Molecular Phylogenetics and Evolution
Vol. 22, No. 1, January, pp. 51– 64, 2002
doi:10.1006/mpev.2001.1038, available online at http://www.idealibrary.com on
Tribal and Subtribal Delimitation and Phylogeny of the Cardueae
(Asteraceae): A Combined Nuclear and Chloroplast DNA Analysis
Núria Garcia-Jacas, Teresa Garnatje, Alfonso Susanna, 1 and Roser Vilatersana
Botanical Institute of Barcelona (C.S.I.C.-Ajuntament de Barcelona), Av. Muntanyans, s.n., E-08038 Barcelona, Spain
Received August 28, 2000; revised March 15, 2001; published online December 5, 2001
Tribal delimitation of Cardueae is controversial,
and the traditional classification in four subtribes
(Echinopsidinae, Carlininae, Carduinae, and Centau-
reinae) has fluctuated widely. Most of the problems
are centered in subtribes Echinopsidinae and Carlini-
nae, often segregated with tribal rank. We therefore
analyzed DNA sequences of the internal transcribed
spaces (ITS) of the nuclear ribosomal DNA genes and
the matK gene of the chloroplast DNA of a broad rep-
resentation of the tribe to examine (1) the phylogeny
of the tribe, (2) the position of Echinopsidinae and
Carlininae, (3) the circumscription of the subtribes
and the position of some conflicting genera, and (4) the
delimitation of some generic complexes in the Cardui-
nae. Phylogenetic analysis of ITS and matK sequence
variation, both separate and combined, strongly sup-
port the monophyly of Cardueae including Carlininae
and Echinopsidinae. The combination of both ge-
nomes suggest that Xeranthemum and its allies should
be included among the Echinopsidinae rather than
the Carlininae, which implies that the capitulum of
Xeranthemum could be interpreted as a syncephaly.
The subtribe Centaureinae forms a well-supported
clade, and their sister clades contain the genera Arc-
tium, Cousinia, Jurinea, and Saussurea from the Car-
duinae. However, some problems persist: Carduinae
are a paraphyletic assemblage, and the subtribal
placement of Berardia, Cardopatium, Cousiniopsis,
and Staehelina remains unresolved. Our results also
indicate that present classification in four subtribes is
unsatisfactory, but it is still the only practical ap-
proach. © 2001 Elsevier Science
Key Words: Asteraceae; Cardueae; ITS; matK; phylog-
eny; tribal delimitation.
INTRODUCTION
The tribe Cardueae (Asteraceae) is usually divided
into four taxonomic entities, but the rank and delimi-
tation of these units are highly problematic. A paper on
1
To whom correspondence should be addressed. Fax: 34 93
4269321. E-mail: asusanna@ibb.csic.es.
51
this matter (Susanna et al., 1995) revised exhaustively
the different classifications. Table 1 summarizes the
different classifications.
The earliest classification by Cassini (1819) recog-
nized three tribes, Echinopeae, Carlineae, and Car-
dueae, the latter with two subtribes, Carduinae and
Centaureinae. Bentham (1873) and Hoffmann (1894),
in contrast, suggested a broadly defined Cardueae com-
prising four subtribes: Echinopsidinae, Carlininae,
Carduinae, and Centaureinae. This treatment has
been generally accepted, but the problem surfaced
again when Wagenitz (1976) suggested the segregation
of the Echinopeae as a separate tribe. Soon after, Dit-
trich (1977) returned to Cassini’s views and segregated
the Echinopeae and the Carlineae. Petit (1988) fol-
lowed Wagenitz (1976) and kept a separate tribe Echi-
nopeae. In view of the contradicting views, the last
general survey of the Asteraceae (Bremer, 1994)
adopted the more conservative, broad tribal concept
and recognized only one tribe.
In a previous molecular analysis based on internal
transcribed spacer (ITS) sequences (Susanna et al.,
1995), we concluded that the Cardueae were monophy-
letic, including Carlininae and Echinopsidinae. The
same conclusion had been reached by Jansen et al.
(1990, 1991), on the basis of chloroplast DNA restric-
tion site data, and by Kim et al. (1992) on the basis of
rbcL sequences. Cladistic analyses of morphological
data in the Compositae (Bremer, 1987, 1994; Karis et
al., 1992) also supported the monophyly of a broadly
defined Cardueae, including Echinopsidinae and Carli-
ninae in addition to Carduinae and Centaureinae.
However, these results did not settle the discussion: in
a cladistic analysis of morphologic characters, Petit
(1997) concluded again that Echinopeae should not be
included in the Cardueae.
Most of the difficulties have originated in the Echi-
nopsidinae, characterized by uniflowered capitula
grouped in second-order heads. This structure has
been the main reason for the segragation of Echinop-
sidinae as a different tribe by Wagenitz (1976), Dittrich
(1977), and Petit (1988, 1997). Homologies of the syn-
florescence of Echinops L. and related genera are ex-
1055-7903/01 $35.00
© 2001 Elsevier Science
All rights reserved.
52 GARCIA-JACAS ET AL.

TABLE 1

Different Subtribal Classifications of the Cardueae

Cassini (1819), Dittrich (1977) Bentham (1873), Hoffmann (1894), Bremer (1994) Wagenitz (1976), Petit (1988, 1997)

Tribe Echinopeae Tribe Cardueae Tribe Echinopeae

Tribe Carlineae Subtribe Echinopsidinae Tribe Cardueae
Tribe Cardueae Subtribe Carlininae Subtribe Carlininae
Subtribe Carduinae Subtribe Carduinae Subtribe Carduinae–Centaureinae
Subtribe Centaureinae Subtribe Centaureinae

tremely difficult to establish (Petit, 1988), as is usually
the case with highly derived syncephalies (Stuessy and
Spooner, 1988). A similar problem is posed by the com-
plicated involucral structures of the Carlininae, which
have been interpreted even as a third-order syncepha-
lia in the genus Carlina L. (Meusel and Kohler, 1960;
Meusel and Kästner, 1994), an interpretation rejected
by Petit (1988). In fact, contradictory results obtained
by different authors in cladistic analyses of morpho-
logic data (e.g., Karis et al., 1992 versus Petit, 1997)
could be partly attributed to different points of view
in addressing the problem of homologies of the recep-
tacular structures of Echinops, Carlina, and related
genera.
Not only the limits of the tribe are problematic. The
boundaries between the classic four subtribes are very
difficult to establish. By example, most of the authors
(Bentham, 1873; Hoffmann, 1894; Dittrich, 1977; Bre-
mer, 1994) included the genera Amphoricarpos Vis.,
Cardopatium Juss., Chardinia Desf., Cousiniopsis
Nevski, Siebera J. Gay, Staehelina L., and Xeranthe-
mum L. among the Carlininae, whereas Petit (1997)
moved Cardopatium and Cousiniopsis to the Echino-
peae (to which he assigned tribal rank) and the rest of
genera to the Carduinae–Centaureinae. There are
some other genera that are very difficult to classify,
such as Berardia Vill.: it was included among the Car-
dueae by Bremer (1994) and moved to the Mutisieae by
Dittrich (1996a).
Finally, there are the problems of generic delimita-
tion posed by some large genera of the tribe: Carduus
L. (90 species), Cirsium Mill. (250 species), Centaurea
L. (400 species), Cousinia Cass. (800 species), Jurinea
Cass. (100 species), and Saussurea DC. (more than 300
species). The natural delimitation of Centaurea (400
species) was recently cleared up by Garcia-Jacas et al.
(2000, 2001), and some apportionments on the limits of
Cirsium and Carduus were made recently by Häffner
and Hellwig (1999), but the rest of the problems per-
sist. Many small genera from central and west Asia
have been described on the basis of splits of Cousinia
and Jurinea (Susanna and Garcia-Jacas, 2001). By
example, the genera Hyalochaete Dittrich & Rech. f.,
Jurinella Jaub. & Spach, and Outreya Jaub. & Spach
are doubful segregates from Jurinea. Also, according to
Duistermaat (1996, 1997) and Petit (1997), the limits
between Arctium L. and Cousinia are unclear and con-
troversial.
Clearly, a molecular approach seemed most applica-
ble for this conflicting group, as the results of cladistic
morphological analysis leads to contradictory results.
Problems encompassed a broad taxonomic range, as we
intended to address questions from the generic to the
tribal level. Thus, we gathered information from dif-
ferent sources by sequencing two regions of the ge-
nome: the nuclear ribosomal DNA (nrDNA) internal
transcribed spacers (ITS1 and ITS2) and the chloro-
plast DNA (cpDNA) gene matK. The ITS region was
extensively demonstrated to be a source of phyloge-
netic information in the Cardueae, in the tribal (Sus-
anna et al., 1995), subtribal (Häffner and Hellwig,
1999; Garcia-Jacas et al., 2001), and generic (Susanna
et al., 1999; Vilatersana et al., 2000) levels. In regard to
matK, if ITS has its major weakness in the resolution
of remote subtribal groups (Susanna et al., 1995), the
matK gene was more applicable to the suprageneric
level. It had been used already in the Asteraceae
(Denda et al., 1999) and in similar taxonomic levels in
other groups such as the Apiaceae (Plunkett et al.,
1996), Cornaceae (Xiang et al., 1998), Myrtaceae
(Gadek et al., 1996), Polemoniaceae (Johnson and Sol-
tis, 1995), and Saxifragaceae (Johnson and Soltis,
1994; Soltis et al., 1996; Brochmann et al., 1998). On
the other hand, the combination of different genomes is
at the present moment one of the best tools for phylo-
genetic reconstruction (Qiu et al., 1999). The combined
approach solves one of the most problematic issues of
molecular phylogenies based on one genome: despite
offering a great number of characters, at the end they
conform to a one-character-based taxonomy (Doyle,
1992).
Our goals in this study were (1) to evaluate the
monophyly of the tribe Cardueae; (2) to compare the
molecular phylogeny with the traditional classification
in four subtribes (Echinopsidinae, Carlininae, Cardui-
nae, and Centaureinae); (3) to examine the boundaries
between the four subtribes and the position of conflict-
ing genera such as Berardia, Cardopatium, Cousiniop-
sis, Staehelina, and the Xeranthemum group; and (4) to
verify the delimitation of some of the large genera of
 COMBINED NUCLEAR–CHLOROPLAST DNA PHYLOGENY OF TRIBE CARDUEAE
the tribe (the Arctium–Cousinia group and the Jurinea
complex).
MATERIALS AND METHODS
Plant Material
Sampling was based on the subtribal classification of
the Cardueae in four subtribes by Bentham (1873),
Hoffmann (1894), and Bremer (1994) with the modifi-
cations suggested by Susanna and Garcia-Jacas
(2001), which recognize 74 genera.
With regard to Centaureinae, only 3 genera were
included, because this subtribe is a very well-defined
monophyletic group and had been the object recently of
a deep survey, with 25 of 33 genera studied (Garcia-
Jacas et al., 2001). As for the rest of the tribe, we
included 31 of 41 genera (according to Susanna and
Garcia-Jacas, 2001) of the subtribes Carduinae, Carli-
ninae, and Echinopsidinae. Two outgroup species were
chosen in the tribe Mutisieae according to previous
sequence analysis (Susanna et al., 1995). Voucher data,
source, and GenBank sequence accession numbers for
the 62 studied species are given in Table 2.
The analysis of ITS sequences used published se-
quences along with new sequences. Sequences of Cen-
taurea involucrata Desf., Cirsium arvense (L.) Scop.,
Cynara humilis L., Jurinea humilis (Desf.) DC.,
Ptilostemon hispanicus (Lam.) Greuter (wrongly pub-
lished as Galactites durieui Spach), and Zoegea mi-
anensis Boiss. were from previous studies (Susanna et
al., 1995; Garcia-Jacas et al., 2000). All the matK se-
quences analyzed are new.
DNA Extraction, Amplification, and Sequencing
Total genomic DNA was extracted, following the
CTAB method of Doyle and Doyle (1987) as modified by
Soltis et al. (1991), from silica-gel-dried leaves collected
in the field or fresh leaves of plants cultivated in the
Botanic Institute of Barcelona. In some cases, herbar-
ium material was used.
cpDNA matK gene strategies. Double-stranded
DNAs of matK were amplified by PCR with trnK-710F
and matK-1848R (Johnson and Soltis, 1995) as PCR
primers. An additional primer, AST-1R, was designed
for this study, as a substitute for primer matK-1848R.
The sequence is as follows: 5⬘ CCGCACACTTGAACG/
CATAACCCAG 3⬘. This primer combination failed to
generate double-stranded products for some taxa.
Therefore, trnK-3914F and trnK-2R (Johnson and Sol-
tis, 1995) were used in place of those primers. The
profile used for amplification included a warm start at
94°C for 2 min, followed by 80°C for 5 min, during
which the polymerase (Ecotaq; Ecogen S. R. L., Barce-
lona, Spain) was added. Forty cycles of amplification
were carried out under the following conditions: 94°C
53
for 1 min 30 s, 48°C for 2 min, and 72°C for 3 min, with
an additional extension step of 15 min at 72°C.
The double-stranded PCR products were cleaned
with a QIAquick PCR Purification Kit (Qiagen Inc.)
and sequenced. In general, four sequencing primers,
trnK-710F, matK-1168R, matK-1848R (Johnson and
Soltis, 1995), and AST-1R were used. Direct sequenc-
ing of the amplified DNA segments was performed with
a Thermo Sequenase II Dye Terminator Cycle sequenc-
ing kit (Amersham), following the protocol recom-
mended by the manufacturer. The nucleotide sequenc-
ing was performed at the Serveis Cientı́fico-Tècnis of
the University of Barcelona on an ABI 377 Automated
DNA Sequencer (Perkin–Elmer). Nucleotide sequences
of matK were edited with Chromas 1.56 (Technelysium
Pty. Ltd.) and easily aligned by hand.
nrDNA ITS region strategies. Double-stranded
DNA of the ITS region was amplified with the 1406F
primer (Nickrent et al., 1994) and ITS4 (White et al.,
1990). In some cases, we used ITS1 (White et al., 1990)
and 17SE (Sun et al., 1994) as forward primers and
26SE (Sun et al., 1994) as reverse primer. The profile
used for amplification included a warm start at 94°C
for 2 min, followed by 80°C for 5 min, during which the
polymerase (Ecotaq; Ecogen S. R. L., Barcelona, Spain)
was added. Thirty cycles of amplification were carried
out under the following conditions: 94°C for 1 min 30 s,
55°C for 2 min, and 72°C for 3 min, with an additional
extension step of 15 min at 72°C.
The PCR products were purified with the QIAquick
PCR Purification Kit (Qiagen Inc.). Both strands were
sequenced with the sequencing primers 1406F, ITS1,
and 17SE as forward primers and ITS 4 and 26SE as
reverse primers. Direct sequencing of the amplified
DNA segments was performed as for the matK region.
Phylogenetic Analysis
DNA sequences were aligned visually by sequential
pairwise comparison (Swofford and Olsen, 1990).
Alignment was checked with ClustalX (Thompson et
al., 1997). Data matrices are available on request from
the corresponding author. Parsimony analysis involved
heuristic searches conducted with PAUP version
4.0b4a (Swofford, 1999) with TBR branch swapping
and character states specified as unordered and un-
weighted. The indels were coded as fifth base, following
the now generally accepted trend of saving the poten-
tial phylogenetic information of shared indels (Sun et
al., 1994; Bain and Jansen, 1995; Samuel et al., 1998).
All most parsimonious trees (MPTs) were saved. To
locate other potential islands of most parsimonious
trees (Maddison, 1991), we performed 100 replications
with random taxon addition and TBR branch swap-
ping. Bootstrap analyses (BS) were performed (Felsen-
stein, 1985), and decay indices (DI) were calculated
(Bremer, 1988; Donoghue et al., 1992) to obtain esti-
54
Origin of the Materials and Herbaria Where the Vouchers are Deposited
Acantholepis orientalis Less.
Alfredia cernua (L.) Cass.
Arctium lappa L.
Arctium minus Bernh.
Atractylis cancellata L.
Atractylis carduus (Forssk.) Christ.
Atractylis humilis L.
Atractylodes japonica Koidz. ex
Kitam.
Berardia subacaulis Vill.
Cardopatium corymbosum (L.)
Pers.
Carduus carlinoides Gouan
Carduus pycnocephalus L.
Carlina acanthifolia All.
Carlina falcata Svent.
Carlina gummifera (L.) Less.
Carlina lanata L.
Carlina macrophylla (Desf.) DC.
Carlina vulgaris L.
Centaurea hajastana Tzvel.
Centaurea involucrata Desf.
Centaurea lingulata Lag.
Chardinia orientalis (L.) O. Kuntze
Cirsium arvense (L.) Scop.
Cirsium echinus (M. Bieb.) Hand.-
Mazz.
Cirsium palustre (L.) Scop.
Cousinia canescens DC.
Cousinia esfandiarii Rech. F. &
Aellen
Cousinia onopordioides Lbd.
Cousiniopsis atractyloides (C.
Winkl.) Nevski
Cynara cornigera Lind.
Cynara humilis L.
Echinops ritro L.
Echinops sp.
Echinops spinosissimus Turra
Echinops viscosus DC.
Galactites tomentosa Moench
Gerbera kunzeana A. Br. &
Aschers.
Hyalochaete modesta Dittrich &
Rech. f.
Jurinea humilis (Desf.) DC.
Jurinea macrocephala DC.
Jurinea sp.
Jurinella moschus (Habl.) Bobrov
GARCIA-JACAS ET AL.
TABLE 2
Uzbekistan: Kyzylkum, 30 Km N of Bukhara, Khassanov IX-1999 (BC) [AF319046, AF319100,
AY013518].
Denmark: Copenhagen Botanical Garden (BC) [AF319047, AF319101, AY013519].
Belgium: Lovaina Botanical Garden (BC) [AF319048, AF319102, AY013520].
Belgium: Lovaina Botanical Garden (BC) [AF319049, AF319103, AY013521].
Spain, Madrid: Torrelaguna, Garcia-Jacas & Susanna 1458 (BC) [AF319050, AF319104,
AY013522].
Egypt: near El Amiriya, Susanna 1856 & Vilatersana (BC) [AF319051, AF319105, AY013523].
Spain, Toledo: Huerta de Valdecarábanos, Susanna 1883 (BC) [AF319052, AF319106].
Japan: Tokyo Botanical Garden (BC) [AF319053, AF319107, AY013524].
France, Alpes Maritimes: Col de la Cayolle, Garnatje 27 & Luque (BC) [AF319054, AF319108,
AY013525].
Greece, Macedonia: between Thermi and Thessaloniki, Roché & Susanna 1951 (BC) [AF319055,
AF319109, AY013526].
Spain, Girona: Abella, Garnatje 11 & Luque (BC) [AF319056, AF319110, AY013527].
Spain, Barcelona: Montjuı̈c, Garnatje & Susanna 1827 (BC) [AF319057, AF319111, AY013528].
Spain, Girona: Abella, Garnatje 8 & Luque (BC) [AF319058, AF319112, AY013529].
Spain, Canary Islands, La Palma: Gallegos, Garnatje 3 & Luque (BC) [AF319059, AF319113,
AY013530].
Switzerland: Genève Botanical Garden (BC) [AF319060, AF319114, AY013531].
Creta, Hania: Phalasarna, Vilatersana 39 (BC) [AF319061, AF319115, AY013532].
Morocco: between Xauen and Oued Laou, Garnatje, Susanna 1893 & Vilatersana (BC)
[AF319062, AF319116].
Switzerland: Zürich, Botanical Garden (BC) [AF319063, AF319117, AY013533].
Armenia, Talin: between villages Pokr Artik and Bagravan, Fajvush, Gabrielyan, Garcia-Jacas,
Guara, Hovannisyan, Susanna 1587, Tamanyan & Vallès (BC) [AF319064, AF319118,
AY013502].
Susanna et al. (1995) for the ITS data [matK: AY013503].
Spain, Madrid: Puerto de Navafrı́a, Garcia-Jacas & Susanna 1462 (BC) [AF058851, AF058876,
AY013505].
Iran, Kordestan: 35 km W of Divandarreh, Garcia-Jacas, Mozaffarian, Susanna 1715 & Vallès
(BC) [AF319065, AF319119, AY013534].
Susanna et al. (1995).
Iran, Azarbayjan-e-Sharghi: 25 Km from Ahad on the road to Kaleibar, Garcia-Jacas,
Mozaffarian, Susanna 1667 & Vallès (BC) [AF319066, AF319120, AY013535].
Spain, Girona: Abella, Garnatje 17 & Luque (BC) [AF319067, AF319121, AY013536].
Iran, Azarbaijan-e-Sharghi: 25 Km from Ahad on the road to Kaleibar, Garcia-Jacas,
Mozaffarian, Susanna 1608 & Vallès (BC) [AF319068, AF319122].
Iran, Mazandaran: between Gachsar and Valiabad, Garcia-Jacas, Mozaffarian, Susanna 1618
& Vallès (BC) [AF319069, AF319123, AY013537].
Iran, Tehran: between Firuzkuh and Semnan, Garcia-Jacas, Mozaffarian, Susanna 1637 &
Vallès (BC) [AF319070, AF319124].
Uzbekistan: Babatak mountains, 40 Km E from Lalmikar, Sukervanik, 18-V-1979 (BC)
[AF319071, AF319125].
Egypt, Alexandria: 2 Km N from the city of Alexandria, Susanna 1840 & Vilatersana (BC)
[AF319072, AF319126, AY013538].
Susanna et al. (1995).
Spain, Tarragona: Xerta, Garnatje & Susanna 1870 (BC) [AF319074, AF319128].
Iran, Hamadan: 5 Km E of Hamadan on the road to Teheran, Garcia-Jacas, Mozaffarian,
Susanna 1721 & Vallès (BC) [AF319073, AF319127].
Germany: Berlin Botanical Garden (BC) [AF319075, AF319129, AY013539].
Germany: Berlin Botanical Garden (BC) [AF319076, AF319130, AY013540].
Spain, Barcelona: Montjuı̈c, Garnatje & Susanna 1867 (BC) [AF319077, AF319131, AY013541].
Germany: Stuttgart Botanical Garden (BC) [AF319078, AF319132, AY013542].
Germany: Berlin Botanical Garden (BC) [AF319080, AF319134].
Susanna et al. (1995).
Iran, Azarbaijan-e-Sharghi: 10 Km northern Qarabchaman, Garcia-Jacas, Mozaffarian,
Susanna 1650 & Vallès (BC) [AF319081, AF319135].
Armenia, Krasnoselsk: Artanish Peninsula, Fajvush, Gabrielyan, Garcia-Jacas, Guara,
Hovannisyan, Susanna 1528, Tamanyan & Vallès (BC) [AF319082, AF319136, AY013543].
Iran, Azarbaijan-e-Sharghi: Sabalan mt., 2800 m, Garcia-Jacas, Mozaffarian, Susanna 1662 &
Vallès (BC) [AF319083, AF319137].
 COMBINED NUCLEAR–CHLOROPLAST DNA PHYLOGENY OF TRIBE CARDUEAE 55

TABLE 2—Continued

Mutisia spinosa R. & P.
Notobasis syriaca (L.) Cass.
Onopordon algeriense Pomel
Onopordon leptolepis DC.
Outreya carduiformis Jaub. &
Spach
Picnomon acarna (L.) Cass.
Ptilostemon afer (Jacq.) Greuter
Ptilostemon chamaepeuce (L.) Less.
Ptilostemon hispanicus (Lam.)
Greuter
Saussurea alpina (L.) DC.
Saussurea discolor (Willd.) DC.
Serratula coronata L.
Silybum marianum (L.) Gaertner
Staehelina baetica DC.
Synurus palmatopinnatifidus
Kitam.
Thevenotia scabra (Boiss.) Boiss.
Tyrimnus leucographus (L.) Cass.
Xeranthemum cylindraceum Sibth.
& Sm.
Xeranthemum inapertum (L).
Miller.
Zoegea mianensis Boiss.
Spain: Barcelona Botanical Garden (BC) [AF319079, AF319133, AY013544].
Egypt, Alexandria: Burg el Arab, Susanna 1844 & Vilatersana (BC) [AF319085, AF319139,
AY013545].
Switzerland: Zürich Botanical Garden (BC) [AY013546].
Iran, Teheran: Sorkhehesar, Garcia-Jacas, Mozaffarian, Susanna 1632 & Vallès (BC)
[AF319086, AF319140, AY013547].
Iran, Teheran: Sorkhehesar, Garcia-Jacas, Mozaffarian, Susanna 1631 & Vallès (BC)
[AF319087, AF319141, AY013548].
Iran, Azarbaijan-e-Gharbi: Orumiyeh, Garcia-Jacas, Mozaffarian, Susanna 1692 & Vallès (BC)
[AF319088, AF319142, AY013549].
Germany: Freiburg Botanical Garden (BC) [AF319088, AF319142, AY013550].
France: Lyon Botanical Garden (BC) [AF319090, AF319144].
Susanna et al. (1995, as Galactites durieui Coss.).
Italia: Cogne Botanical Garden (BC) [AF319091, AF319145].
Switzerland: Meyrin Botanical Garden (BC) [AF319092, AF319146].
Austria: Vienna Botanical Garden (BC) [AF319093, AF319147, AY013513].
Spain, Madrid: Torrelaguna, Garcia-Jacas & Susanna 1461 (BC) [AF319094, AF319148,
AY013551].
Spain, Málaga: Sierra Bermeja, Garcia-Jacas & Susanna 1826 (BC) [AF319095, AF319149].
Japan: the Nippon Shinyaku Institute for Botanical Research (BC) [AF319096, AF319150,
AY013551].
Uzbekistan: Kyzylkum, 50 km northern town by Zhdavan, Khassanov, IX-1999 (BC)
[AY013553].
Spain, Tarragona: La Vilella Baixa, R. Pascual, VI-1998 (BC) [AF319097, AF319151,
AY013554].
Switzerland: Genève Botanical Garden (BC) [AF319098, AF319152, AY013556].
Spain, Madrid: Torrelaguna, Garcia-Jacas & Susanna 1456 (BC) [AF319099, AF319153,
AY013555].
Garcia-Jacas et al. (2000) for the ITS data [matK: AY013517].

Note. ITS 1 and 2 (beginning with AF) and matK (beginning with AY) GenBank Accession Nos. between brackets. Previous publications
refer to ITS sequences.

mates of support for each monophyletic group. Three RESULTS

parsimony analyses were performed, with three differ-
ent data sets: the ITS data, the matK data, and the
combined ITS and matK data.
Due to the unpracticability of performing bootstrap
analysis in the usual way with PAUP 4.0b4a in the
cpDNA (matK) matrix, we used the approach by Lidén
et al. (1997) using 1000 replicates, random taxon addi-
tion with 20 replicates per replicate, and no branch
swapping. Results obtained with this method are very
similar to those obtained with other approaches (Mort
et al., 2000).
With regard to decay analysis, the search for trees
three steps longer than the shortest trees was unprac-
tical in all cases. Therefore, the next decay analyses
were conducted with the clade-constraint approach as
discussed in Morgan (1997).
The nrDNA ITS and cpDNA matK data sets were
tested for congruence with the partition homogeneity
test (Farris et al., 1995) as implemented in PAUP
4.0b4a, before the data sets were combined. The parti-
tion homogeneity test was conducted with 1000 repli-
cates, heuristic search option with simple addition se-
quence, TBR, and MULPARS.
Nuclear Ribosomal DNA ITS
The ITS1 and ITS2 alignment of 60 taxa consisted of
515 positions and contained 199 phylogenetically infor-
mative substitutions and 156 phylogenetically infor-
mative indels. Mean pairwise distances (as calculated
by PAUP) within ingroup varied from 0% (between
Echinops viscosus DC. and Echinops spinosissimus
Turra) to 31.2% (between Xeranthemum inapertum
(L.) Miller and Carlina vulgaris L.). Pairwise distance
between ingroup and outgroup varied from 25.7% (be-
tween Gerbera kunzeana A. Br. & Aschers. and Carlina
lanata L.) to 36.2% (between Xeranthemum inapertum
and Gerbera kunzeana).
The parsimony analysis yielded eight MPTs of 2073
steps in one island. The strict consensus of all the trees
is shown in Fig. 1; the consistency index (CI) excluding
uninformative characters was 0.3726, the retention in-
dex (RI) was 0.6378, and the homoplasy index (HI) was
0.6274 (Table 3).
The strict consensus of the eight MPTs produced
from the ITS analysis (Fig. 1) support monophyly of the
Cardueae (BS ⫽ 100%, DI ⫽ 20). The subtribe Carli-
56 GARCIA-JACAS ET AL.

FIG. 1. Strict consensus tree of the eight most parsimonious trees generated by the ITS matrix. Length: 2073 steps; consistency index
excluding uninformative characters: 0.3726; retention index: 0.6378; homoplasy index: 0.6274. Subtribal assignments follow Bremer (1994).
CARD, Carduinae; CARL, Carlininae; CENT, Centaureinae; ECHI, Echinopsidinae; OUTG, outgroup. Berardia was unasigned to any
subtribe. Ono; Onopordon.

ninae was sister to the remaining Cardueae with weak
support (BS ⫽ 58%, DI ⫽ 2). This clade had strong
support (BS ⫽ 92%, DI ⫽ 3) including the genera
Atractylis L., Atractylodes DC., and Carlina. The next
clade was formed by Cardopatium corymbosum (L.)
Pers. and Cousiniopsis atractyloides (C. Winkl.)
Nevski, with weak support (BS ⫽ 57%, DI ⫽ 1). The
next branches had no support from the bootstrap or the
decay index. However, there were some groups with
strong support, such as the clade formed by Alfredia
Cass., Synurus Iljin and Onopordon L. (BS ⫽ 92%,
DI ⫽ 5). Another clade was formed by the genera
Chardinia and Xeranthemum (BS ⫽ 100%, DI ⫽ 15).
Next, there were the genera Cynara L. (BS ⫽ 100%,
DI ⫽ 15) and Galactites Moench and Ptilostemon Cass.
(BS ⫽ 100%, DI ⫽ 36). The clade formed by Echinops
and Acantholepis Less. was the next group with very
high support (BS ⫽ 100%, DI ⫽ 30). Next was the clade
formed by Cirsium, Notobasis Cass., Picnomon Adans.,
Silybum Adans., Carduus, and Tyrimnus Cass. (BS ⫽
 COMBINED NUCLEAR–CHLOROPLAST DNA PHYLOGENY OF TRIBE CARDUEAE
TABLE 3
Comparison of Results from the ITS, matK, and
Combined Data Sets
Data set ITS matK Combined
Total characters 515 1011 1542
Informative characters 355 126 443
Number of MPTs 8 1887 4
Number of steps 2073 271 1915
Consistency index 0.3726 0.5679 0.4389
Retention index 0.6378 0.7384 0.6119
Homoplasy index 0.6274 0.4321 0.5611
Range of divergence, 0–31.2 0–6.8 0.8–13.5
ingroup (%)
93%, DC ⫽ 8). Finally, there was the clade of the
Centaureinae and their sister clades, with very low
support (BS ⫽ 64%, DI ⫽ 3). Within this clade, there
were the Centaureinae with strong support (BS ⫽
100%, DI ⫽ 18), and three more clades: the genus
Saussurea (BS ⫽ 100%, DI ⫽ 15), the clade with Arc-
tium and Cousinia with weak support (BS ⫽ 62%, DI ⫽
2), and finally the clade formed by Hyalochaete, Juri-
nea, Jurinella, and Outreya (BS ⫽ 100%, DI ⫽ 18).
Chloroplast DNA matK
The matK alignment of 44 taxa consisted of 1011
positions and contained 104 phylogenetically informa-
tive substitutions and 22 phylogenetically informative
indels. Mean pairwise distances (as calculated by
PAUP) within ingroup varied from 0% (between
Carlina vulgaris and Carlina lanata) to 6.8% (between
Xeranthemum inapertum and Echinops spinosissi-
mus). Pairwise distance between ingroup and outgroup
varied from 6.4% (between Outreya carduiformis Jaub.
& Spach and Mutisia spinosa R. & P.) to 25.7% (be-
tween Gerbera kunzeana and Carlina lanata).
The parsimony analysis yielded 1887 MPTs of 271
steps in one island. The strict consensus of all the trees
is shown in Fig. 2; the CI excluding uninformative
characters was 0.5679, the RI was 0.7384, and the HI
was 0.4321 (Table 3).
The strict consensus of the 1887 MPTs produced
from the matK analysis (Fig. 2) supports monophyly of
the Cardueae (BS ⫽ 96%, DI ⫽ 6). There was a poly-
tomy, basal to the weakly supported Carduinae (BS ⫽
61%, DI ⫽ 2). This grade was formed by the Carlininae
(BS ⫽ 82%, DI ⫽ 2) with Atractylis, Atractylodes,
Carlina, and Thevenotia DC., the Echinopsidinae with
weak support (BS ⫽ 63%, DI ⫽ 2), the genera Berardia
and Cardopatium, and, finally, the group formed by
Chardinia and Xeranthemum (BS ⫽ 92%, DI ⫽ 4). The
rest of the analysis had some groups with strong sup-
port. Within the Carduinae, there were the clade
formed by Carduus, Notobasis, Picnomon, Tyrimnus,
Cirsium, and Silybum (BS ⫽ 87%, DI ⫽ 3), the clade
that included Jurinea and Outreya (BS ⫽ 93%, DI ⫽ 4),
57
and the monotypic clade formed by Arctium (BS ⫽
99%, DI ⫽ 7). However, there was one clade with low
support: the clade formed by the genera Alfredia and
Synurus (BS ⫽ 61%, DI ⫽ 1). Finally, the clade of
the Centaureinae had very low support (BS ⫽ 58%,
DI ⫽ 1).
Combined nrDNA ITS and cpDNA matK
The P value resulting from the partition homogene-
ity test (P ⫽ 0.245) indicates that data partitions are
random, and there is good congruence between nrDNA
ITS and cpDNA matK data sets, at a significance three-
shold of P ⫽ 0.05 (Farris et al., 1995).
The combined ITS–matK alignment of 42 taxa con-
sisted of 1542 positions with 286 phylogenetically in-
formative substitutions and 157 phylogenetically infor-
mative indels. Mean pairwise distances (as calculated
by PAUP) within ingroup varied from 0.8% (between
Carlina lanata and Carlina falcata Svent.) to 13.5%
(between Xeranthemum inapertum and Carlina vul-
garis). Pairwise distance between ingroup and out-
group varied from 10.1% (between Gerbera kunzeana
and Carlina lanata) to 15.2% (between Xeranthemum
inapertum and Gerbera kunzeana).
The parsimony analysis yielded 4 MPTs of 1915
steps in one island. The strict consensus of all the trees
is shown in Fig. 3; the CI excluding uninformative
characters was 0.4389, the RI was 0.6119, and the HI
was 0.5611 (Table 3).
The strict consensus of the four MPTs produced from
the combined analysis (Fig. 3) support the monophyly
of the Cardueae (BS ⫽ 100%, DI ⫽ 31) as in the ITS
and matK single analyses. The subtribe Carlininae
was sister to the remaining Cardueae with weak sup-
port (BS ⫽ 58%, DI ⫽ 2), as in the ITS analysis. The
Carlininae had strong support (BS ⫽ 97%, DI ⫽ 5)
including Atractylis, Atractylodes, and Carlina. The
next monotypic clade was Cardopatium corymbosum,
sister to the rest of the weakly supported Echinopsidi-
nae ⫹ Carduinae (BS ⫽ 66%, DI ⫽ 2). The clade formed
by the Echinopsidinae was the next sister group to the
rest of the Cardueae with weak support (BS ⫽ 55%,
DI ⫽ 1). The Echinopsidinae was well supported (BS ⫽
83%, DI ⫽ 4); within this clade there were two
branches, one with the genera Echinops and Acantho-
lepis (BS ⫽ 100%, DI ⫽ 34) and the other with the
genera Chardinia and Xeranthemum (BS ⫽ 100%,
DI ⫽ 16).
The next branches had no support from the bootstrap
or the decay index, and this part of the tree can be
considered a polytomy. The only groups with support
were the clade formed by Alfredia, Synurus, and Ono-
pordon (BS ⫽ 97%, DI ⫽ 4) and the clade formed by
Carduus, Tyrimnus, Silybum, Cirsium, Notobasis, and
Picnomon (BS ⫽ 100%, DI ⫽ 12). Finally, there was the
clade of the Centaureinae and related clades, with very
low support (BS ⫽ 64%, DI ⫽ 2). Within this clade,
58 GARCIA-JACAS ET AL.

FIG. 2. Strict consensus tree of the 1887 most parsimonious trees generated by the matK matrix. Length: 271 steps; consistency index
excluding uninformative characters: 0.5679; retention index: 0.7384; homoplasy index: 0.4321. Subtribal assignments follow Bremer (1994). CARD,
Carduinae; CARL, Carlininae; CENT, Centaureinae; ECHI, Echinopsidinae; OUTG, outgroup. Berardia was unasigned to any subtribe.

there are two groups: the Centaureinae with strong
support (BS ⫽ 100%, DI ⫽ 17) and two clades, one with
Arctium and Cousinia (BS ⫽ 73%, DI ⫽ 3) and another
one with Jurinea and Outreya (BS ⫽ 100%, DI ⫽ 28).
DISCUSSION
Relative Utility of ITS and matK Regions in the
Cardueae
Neither the ITS nor the matK alone have fully com-
pleted the objectives of our study. ITS analysis (Fig. 1)
shows excellent resolution at the generic level; how-
ever, it leaves unresolved the relationships between
most of the major groups. The consistency index is
mediocre (CI ⫽ 0.3726), and the homplasy index is high
(HI ⫽ 0.6274). The high homoplasy in the ITS data is
confirmed by the high difference between the consis-
tency index and the retention index (Table 3) (Plunkett
and Downie, 1999). However, one of the reasons for the
high homoplasy could be the size of the matrix (60
taxa), according to Archie (1989). In any case, it is
obvious that ITS alone, as expected, cannot fully re-
 COMBINED NUCLEAR–CHLOROPLAST DNA PHYLOGENY OF TRIBE CARDUEAE 59

FIG. 3. Strict consensus tree of the four most parsimonious trees generated by the combined matK–ITS matrix. Length: 1915 steps;
consistency index excluding uninformative characters: 0.4389; retention index: 0.6119; homoplasy index: 0.5611. Boxes with subtribal
assignments are from Susanna and Garcia-Jacas (2001). Ono; Onopordon; Jur; Jurinea.

solve the relationships between the widely diverging
basal groups of the tribe Cardueae.
On the other hand, matK analysis shows good reso-
lution at the generic level in many cases, but does not
support some indisputable genera, e.g., Echinops (Fig.
2), and the support for another indisputable clade such
as the Centaureinae (Fig. 2) is practically null (BS ⫽
58%, DI ⫽ 1). The homoplasy index and the consistency
index are better than the ITS values (CI ⫽ 0.5679;
HI ⫽ 0.4321), but the number of informative charac-
ters is very low (Table 3). It also leaves the basal
groups in an unresolved grade, a rather unexpected
result, as matK was supposed to be more adequate for
higher taxonomic levels.
Instead, the combination of both genomes shows bet-
ter resolution, both at the generic and the subtribal
level. This result is a confirmation of the superiority of
the combined approach for phylogenetic studies (Qiu et
al., 1999).
The Delimitation of the Cardueae
Sequence analysis of chloroplast and nuclear DNA,
both individually and combined, strongly support the
monophyly of the Cardueae [BS ⫽ 100%, DI ⫽ 20 (Fig.
60 GARCIA-JACAS ET AL.
1); BS ⫽ 96%, DI ⫽ 6 (Fig. 2); BS ⫽ 100%, DI ⫽ 31 (Fig.
3)]. The broad concept favored by Bentham (1873),
Hoffmann (1894), and Bremer (1994) looks accurate,
according to the new molecular evidence. The com-
bined analysis classifies the tribe in, first, a basal grade
formed successively by the subtribe Carlininae, the
genus Cardopatium, and the subtribe Echinopsidinae
(including the Xeranthemum group, usually placed
among the Carlininae), and second, the polytomy of the
subtribe Carduinae, which includes the subtribe Cen-
taureinae (Fig. 3). Phylogeny suggested by the ITS
alone (Fig. 1) coincides in the position of the Carlininae
and the genus Cardopatium (with Cousiniopsis), but
places Echinopsidinae (without the Xeranthemum
group) as nested among the Carduinae. With regard to
matK alone, the basal part of the tree is an unresolved
grade with Carlininae, Echinopsidinae, Cardopatium,
Berardia, and the Xeranthemum group (Fig. 2). We
shall discuss each clade separately.
The Carlininae. The Carlininae are consistently
placed as sister to the rest of the Cardueae by the ITS
alone and the combined analysis, with the same low
support (BS ⫽ 58%, DI ⫽ 2; Figs. 1 and 3) as that in our
previous analysis (Susanna et al., 1995). The analysis
of the matK region leaves this part of the tree unre-
solved (Fig. 2), but this result is compatible with the
others. Probably, the tribe Carlininae constitutes the
oldest stock of the tribe, which, according with its dis-
tribution, would indicate an east Mediterranean origin
for the Cardueae (Meusel and Kästner, 1990, 1994;
Jäger, 1987). An interesting character that reinforces
the basal position of the Carlininae is the presence of
true ligules in Atractylis (Dittrich, 1977; even doubt-
ing—“almost ligulate”—whether they are true ligules),
a character shared with many members of the tribe
Mutisieae.
According to our results, circumscription of Carlini-
nae must be broadly redefined. The subtribe must in-
clude only the genera Carlina, Atractylis, Atractylodes
[BS ⫽ 92%, DI ⫽ 3 in the ITS analysis (Fig. 1); BS ⫽
97%, DI ⫽ 5 in the combined analysis (Fig. 3)], and
Thevenotia (BS ⫽ 82%, DI ⫽ 2 in the matK analysis,
Fig. 2). With regard to Tugarinovia Iljin, this genus
was not included in our analysis, but there are strong
doubts on its relationship to the rest of the group,
contrary to Dittrich et al. (1987). The exclusion from
the Carlininae of the Xeranthemum group and the
genera Cardopatium and Cousiniopsis was already
pointed out by Petit (1988, 1997), who sent Cardopa-
tium and Cousiniopsis to the Echinopeae (which he
treated as a separate tribe) and Xeranthemum to the
Carduinae. Homologies of the complex involucral pat-
tern of bracts of Carlina and the receptacular scales
of the Xeranthemum group were obviously misinter-
preted by previous authors.
Our results also suggest interesting considerations
on the evolution of some characters in the group. With
regard to life forms, Carlina falcata, from the Canary
Islands, is an unarmed and shrubby plant, two very
unusual features in a group formed mainly by spiny
perennial herbs. This led Meusel and Kästner (1994) to
suggest that the Canarian species of Carlina were the
oldest stock of the genus. However, all the analyses
group in a very robust clade Carlina falcata and the
widespread European spiny herbs Carlina lanata and
C. vulgaris [BS ⫽ 73%, DI ⫽ 2 in the ITS analysis (Fig.
1); BS ⫽ 100%, DI ⫽ 6 in the matK analysis (Fig. 2);
BS ⫽ 93%, DI ⫽ 6 in the combined analysis (Fig. 3)].
We must conclude that the shrubby habit is a second-
ary adaptation in insular species of Carlina, as sug-
gested by Carlquist (1976) for island species of the
Asteraceae and recently verified in another Canarian
genus of the Cardueae (Cheirolophus Cass.; cf. Sus-
anna et al., 1999). The robust clade of morphologically
very different Carlina falcata, C. lanata, and C. vul-
garis also indicates that, at least in Canarian plants,
extensive changes in habit and morphology, such as
the secondary loss of the spines, are not correlated to
DNA divergence, a confirmation of the recent origin of
some Macaronesian plants (Susanna et al., 1999). It
also illustrates the unsuitability of adaptative charac-
ters in the systematics of this group.
With regard to involucral morphology, two species of
Carlina from the west Mediterranean and North Af-
rica, Carlina gummifera (L.) Less. and C. macrophylla
(Desf.) DC., were either classified in the genus Atrac-
tylis (De Candolle, 1838) or segregated to a different
genus, Chamaeleon Cass. The main difference with
Carlina is the absence of the inner row of radiant,
showy involucral bracts usually associated to Carlina
(Petit, 1987; Meusel and Kästner, 1990, 1994). ITS
analysis groups both species in a very strong clade
(BS ⫽ 100%, DI ⫽ 14), but this clade is nested within
Carlina with even higher support (BS ⫽ 100% and
DI ⫽ 44; Fig. 1). On the other hand, both matK and the
combined analyses include Carlina gummifera in the
Carlina clade with the highest support (BS ⫽ 81%,
DI ⫽ 2; BS ⫽ 100%, DI ⫽ 49; Figs. 2 and 3, respec-
tively). These results suggest that both species must
remain in Carlina, a view strongly supported by the
structure of the involucre: Carlina macrophylla and C.
gummifera share with the rest of species of Carlina the
peculiar and exclusive structure of receptacular scales
(Petit, 1987). The lack of the showy bracts could be a
primitive character, as C. gummifera is basal to the
rest of the genus in the matK and combined analyses
(Figs. 2 and 3). Alternatively, the loss of the bracts
could be interpreted as a secondary loss as small,
unconspicuous bracts are present in Carlina gummifera
(Susanna and Garcia-Jacas, 2001). We favor this sec-
ond hypothesis because secondary reductions are very
frequent in the tribe (and they have often contributed
 COMBINED NUCLEAR–CHLOROPLAST DNA PHYLOGENY OF TRIBE CARDUEAE
to the difficulty to the classification in many genera; cf.
Vilatersana et al., 2000).
The genera Cardopatium and Cousiniopsis. The
placement of Cardopatium and Cousiniopsis is a prob-
lem that our analysis has failed to solve. These two
monotypic genera have been classified usually among
the Carlininae (Bentham, 1873; Hoffmann, 1894; Dit-
trich, 1977; Bremer, 1994). Petit (1997) moved them to
the Echinopeae, which he considered a separate tribe,
a view not supported by our results. The analysis of the
ITS region (Fig. 1) suggests a weak relationship be-
tween Cardopatium and Cousiniopsis, as was already
pointed out by Bremer (1994).
The Echinopsidinae. In view of the strong support
of both genomes for the position of Echinops within the
tribe Cardueae, the secondary heads of Echinops must
be regarded from a rather different point of view.
Wagenitz (1976), Dittrich (1977), and Petit (1988,
1997) have considered syncephaly a trait without par-
allel in the rest of the groups of the Cardueae sensu
lato. However, we should rather conclude that the com-
pound inflorescence of Echinops is an extreme adapta-
tion without high systematic relevance. In fact, the
trend toward the grouping of small, few-flowered heads
in compound inflorescences is common across all the
subtribes of the Cardueae: in Carlininae (Atractylis
polycephala Coss. has only three flowers per head), in
Carduinae (Cousinia congesta Bunge, Cousinia triflora
Schrenk, Cirsium congestum Fisch. & C. A. Mey.), and
even in the Centaureinae (Centaurea aggregata Fisch.
& C. A. Mey.). The epithets of all these taxa are very
explicit.
The inclusion of Xeranthemum and Chardinia—tra-
ditionally classified among the Carlininae—in a robust
clade (BS ⫽ 83%, DI ⫽ 4) within the Echinopsidinae,
even if only in the combined analysis (Fig. 3), is a
rather surprising result: no one had suspected a sister
relationship between both groups. Furthermore, this
inclusion is not supported by ITS and matK alone. A
priori, it is very difficult to connect usually perennial,
robust, spiny species of Echinops with predominantly
annual, delicate, unarmed plants of the Xeranthemum
complex. However, some characters could have given a
hint. The only species of the monotypic genus Acant-
holepis, A. orientalis Less., unanimously considered
very closely related to Echinops by its compound sec-
ond-order inflorescence, is an unarmed, frail annual.
Vegetative similarities between Acantholepis and the
Xeranthemum group are obvious, especially in the un-
armed, entire, linear-lanceolate leaves with velvety in-
dument, but resemblances between the Echinops and
Xeranthemum groups are not only vegetative. As is
obvious from Dittrich (1977, Figs. 7a and 7b; 1996b),
both groups share a similar achene anatomy: they have
a parenchymatic pericarp densely covered with multi-
cellular hairs (both characters shared by the Carlini-
61
nae), and the scaly pappus is directly attached to the
pericarp.
Finally, the most important shared trait was hith-
erto neglected. The receptable of Xeranthemum and
related genera has long tapering scales, longer than
florets, which have been interpreted usually as homol-
ogous to the receptacular bracts of Carlina (true bracts
sensu Petit, 1988; Stuessy and Spooner, 1988) by most
authors (Kruse and Meusel, 1972). This was the main
reason for the inclusion of the Xeranthemum complex
in the Carlininae (Bentham, 1873; Hoffmann, 1894;
Dittrich, 1977; Bremer, 1994). However, they can be
interpreted in a very different way: the bracts of Xer-
anthemum could be the invoucral bracts of a primary
head, a view supported by the disposition of the scales
(see Kruse and Meusel, 1972; Petit, 1987). According to
this interpretation, the involucre of Xeranthemum and
related genera must be considered a second-order sin-
cephaly, the same as that in Acantholepis and Echi-
nops.
With regard to chemistry, Echinops and Xeranthe-
mum are connected by the presence of tiophenes that
are unique in all the Cardueae (Wagner, 1977; Bremer,
1994). However, this chemical character has been only
partially explored, and no strong conclusions can be
drawn yet.
The problem of Berardia. The position of the alpine
genus Berardia supports its inclusion among the Car-
dueae. Dittrich (1996a), on the basis of achene anat-
omy, moved Berardia to the tribe Mutisieae, where it
would be aberrant from a biogeographic standpoint:
there are no European representatives of this mainly
tropical tribe. However, Berardia remains isolated in
the Cardueae: it is a monotypic genus from the Alps
without any morphologic affinities to any other genus
of the tribe (Dittrich, 1977, 1996a; Bremer, 1994).
Some striking parallelisms between Berardia and spe-
cies of Saussurea, Jurinea, and related genera from the
mountains of central Asia are only the typical adapta-
tions of plants to high mountains: acaulescence, dense
wooly indument, and stout woody rootstock.
The subtribe Carduinae. The problem posed by the
paraphyletic (if we exclude the Centaureinae) assem-
blage of the Carduinae (Susanna et al., 1995; Häffner
and Hellwig, 1999) remains unresolved. However,
some results are relevant for the systematics of the
subtribe.
The first interesting group is the “Onopordon” group
(Susanna and Garcia-Jacas, 2001), formed by Alfredia,
Synurus, and Onopordon [BS ⫽ 92%, DI ⫽ 5 in the ITS
analysis (Fig. 1); BS ⫽ 97%, DI ⫽ 4 in the combined
analysis (Fig. 3)]. Synurus was included among the
Centaureinae by Dittrich (1977) and Bremer (1994).
Recently, Häffner and Hellwig (1999), on the basis of
ITS sequence analysis, moved it to the Carduinae.
However, the strongly supported association of Alfre-
62 GARCIA-JACAS ET AL.
dia and Synurus (BS ⫽ 100%, DI ⫽ 15 both in the ITS
and in the combined analyses; Figs. 1 and 3) has a
deeper basis: they share the habit, the form and indu-
ment of the leaves, the floral morphology, and the
structure of the achenes. Synurus must be considered a
mere synonym of Alfredia (Susanna and Garcia-Jacas,
2001). Onopordon and Alfredia share a very uncom-
mon feature in the tribe: the receptacle is naked,
honey-combed, and without scales or bristles.
The next important natural group within the Car-
duinae is the “Carduus” group (Susanna and Garcia-
Jacas, 2001), formed by the mainly rose-flowered spiny
plants classically grouped under the common name
“thistles.” This group includes two large genera (Car-
duus and Cirsium) and many other smaller genera
represented here by Cynara, Galactites Moench, Noto-
basis, Picnomon, Ptilostemon, Silybum, and Tyrimnus.
The group formed by Carduus, Cirsium, Notobasis,
Picnomon, Silybum, and Tyrimnus was especially
strongly supported by all the analyses [BS ⫽ 93%, DI ⫽
8 in the ITS analysis (Fig. 1); BS ⫽ 87%, DI ⫽ 3 in the
matK analysis (Fig. 2); BS ⫽ 100%, DI ⫽ 12 in the
combined analysis (Fig. 3)]. Similarities in morphol-
ogy, habit, and biogeography among all these genera
are great, and the group can be considered a natural
one. Häffner and Hellwig (1999), whose ITS analysis
focused on this complex, arrived at the same conclu-
sion. However, our analysis includes only a few repre-
sentatives of the two largest genera, Carduus and Cir-
sium, and we cannot draw further conclusions on their
delimitation, which is problematic according to
Häffner and Hellwig (1999). However, there is a sig-
nificant difference between the results of our analysis
in Cirsium and the results obtained in another very
large genus, Cousinia. Species of Cirsium are grouped
in a clade with low support, whereas species of
Cousinia form a very strong clade (Fig. 1). Sequence
divergence among species of Cousinia is lower than
that in Cirsium, which indicates that Cousinia is a
more recent genus with an explosive diversification
(800 species!). This is concordant with the geographic
distribution: Cirsium has an Eurasian, African, and
North American distribution, whereas Cousinia has a
much more restricted distribution centered in the
Irano-Turanian region (Kazmi, 1963; Dittrich, 1977;
Häffner and Hellwig, 1999; Susanna and Garcia-Jacas,
2001).
Another clade is formed by the Centaureinae and
some related groups, among which the sister group to
the Centaureinae should be looked for, as was already
pointed out by Susanna et al. (1995), Petit (1997), and
Häffner and Hellwig (1999). As the Centaureinae have
been the object of a deeper analysis elsewhere (Garcia-
Jacas et al., 2001), we shall comment only that, as in
previous results, they form a robust monophyletic
clade.
The groups that are more closely related to the Cen-
taureinae are the clade which includes the genera Arc-
tium and Cousinia, the clade formed by Jurinea and
Outreya (Fig. 3), plus Hyalochaete, Jurinella, and Sau-
ssurea in the ITS tree (Fig. 1). However, the relation-
ships of these genera to the subtribe Centaureinae are
only very weakly supported by the ITS and the com-
bined analysis and not supported at all by the matK
analysis.
Other conclusions can be drawn from this clade, such
as the association of Arctium and Cousinia in a clade
with only moderate support [BS ⫽ 62%, DI ⫽ 2 in the
ITS analysis (Fig. 1); BS ⫽ 73%, DI ⫽ 3 in the com-
bined analysis (Fig. 3)] and not supported by the matK
sequences (Fig. 2). Relationships between both genera
have been signaled from old (see Duistermaat, 1996,
1997; Petit, 1997). However, the problem of Arctium
“getting entangled to Cousinia” (Duistermaat, 1997) is
easy to solve: we only have to move the “Arctioid”
Cousinia species to Arctium.
The last clade from the combined analysis that we
shall comment on is formed by Outreya and Jurinea
(BS ⫽ 100%, DI ⫽ 28; Fig. 3). In the ITS tree, this clade
includes also Hyalochaete and Jurinella with very high
support (BS ⫽ 100%, DI ⫽ 18; Fig. 1). The strong
support for this group confirms the suggestions by Su-
sanna and Garcia-Jacas (2001): on strict morphological
grounds, Hyalochaete, Jurinella, and Outreya cannot
be segregated from Jurinea (significantly, all three
were originally described within Jurinea).
The Subtribal Classification: A Recapitulation
Some final conclusions can be drawn on the subtribal
classification. Support for the basal branches is very
low or nonexistent in all the analyses (Figs. 1–3). More-
over, two genera, Cardopatium and Cousiniopsis, can-
not be included in any subtribe, and the positions of
Staehelina (Fig. 1) and Berardia remain uncertain
(Figs. 1–3). These results have forced us to be very
cautious when suggesting a subtribal classification in a
recent synthesis of the tribe (Susanna and Garcia-
Jacas, 2001).
Another problem is that the segregation of the Cen-
taureinae with subtribal rank leaves the subtribe Car-
duinae paraphyletic, as has been repeatedly pointed
out (Bremer, 1994; Susanna et al., 1995; Petit, 1997;
Häffner and Hellwig, 1999). However, the cladistically
correct solution has a major drawback: if we made a
natural Carduinae ⫹ Centaureinae, the resulting sub-
tribe would include the genera Centaurea (400 spp.),
Cousinia (800 spp.), Saussurea (more than 300 spp.),
Jurinea (100 spp.), Carduus (90 spp.), and Cirsium
(250 spp.). This totals nearly 2000 species of the ca.
2500 species of the Cardueae, 80% of the total. Proba-
bly, the Centaureinae ⫹ Carduinae are a natural group
(even if unsupported by molecular data), but to keep
them united in a subtribe would be extremely unprac-
tical.
 COMBINED NUCLEAR–CHLOROPLAST DNA PHYLOGENY OF TRIBE CARDUEAE
Finally, at the present state of knowledge, we think
that the most conservative approach is the only prac-
tical solution: keep the four classical subtribes, with
the broad modifications of delimitation suggested by
Susanna and Garcia-Jacas (2001) and reflected in
Fig. 3.
ACKNOWLEDGMENTS
Financial support from the Dirección General de Enseñanza Su-
perior, Spain, Projects PB 93/0032 and PB 97/1134, and grant
1999SGR0032, generalitat de Catalonia, are gratefully acknowl-
edged. We thank N. Gabrielyan, L. Kapustina, F. Khassanov, and V.
Mozaffarian for their assistance in the field work and help in the
identification of some difficult materials. The collaboration of the
botanical gardens listed in Table 2 is also acknowledged. We also
thank Vicki A. Funk, Smithsonian Institution and an anonymous
reviewer for many helpful commentaries for improving the text.
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