiriya and Burg-el-Arab, 20 km DQ319141 south of Burg-el-Arab, Susanna 1838 & Vilatersana, 7.6.1998 (BC). Centaurea paphlagonica Turkey endemic Garcia-Jacas et al. (2000) DQ319142 (Bornm.) Wagenitz Centaurea patula Boiss. Iran, Turkey Garcia-Jacas et al. (2000) DQ319143 Centaurea pectinata L. W Mediterranean Spain, Barcelona: Montseny, Santa Fe to Sant Marcal,¸ DQ319144 1300–1400 m, Garcia-Jacas & Susanna 1469, 6.7.94 (BC) Centaurea pinae Pau var. Iberian Peninsula Spain, Teruel: Puerto Ragudo, 900 m, Blanca, Socorro & AM114310 pinae endemic Valle 6768, 15.7.1978 (GDAC) Garcia-Jacas et al. — Molecular Analysis of the Centaurea jacea Group 745

T ABLE 1. Continued

Species Range Voucher ITS accession

Centaurea polyacantha Iberian Peninsula, Susanna et al. (1995) DQ319147 Willd. Morocco Centaurea polypodiifolia Caucasus, Iran, Iraq, Garcia-Jacas et al. (2000) DQ319148 Boiss. Syria, Turkey Centaurea proto-gerberi Ukraine endemic Ukraine, Luganskaya: Stanichno-Lugansk, Romashchenko, 5.9.2002 (BC) DQ319149 Klokov Centaurea pseudoleucolepis Ukraine endemic Ukraine, Donetzkaya: Kamennye Mogily national reservation, DQ319150 Kleopow Romashchenko, 1.8.2002 (BC) Centaurea pterocaula Trautv. Caucasus Garcia-Jacas et al. (2000) DQ319151 Centaurea ptosimopappa Turkey endemic Turkey, Seyhan-Hatay: Nur Dag, Gavur Dag, near Osmaniye DQ319152 Hayek 1000–1350 m, Wagenitz & H. J. Beug, 11.10.1957 (B) Centaurea ptosimopappoides Turkey endemic Turkey, Adana: Adana-Karsanti, Pos Ormonlari, Ormanalti, DQ319153 Wagenitz A. Savran, 25.7.02 (BC) Centaurea pullata L. Iberian Peninsula, Morocco, Ouarzazate: Dj. Mgoun, Amesker, Standinger & Finckh, DQ319154 Morocco 2.04.2001 (BC) Centaurea pungens Pomel Algeria, Morocco Morocco, Bouarfa: Bouarfa-Figuig, 65 km from Figuig, Garnatje, DQ319155 Susanna 1781 & Vilatersana, 16.6.1997 (BC) Centaurea resupinata Coss. Iberian Peninsula Spain, Albacete: between Elche de la Sierra and Hellı´n, Cenajo dam, AM114288 subsp. resupinata endemic Blanca & Varo 6714, 6.7.1977 (GDAC) Centaurea rhizantha Caucasus, Iran, Garcia-Jacas et al. (2000) DQ319157 C. A. Mey. Turkey Centaurea rigida Turkey endemic Turkey, Erzurum: 62 km from Erzurum on the road to Bingo¨l, 10 km from DQ319158 Banks & Sol. Cat,¸ Ertug˘rul, Garcia-Jacas, Susanna 2377 & Uysal, 6.8.2002 (BC) Centaurea saligna (C. Koch) Turkey endemic Turkey, Erzurum: between Karayazi and Go¨ksu, 2200 m, Uysal 891, DQ319159 Wagenitz 31.7.2004 (KNYA) Centaurea sarandinakiae Ukraine endemic Ukraine, Crimea: Planerskoe, Kara-Dag˘ mountain, Romashchenko, DQ319160 N. B. Illar 16.8.2002 (BC) Centaurea sclerolepis Boiss. Turkey endemic Turkey, Diyarbakir: between Diyarbakir and Silvan, 38 km from DQ319161 Diyarbakir, 751 m, Uysal 898, 1.8.2004 (KNYA) Centaurea semijusta Juz. Ukraine endemic Ukraine, Crimea: Simferopol, Chatyr-Dag˘ mountain, Romashchenko, DQ319162 1.9.2002 (BC) Centaurea solstitialis L. Widespread (weed) Spain, Tarragona: road from Xerta to Port del Compte, 5 km from cross- DQ319163 road, Garnatje & Susanna 1875, 6.7.1998 (BC) Centaurea spectabilis (Fisch. Caucasus, Turkey Turkey, Van: Van, between Van and Gu¨rpinar, Kurubas¸ pass, DQ319164 & C. A. Mey.) Sch. Bip. 2237 m, Ertug˘rul 2941, 5.7.2003 (KNYA) Centaurea spinosa L. Aegean Greece, Thrakia: Nomos Evrou, Samothraki, 2 m, Raus/Sch 18942, Berlin DQ319165 Botanical Garden, Index Seminum 1997 Centaurea stapfiana Turkey endemic Turkey, Diyarbakir: between Diyarbakir and Silvan, Yesilko¸ ¨y village, DQ319166 (Hand.-Mazz.) Wagenitz hills of Kendal, 627 m, Uysal 900, 1.8.2004 (KNYA) Centaurea sterilis Stev. Ukraine endemic Ukraine, Crimea: Planerskoe, Kara-Dag˘ mountain, Romashchenko, DQ319167 16.8.2002 (BC) Centaurea steveni M. Bieb. Caucasus, Iran Garcia-Jacas et al. (2000). DQ319168 Centaurea sulphurea Willd. W Mediterranean Morocco, Khenifra: Aguelmame Azigza, Standinger & Finckh, DQ319169 6.6.2001 (BC) Centaurea thracica (Janka) Aegean, Balkans, Turkey, Balikasir: between Balikesir and Bursa, near Harmancık, 1005 m, DQ319171 Hayek Greece, Turkey Uysal 564, 30.6.2004 (KNYA) Centaurea tomentella Turkey endemic Turkey, Malatya: 4 km NW Dog˘ansehir to Polat, 1000 m, Ertug˘rul, DQ319172 Hand.-Mazz. Garcia-Jacas, Susanna 2353 & Uysal, 6.8.2002 (BC) Centaurea vankovii Klokov Ukraine endemic Ukraine, Crimea: Alupka, Ai-Petri mountain, Romashchenko, 30.8.2002 DQ319173 (BC) Centaurea virgata Lam. Turkey endemic Turkey, Mug˘la: Ko¨yceg˘iz district, Sandras Dag˘ range 13 km from Ag˘la, DQ319174 1700 m, Ertug˘rul, Garcia-Jacas, Susanna 2252 & Uysal, 29.7.2002 (BC) Centaurea wiedemanniana Turkey endemic Turkey, Bilecik: Selimiye, between Osmaneli and Bilecik, DQ319175 Fisch. & C. A. Mey. 100 m, Davis & Coode, 1.7.1962 (E) Centaurea xylobasis Rech. f. Greece (Islands) Greece, Samos: Mt. Kerki, 1000–1400 m, Runemark & Nordenstam, DQ319176 2.8.1960 (E) Outgroup Centaurea cyanus L. Cosmopolitan Susanna et al. (2006). AY826254 Centaurea mollis E Europe Ukraine, Podolia: Lysa Hora, 2 km E of Vilshanitsa near Zolochiv, DQ319133 Waldst. & Kit. Boratyn˜ski & Romo 0506D, 5.6.2000 (BC) Centaurea napulifera E Europe Romania, Constanta: Dobrogea, N from Cheia village, Cheia gorges of the DQ319136 Rochel subsp. thirkei Casimcea river, 130 m, Badarau 2.4.1996 (BC) (Sch. Bip.) Dosta´l Centaurea pinardii Boiss. Balkans, Turkey Turkey, Burdur: 4 km from Burdur on the road to Sparta, Askeriye DQ319146 outskirts, 950 m, Ertug˘rul, Garcia-Jacas, Susanna 2244 & Uysal, 28.7.2002 (BC) Centaurea tchihatcheffi Turkey endemic Turkey, Ankara: Go¨lbasi, lake shore, 950 m, M. Vural, 30-5-2002 (BC) DQ319170 Fisch. & C. A. Mey. 746 Garcia-Jacas et al. — Molecular Analysis of the Centaurea jacea Group following conditions: 94 C for 1 min 30 s, 57 C for 2 min T ABLE 2. Results from the ITS region and 72 C for 3 min, with an additional extension step of 15 min at 72 C. Some species were amplified using 1046F Data set ITS region (Nickrent et al., 1994) as the forward primer and ITS4 (White et al., 1990) as the reverse primer. The profile used Number of taxa 103 Length range (bp) 629–635 for amplification was the same as above, apart from the Total characters 644 annealing temperature (55 C). The PCR products were Number of informative characters 148 purified with the QIAquick PCR Purification Kit (Qiagen Number of most-parsimonious trees 2 31 208 Inc., Valencia, CA, USA). Both strands were sequenced Number of steps 422 Consistency index (CI) 0.4521 with 17SE and 1046F as forward primers and 26SE and Retention index (RI) 0.8471 ITS4 as reverse primers. Direct sequencing of the amplified Range of divergence, ingroup (%) 0–9 DNA segments was performed using a BigDye Terminator Range of divergence, ingroup–outgroup (%) 5–10 Cycle Sequencing v3.1 (PE Biosystems, Foster City, CA, USA), following the manufacturer’s protocol and analysed Consistency and retention indices and divergence ranges were calculated on an ABI PRISM 3730 DNA analyser (PE Biosystems). excluding non-informative characters. The sequences were edited using Chromas 1.56 (Tech- nelysium Pty, Tewantin, Australia). RESULTS Phylogenetic analysis The numerical results of the analysis of the ITS region are detailed on Table 2. The topology of the trees produced Sequences were aligned visually by sequential pairwise by parsimony and Bayesian approaches was coincident, comparison (Swofford and Olsen, 1990). The data matrix is and Fig. 1 shows the parsimony Bayesian majority-rule available on request from the corresponding author. consensus tree with the addition of the Bayesian and the Parsimony analysis involved heuristic searches con- bootstrap support values (PP, BS). One of the most- ducted with PAUP version 4.0b4a (Swofford, 1999) using parsimonious trees is shown in Fig. 2. tree-bisection-reconnection (TBR) branch swapping with The strict Bayesian majority-rule consensus tree produced character states specified as unordered and unweighted. from the ITS analysis (Fig. 1) supports the monophyly of the The indels were treated as missing data. All most- Jacea group (BS = 85 %, PP = 100). Within this clade, there parsimonious trees were saved. To locate other potential are two main evolutionary branches: the first branch, which islands of most-parsimonious trees (Maddison, 1991), 100 we will name ‘the circum-Mediterranean/Eurosiberian replications were performed with random taxon addition, clade’, encompasses the sections with a wide distribution also with TBR branch swapping. Bootstrap analysis (BS) in Europe and western Asia (BS = 83 %, PP = 099); the was performed (Felsenstein, 1985) with 100 replicates with second branch is composed of two clades: one clade, which simple taxon addition and TBR branch swapping. will be named the ‘western Mediterranean clade’, is formed Bayesian inference estimation was calculated using by the representatives of sections of western Mediterranean MrBayes 3.01 (Ronquist and Huelsenbeck, 2003). The distribution (BS = 75 %, PP = 100); and the other, which will best-available model of molecular evolution required for be named the ‘eastern Mediterranean clade’, comprises the Bayesian estimations of phylogeny was selected using sections which have an eastern Mediterranean and Irano- hierarchical likelihood ratio tests and Akaike information Turanian distribution (BS = 100 %, PP = 100). criteria as implemented in the software MrModeltest 1.1b (Nylander, 2002), which considers only nucleotide sub- stitution models that are currently implemented in PAUP and MrBayes 3.01 (Huelsenbeck and Ronquist, 2001; DISCUSSION Ronquist and Huelsenbeck, 2003). The symmetrical model, In view of the confirmed close relationship between with equal base frequencies with some sites assumed to be geographical distribution and phylogenetic patterns as invariable and variable sites assumed to follow a discrete inferred from nuclear DNA, considered here are the three gamma distribution (SYM+I+G; Zharkikh, 1994), was major clades: the widely distributed clade, the western selected as the best-fit model of nucleotide substitution for clade and the eastern clade. the dataset using both hierarchical likelihood ratio tests and Akaike information criteria. Bayesian inference analyses The circum-Mediterranean/Eurosiberian clade were initiated with random starting trees and were run for 2 · 106 generations. Four Metropolis-coupled Monte In all analyses this clade was placed as sister to the Carlo Markov chains were sampled every 100 generations, eastern and western Mediterranean clades (Fig. 1), without resulting in 20 000 sample trees. A critical aspect of the Bayesian support but with high bootstrap support (BS = Bayesian analysis is to ensure that the Markov chain has 94 %). This group has a wide distribution from central– reached stationarity. All sample points prior to stationarity western Asia to the Iberian Peninsula, with many narrow are essentially random and are discarded as ‘burn-in’ 2000 endemics at the eastern and western extremes of the sample trees because, usually, they do not contain useful distribution. parameter estimates. Internodes with posterior probabilities As has been pointed out in a previous report (Garcia- >95 % were considered statistically significant. Jacas et al., 2000), some sections are consistently Garcia-Jacas et al. — Molecular Analysis of the Centaurea jacea Group 747

1·00 Centaurea albonitens 86 Centaurea aucheri Centaurea amadanensis Centaurea fenzlii 0·99 Centaurea sclerolepis 71 Centaurea kurdica 0·96 Centaurea imperialis 71 Centaurea nemecii 1·00 0·99 Centaurea aladaghensis 0·74 94 Centaurea cataonica Cynaroides 82 Centaurea tomentella – Centaurea antitauri 0·60 Centaurea spectabilis – Centaurea macrocephala Centaurea stapfiana 1·00 Centaurea behen S1562 0·89 0·8 100 Centaurea behen S1568 1·00 – 51 Centaurea rigida 100 Centaurea polypodiifolia 1·00 Centaurea rhizantha Centaurea steveni Microlophus 100 Centaurea armena Centaurea sqligna 1·00 Centaurea glastifolia 100 0·99 Centaurea pterocaula 1·00 Centaurea thracica 69 Centaurea odyssei 53 Centaurea xylobasis Centaurea isaurica 1·00 Centaurea cheirolepidoides 0·65 Centaurea drabifolia Eastern 100 Centaurea hermanii – Centaurea nivea Mediterranean Centaurea cankiriense Clade Centaurea derderiifolia P1 1·00

Centaurea ensiformis Middle East Turkey, Caucasus, Iran, 88 Centaurea kotschyi 1·00 Centaurea deflexa 100 Centaurea lycopifolia 1·00 Centaurea paphlagonica 1·00 Centaurea ptosimopappoides 100 Centaurea cheirolopha 88 Centaurea chrysantha Centaurea ptosimopappa Centaurea bruguierana 1·00 Centaurea calcitrapa 0·55 100 1·00 0·97 Centaurea glomerata 1·00 64 Centaurea pallescens 94 100 East

89 Centaurea iberica Middle

Centaurea alexandrina Calcitrapa 1·00 0·96 Centaurea aspera 0·82 100 Centaurea napifolia – 61 Centaurea involucrata 1·00 Centaurea polyacantha 1·00 67 Centaurea pullata 1·00 Centaurea pungens 93 Centaurea diluta Italy Iberian

75 67 N Africa, 1·00 Centaurea sulphurea Peninsula, – 1·00 Centaurea melitensis 85 Centaurea solstitialis Western 100 0·98 Centaurea aggregata Mediterranean Centaurea cadmea – Centaurea wiedemanniana Clade Centaurea aziziana 0·98 Centaurea proto–gerberi 68 Centaurea sterilis Centaurea deusta Centaurea virgata Centaurea calolepis 1·00 Centaurea cariensis 100 Centaurea spinosa 0·91 Centaurea diffusa 71 Centaurea pseudoleucolepis Centaurea vankovii 1·00 Centaurea donetzica Centaurea semijusta 71 Acrolophus-Phalolepis 0·88 Centaurea sarandinakiae – Centaurea corymbosa Centaurea alba subsp. costae Centaurea alba subsp. latronum 0·98 Centaurea resupinata Centaurea gadorensis 86 Centaurea avilae Centaurea monticola 0·75 1·00 – Centaurea carratracensis 100 Centaurea boissieri Centaurea jaennensis

Centaurea pinae Willkommia Centaurea debdouensis Centaurea benedicta 0·99 1·00 Centaurea jacea 83 0·57 Centaurea nigra 100 Iran Turkey, Africa, North Europe, – 1·00 0·67 Centaurea linifolia 1·00 – Centaurea hyssopifolia Circum-Mediterranean 100 Centaurea exardta and Eurosiberian Clade 71 Centaurea pectinara Jacea-

Centaurea inexpectata Lepteranthus Centaurea patula 1·00 0·53 Centaurea napulifera 100 Centaurea mollis Outgroup – Centaurea cyanus 1·00 Centaurea tchihatcheffii 100 Centaurea pinardii

F IG. 1. Majority-rule consensus tree based on Bayesian Monte Carlo Markov chains. Numbers above branches are Bayesian posterior probability percentages; numbers below branches are bootstrap percentages. P1, Cheirolepis-Pseudoseridia polytomy. associated in well-supported clades. The most important four sections are morphologically similar, and, as noted is the complex made up of the sections Acrolophus, by Wagenitz (1989), the separation of Acrolophus and Maculosae, Phalolepis and Pseudophalolepis (BS = 100 %, Phalolepis is clearly artificial. The species of this group PP = 10), named Acrolophus-Phalolepis in Fig. 1. These are either effective colonizers, e.g. C. diffusa, or narrow 748 Garcia-Jacas et al. — Molecular Analysis of the Centaurea jacea Group

Centaurea albonitens Centaurea aucheri Centaurea amadanensis Centaurea fenzlii Centaurea sclerolepis Centaurea kurdica Centaurea imperialis Centaurea nemecii Centaurea aladaghensis Centaurea cataonica Centaurea tomentella Centaurea antitauri Centaurea spectabilis Centaurea macrocephala Centaurea behenS1562 Centaurea behenS1568 Centaurea rigida Centaurea polypodiifolia Centaurea stapfiana Centaurea thracica Centaurea odyssei Centaurea xylobasis Centaurea isaurica Centaurea cheirolepidoides Centaurea drabifolia Centaurea hermanii Centaurea nivea Centaurea cankaviensis Centaurea derderiifolia Centaurea ensiformis Centaurea kotschyi Centaurea deflexa Centaurea lycopifolia Centaurea paphlagonica Centaurea rhizantha Centaurea steveni Centaurea armena Centaurea saligna Eastern Centaurea glastifolia Mediterranean Centaurea pterocaula Centaurea ptosimopappoides Clade Centaurea cheirolopha Centaurea chrysantha Centaurea ptosimopappa Centaurea bruguierana Centaurea calcitrapa Centaurea alexandrina Centaurea glomerata Centaurea pallescens Centaurea iberica Centaurea aspera Centaurea napifolia Centaurea involucrata Centaurea pungens Centaurea polyacantha Centaurea pullata Western Centaurea diluta Centaurea sulphurea Mediterranean Centaurea melitensis Clade Centaurea solstitialis Centaurea aggregata Centaurea cadmea Centaurea wiedemanniana Centaurea aziziana Centaurea proto-gerberi Centaurea sterilis Centaurea deusta Centaurea virgata Centaurea calolepis Centaurea cariensis Centaurea spinosa Centaurea diffusa Centaurea pseudoleucolepis Centaurea vankovii Centaurea donetzica Centaurea semijusta Centaurea sarandinakiae Centaurea corymbosa Centaurea alba subsp. costae Centaurea alba subsp. latronum Centaurea resupinata subsp. resupinata Centaurea gadorensis Centaurea avilae Centaurea monticola Centaurea carratracensis Circum-Mediterranean Centaurea boissieri subsp. boissieri Centaurea jaennensis and Eurosiberian Clade Centaurea pinae subsp. pinae Centaurea debdouensis Centaurea patula Centaurea jacea Centaurea nigra Centaurea linifolia Centaurea hyssopifolia Centaurea exarata Centaurea pectinata Centaurea inexpectata Centaurea benedicta Centaurea napulifera Centaurea mollis Centaurea cyanus Centaurea tchihatcheffii Centaurea pinardii 5 changes

F IG. 2. One of the most-parsimonious trees resulting from the ITS analysis. Garcia-Jacas et al. — Molecular Analysis of the Centaurea jacea Group 749 endemics, e.g. C. sarandinakiae N. B. Illar and (1973) to section Maculosae together with C. corymbosa C. pseudoleucolepis Kleopow. (as has been seen, C. corymbosa was placed in the present The section Pseudophalolepis was described by analyses in the Acrolophus-Phalolepis group). The present Klokov et al. (1963) to include certain species from results do not appear to support recognition of Jacea the Ukraine, on rather weak morphological grounds. The (represented in the present analyses by C. inexpectata, C. representative species of this section, C. donetzica jacea and C. nigra) and Lepteranthus (represented in the Klokov, C. proto-gerberi Klokov and C. pseudoleucolepis, present analyses by C. hyssopifolia, C. linifolia and C. do not form a natural group and appear in different clades pectinata) as separate sections. Likewise, there are no in the present analysis. For example, C. donetzica is important morphological differences between Jacea and included in a group with three other Ukrainian and Lepteranthus, which were originally considered as separate Crimean microendemics of the section Phalolepis, namely sections only because of the slightly more lacerate bract C. sarandanakiae, C. semijusta Juz. and C. vankovii appendices of species of Lepteranthus (Hayek, 1901). Klokov, with moderate bootstrap support (BS = 71 %) and Centaurea inexpectata, the easternmost representative of high Bayesian support (PP = 100). These results argue this group studied here (it is a Turkish endemic), is placed strongly against the maintenance of Pseudophalolepis as as sister to the remaining species with moderate support a separate section, as is evident from morphology. (BS = 71 %, PP = 100). Similarly, there do not appear to be good grounds for The position of C. benedicta (L.) L. reflects a conflict maintaining section Maculosae (Dosta´l, 1973), since in between macromorphology and phylogenetic relationships the present analysis the two representatives of this section as revealed by pollen type and DNA sequence analyses. It were deeply nested within other sections. Centaurea shows some striking differences (especially in achene corymbosa Pourr. was placed in a polytomy with species morphology) that until recently were considered adequate of Acrolophus and Phalolepis, with which it shares a grounds for consideration as a monotypic genus (as Cnicus similar morphology. Centaurea exarata Boiss. ex Coss. fell benedictus L.). However, the Jacea pollen type (Wagenitz, in section Jacea (clade Jacea-Lepteranthus in the Fig. 1), 1955) found in this species was the first clue to its true although in this latter case the inclusion is not supported relationships, which were later confirmed with DNA by morphology, since the morphology of C. exarata is sequencing (Garcia-Jacas et al., 2000, 2001). Centaurea unlike that of all other members of the genus. However, patula DC. shows a unique characteristic within the genus the chromosome number of C. exarata, x = 11 (Valde´s- Centaurea: its chromosome number is x = 7, the lowest Bermejo, 1980), coincides with the basic number found found to date in the genus (Garcia-Jacas et al., 1996). in sect. Jacea, a fact that favours its inclusion in this section. Section Willkommia, composed of Iberian and North The western Mediterranean clade African endemics and morphologically similar to section This group comprises the only sections found exclu- Acrolophus, forms another robust clade with strong sively in the western Mediterranean, Melanoloma and bootstrap (100 %) and Bayesian (PP = 100) support. It is Seridia (C. aspera L., C. involucrata Desf., C. polyacantha placed with the Acrolophus-Phalolepis group, but with Willd. and C. pullata L.), together with other sections weak statistical support (Fig. 1). The present results made up of widely distributed colonizing species (i.e. the indicate that section Willkommia should be maintained, Hymenocentron-Mesocentron group, which includes some especially in view of the lack of support for its inclusion well-known weeds, e.g. C. diluta Aiton, C. melitensis L., within the Acrolophus-Phalolepis group. C. solstitialis L. and C. sulphurea Willd.). This clade A surprising result of the present analysis is the has 75 % bootstrap support and high Bayesian support (PP = placement of the two Iberian representatives of Phalolepis, 100). It is the clade in which natural classification C. alba L. subsp. alba and C. alba subsp. latronum (Pau) of sections is most difficult, no doubt due to frequent Dosta´l. Both are placed within the same clade as section intersectional hybridizations (C. aspera · C. pullata is a Willkommia.Sua´rez-Santiago (2005) recently demon- common hybrid in the southern Iberian peninsula). strated the existence of ancient hybridizations between This clade also includes C. pungens Pomel, a North the sections Phalolepis and Acrolophus and section African species that has traditionally been placed in section Willkommia: the nuclear genome of the Iberian species of Calcitrapa. The present results suggest that this species the sections Acrolophus and Phalolepis does not bear forms part of the Hymenocentron-Melanoloma-Seridia any relation to that of other species of these sections, but group, with which it shares a basic chromosome number shows considerable similarities with the nuclear genome of x = 11, unlike the species of the section Calcitrapa with of species of section Willkommia. x = 10 (Romashchenko et al., 2004). Another complex is made up of sections Jacea and Lepteranthus, all sampled species of which (C. hyssopifolia Vahl, C. jacea L., C. inexpectata Wagenitz, C. linifolia L., The eastern Mediterranean clade C. nigra L. and C. pectinata L.) are placed in a single clade This group is by far the largest in the genus Centaurea, with moderate bootstrap support (71 %) and strong comprising about 200 species in the Caucasus, Greece, Bayesian support (PP = 100). This clade also included Iran and Turkey. As has been pointed out previously C. exarata Coss., which according to Cosson (1850) (Garcia-Jacas et al., 2000), section Calcitrapa, widely belongs to section Acrolophus and according to Dosta´l distributed in the western Mediterranean region, falls in this 750 Garcia-Jacas et al. — Molecular Analysis of the Centaurea jacea Group group (clade Calcitrapa in Fig. 1) as sister to the remaining x = 8, shared with C. steveni. The present results indicate taxa with strong support (bootstrap 100 %, PP = 100). that section Rhizocalathium should be maintained, but with As in the western Mediterranean clade, the present modified limits. However, the status of section Pseu- results suggest a series of section groupings. The first doseridia is highly dubious. Centaurea cheirolepidoides, comprises the sections Calcitrapa, Seridioides and C. hermanii and C. lycopifolia are included in a major Tetramorphaea (the Calcitrapa clade in Fig. 1). The polytomy with the species of section Cheirolepis, C. steveni species of these three sections make up a clade with strong falls with the species of section Rhizocalathium support (bootstrap 100 %, PP = 100). Within this clade, and, finally, another representative of Pseudoseridia, C. bruguierana Hand.-Mazz., the only species described C. cheirolopha (Fenzl) Wagenitz, forms a clade with to date in section Tetramorphaea, is sister to the species C. ptosimopappoides Wagenitz (section Ptosimopappa) of the section Calcitrapa. In the analyses of Garcia-Jacas and C. chrysantha. et al. (2000), the species of sections Calcitrapa and Tetramorphaea were grouped together with strong boot- strap support. In the present study, with consideration of Section Microlophus more species of the section Calcitrapa, the association The present analysis included four species of section of these two sections is confirmed. Also placed within Microlophus: C. behen L., C. polypodiifolia Boiss., this clade was C. glomerata Vahl, included by Wagenitz C. rigida Willd. and C. thracica (Janka) Hayek. Only and Hellwig (1996) in the section Seridioides, and in the two of these species, C. polypodiifolia and C. rigida, both present study placed with C. pallescens Del. with weak with the same basic chromosome number x = 8, form a bootstrap support (64 %) and moderate Bayesian support group with strong support (BS = 100 %, PP = 100). (PP = 097). The present results thus do not provide any Centaurea behen L., for which two Armenian populations support for the section Seridioides. were sampled, was placed with this group, but without statistical support (clade named Microlophus in Fig. 1). Sections Cynaroides and Paraphysis Centaurea behen has an unusual chromosome number for the Jacea group, 2n = 34 (Romashchenko et al., 2004) These are the only two sections of the genus Centaurea and for subtribe Centaureineae. This chromosome number with capitula grouped in a spike-like inflorescence; both could be explained if this species were a allopolyploid sections also have highly characteristic entire hastate hybrid between a species with x = 8 and another with x = 9. leaves with velvety indumentum. The species of these two According to ITS phylogeny, the lack of relationship sections fall into two distinct clades: one comprised of a between the samples of C. behen and the remaining species polytomy involving various species of section Cynaroides of the eastern clade suggests that it may have a hybrid (C. kurdica Reichardt, C. fenzlii Reichardt, C. imperialis origin. Centaurea thracica, with x = 9, is placed in a Hausskn. and C. sclerolepis Boiss.) and the two species clade with the species of the complex Cheirolepis- of section Paraphysis (C. amadanensis Sch. Bip. and Pseudoseridia-Pteracantha-Plumosipappus, which like- C. nemecii Na´be˘lek) with bootstrap support of 71 % and wise have a chromosome number of x = 9. These species PP = 099. The other clade grouped the remaining species are grouped with C. odyssei Wagenitz and C. xylobasis of section Cynaroides (C. aladaghensis Wagenitz, Rech. f. of section Pteracantha. This entanglement of C. cataonica Boiss. and C. tomentella Hand.-Mazz.) with sections confirms the difficulties of establishing a sectional strong support (BS = 94 %, PP = 100). The two clades fall classification based on bract appendices, as previously together (named Cynaroides in Fig. 1), but with weak suggested by Ertug˘rul et al. (2004). More studies are support (BS = 71 %, PP = 096). needed in section Microlophus, especially on the correla- On morphological and karyological grounds it seems tion between karyology and systematics. clear that the species of section Cynaroides (including the species of section Paraphysis) are closely related. It is thus surprising that the present analysis separates the species The of this group into two different clades. The nine ITS Cheirolepis-Pseudoseridia-Pteracantha-Plumosipappus complex sequences studied only show ten differences between the two clades, of which four are autoapomorphies, one is These four sections were placed in a single clade within shared by two species and the remaining five are in line a polytomy (marked P1 in Fig. 1) with a bootstrap value with the separation of these species into two independent of 88 % and PP = 100. The species are well character- clades. ized morphologically, but not at the molecular level: the sequences show practically no differences, and the differences that do exist are mostly autoapomorphies. This Sections and Pseudoseridia Rhizocalathium probably reflects a recent and rapid speciation, so that there The two species of section Rhizocalathium included in are morphological differences but practically no genetic the study, C. armena Boiss. and C. rhizantha C.A. Mey., differences. The same occurs in other groups of the are linked to C. steveni M. Bieb. from section Pseu- Centaureineae in which speciation is thought to have been doseridia with strong support (BS = 100 %, PP = 100). The recent and rapid, such as Cheirolophus Cass. (Susanna species of section Rhizocalathium have an unusual basic et al., 1999) or Centaurea section Willkommia (Sua´rez- chromosome number for the sections in the Jacea group, Santiago, 2005). Garcia-Jacas et al. — Molecular Analysis of the Centaurea jacea Group 751

Data from ITS thus support merging all these sections Sections Chartolepis, Grossheimia and into a single section, and morphology supports this option Pseudophaeopappoides (Ertug˘rul et al., 2004). Although there are differences in Apart from the fact that these sections belong to the pappus and bract morphology, these differences lie within eastern clade, the present analysis does not reveal any the range of variability that can be found in other sections relationships among these three sections. In the case of such as Melanoloma and Seridia (Ertug˘rul et al., 2004). section Pseudophaeopappoides, this is unfortunate, In this clade are also found C. odyssei and C. xylobasis because the only species described in this section is the (section Pteracantha) and C. thracica (section Microlo- puzzling C. antitauri Hayek, the affinities and relationships phus), which are grouped together with low bootstrap of which are unclear. Sections Chartolepis (Centaurea support but high Bayesian support (PP = 0 99). In contrast, glastifolia L. and C. pterocaula Trautv.) and Grossheimia other species that have previously been included in section (C. macrocephala Puschk.) have no close relatives Pseudoseridia were placed in other clades, as noted according to the present analyses. Centaurea glastifolia above. Centaurea saligna, traditionally placed in section and C. macrocephala have given rise to a fertile hybrid in Cheirolepis, is not grouped with the rest of taxa of this Armenia, C. caroli-henrici Gabrieljan & Dittrich (Dittrich section. According to other studies (T. Uysal, pers. and Gabrieljan, 1993). comm.), C. saligna may be located as one of the first branches in the clade Cheirolepis-Pseudoseridia- Position of certain species in the eastern clade Pteracantha-Plumosipappus. One of the goals of this study was to clarify the doubtful Section Ptosimopappus position of C. chrysantha within section Acrocentron and the relationships of four species of difficult sectional The present analysis includes the only two species assignment (C. antitauri, C. cankiriensis, C. ensiformis and described in this section, C. ptosimopappa Hayek and C. isaurica). All of them were placed in the eastern clade, C. ptosimopappoides. These two species are not grouped as expected, and their affinities seem clear except in the together in the analysis, indicating that this is not a case of the isolated C. antitauri. natural section. Species in section Ptosimopappus have an unusual shrubby habit, and on the basis of this were at Centaurea chrysantha one time considered as a separate genus, Ptosimopappus Boiss. Wagenitz (1975) had already pointed out that The present results confirm the conclusion based on C. ptosimopappoides is morphologically intermediate chromosome studies, indicating a basic chromosome between Ptosimopappus and Microlophus. The present number of 2n = 18, that this species is related to species results rule out any relationship between C. ptosimopappa of the eastern sections of the Jacea group (Romashchenko and C. ptosimopappoides, and confirm the isolation of et al., 2004). Centaurea chrysantha clearly falls into the a monotypic section Ptosimopappus (inluding only Jacea group, and specifically into the clade of eastern C. ptosimopappa), which in the present analysis is not species. Within this clade, it is sister to two species usually exclusively related to any other. classified in two different sections (C. ptosimopappoides from section Ptosimopappus and C. cheirolopha from Section Phaeopappus section Pseudoseridia), with good support (BS = 88 %, PP = 100; Fig. 1). As has been seen, C. ptosimopappoides Four representatives of this section were included, is unrelated to section Ptosimopappus, and section namely C. albonitens Turr., C. aucheri (DC.) Wagenitz, Pseudoseridia cannot be mantained. Thus, C. cheirolopha, C. spectabilis and C. stapfiana (Hand.-Mazz.) Wagenitz. C. chrysantha and C. ptosimopappoides, upon close This section has often been recognized as a distinct genus, morphological examination, could together constitute a Tomanthea, especially by the Russian school. Gabrieljan new section. and Aghababian (1990) considered that it should be divided into two different genera, Chrysopappus Takht. [C. Centaurea cankiriensis stapfianus (Hand.-Mazz.) Takht.] and Tomanthea (remain- ing species). The generic distinction of Tomanthea was This species was described indicating that it should be rejected on molecular grounds by Garcia-Jacas et al. placed in section Cheirolepis. The present results confirm (2000). Chrysopappus also belongs in Centaurea (Fig. 1). that it forms part of the Cheirolepis-Pseudoseridia complex In the present results, the section appears to be fragmented (Fig. 1) together with C. drabifolia and C. kotschyi, with (Fig. 1): only C. albonitens and C. aucheri were linked which it shares bract appendix morphology (T. Uysal, pers. with strong support (BS = 86 %, PP = 100). On the one comm.). hand, this fragmentation could support the views of Centaurea ensiformis Gabrieljan and Aghababian (1990) that Chrysopappus is distinct, if only at the sectional rank. On the other hand, This is a Turkish endemic that occurs on serpentine soils, morphological connections between all the species of and its morphological characters do not allow clear section Phaeopappus are rather clear (Wagenitz, 1963) assignment to any of the existing sections (Davis, 1956; and, as in the above-mentioned cases of inconsistencies Wagenitz, 1975). The present results indicate a relationship between morphology and molecular data, a possible with the Cheirolepis-Pseudoseridia complex (Fig. 1). explanation for this is past hybridization. A recent morphological study (T. Uysal, pers. comm.) 752 Garcia-Jacas et al. — Molecular Analysis of the Centaurea jacea Group indicates relationships with C. cankiriensis, which is (Font et al., 2002), hybridization may have played, and compatible with the present results. may still play, a major role in the evolution of this group.

Centaurea isaurica This species was placed by the present analysis in the ACKNOWLEDGEMENTS Cheirolepis-Pseudoseridia complex (Fig. 1), as suggested Financial support from the Spanish Ministery of Education by Wagenitz (1975). On morphological grounds, its and Science (Projects BOS2001-3041-C02-02 and relationships with C. drabifolia and C. kotschyi are CGL2004-04563-C02-01/BOS) and the Generalitat de obvious, as noted by Ertug˘rul et al. (2004). Catalunya (‘Ajuts a Grups de Recerca Consolidats’ GREB 2001SGR00125) is gratefully acknowledged. Kostyantyn Romashchenko benefited from a grant from CONCLUDING REMARKS the Spanish Agency for International Cooperation, Ministry The present study confirms, with a much more compre- of Foreign Affairs. Tuna Uysal’s stay at the Botanic hensive sample, the existence of three major clades Institute of Barcelona was funded by Tukish Tubitak. (circum-Mediterranean/Eurosiberian, western Mediterran- Vı´ctor N. Sua´rez-Santiago benefited from a grant from the ean and eastern Mediterranean) in the Jacea group of Spanish Ministery of Education and Science (associated Centaurea, and for the first time indicates that the first of with Project PB98-1288). We thank Dr S. Badarau, these is sister to the other two. In the eastern Mediterranean Romania, and M. Dr Vural, Turkey, for providing material clade, it is now clear that section Calcitrapa (with most of of C. napulifera and C. tchihatcheffii. The nucleotide the species in the Middle East) is sister to all other sections sequencing was performed at the ‘Serveis Cientı´fico- in this group. Te`cnics’ of the University of Barcelona. Besides confirming the three natural geographic groups, the present results indicate the need for profound changes to the sectional delimitation of this genus in order to LITERATURE CITED reconcile classification and molecular systematics. Sections Boissier E. 1875. 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