generic delimitation and phylogenetic relationships within the

MOLECULARPHYLOGENETICSAND

Molecular Phylogenetics and Evolution 32 (2004) 951–977

EVOLUTION

www.elsevier.com/locate/ympev

Generic delimitation and phylogenetic relationshipswithin the subtribe Chironiinae (Chironieae: Gentianaceae),with special reference to Centaurium: evidence from nrDNA

and cpDNA sequences

Guilhem Mansiona,* and Lena Struweb

a Laboratoire de Botanique Evolutive, Universit�e de Neuchatel, CH-2007 Neuchatel, Switzerlandb Department of Ecology, Evolution, and Natural Resources, Rutgers University, New Brunswick, NJ 08901, USA

Received 21 October 2003; revised 7 March 2004

Available online 4 June 2004

Abstract

To better understand the evolutionary history of the genus Centaurium and its relationship to other genera of the subtribe

Chironiinae (Gentianaceae: Chironieae), molecular analyses were performed using 80 nuclear ribosomal ITS and 76 chloroplast

trnLF (both the trnL UAA intron and the trnL–F spacer) sequences. In addition, morphological, palynological, and phytochemical

characters were included to a combined data matrix to detect possible non-molecular synapomorphies. Phylogenetic reconstructions

support the monophyly of the Chironiinae and an age estimate of ca. 22 million years for the subtribe. Conversely, both molecular

data sets reveal a polyphyletic Centaurium, with four well-supported main clades hereafter treated as separate genera. The primarily

Mediterranean Centaurium s.s. is closely related to southern African endemics Chironia and Orphium, and to the Chilean species

Centaurium cachanlahuen. The resurrected Mexican and Central American genus Gyrandra is closely related to Sabatia (from eastern

North America). Lastly, the monospecific genus Exaculum (Mediterranean) forms a monophyletic group together with the two new

genera: Schenkia (Mediterranean and Australian species) and Zeltnera (all other indigenous American centauries). Several bio-

geographical patterns can be inferred for this group, supporting a Mediterranean origin followed by dispersals to (1) North

America, Central America, and South America, (2) southern Africa (including the Cape region), and (3) Australia and Pacific

Islands.

� 2004 Elsevier Inc. All rights reserved.

Keywords: Biogeography; Character evolution; Centaurium; Gentianaceae; Gyrandra; Molecular clock; Phylogeny; Schenkia; Zeltnera

1. Introduction

Early classifications of the Gentianaceae were estab-lished by Endlicher (1838), Grisebach (1839), Bentham

and Hooker (1876), Knoblauch (1894), and Gilg (1895).

Despite important systematic progress achieved either

with traditional data (e.g., Broome, 1973; M�esz�aros,1994, 1996; Struwe et al., 1994; Zeltner, 1970) or mo-

lecular approaches (e.g., Chassot et al., 2001; Yuan

and K€upfer, 1995; Yuan et al., 1996, 2003), only the

* Corresponding author. Fax: +41-32-718-3001.

E-mail address: [email protected] (G. Mansion).

1055-7903/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.ympev.2004.03.016

infrafamilial classification of Gilg (1895), based on

pollen characters and gross morphology, was used as a

reference during the 20th century. With the increase inmolecular tools and results, a new classification was

proposed, which combined phylogenetic approaches

and traditional data (Struwe et al., 2002). In this treat-

ment, the gentian family (87 genera and ca. 1615–1688

species) has been organized into six monophyletic tribes

(Chironieae, Exaceae, Gentianeae, Helieae, Potalieae,

and Saccifolieae) based on cladistic analyses of trnL

(UAA) intron and matK sequence data. The tribeChironieae is an important assemblage of 23 genera and

ca. 160 species (Struwe et al., 2002). This tribe was

further subdivided in three monophyletic subtribes, the

mail to: [email protected]

952 G. Mansion, L. Struwe / Molecular Phylogenetics and Evolution 32 (2004) 951–977

Canscorinae, Chironiinae, and Coutoubeinae, on thebasis of pollen type (monads vs. tetrads), corolla tube

length, and anther spiralization.

Within the subtribe Chironiinae, the genus Centau-

rium (commonly called centaury) comprises mostly

short-lived (annual or biennial, rarely perennial) her-

baceous species and is characterized by the coiling of the

anthers after pollen release. The taxa are widely dis-

tributed in wet and disturbed habitats worldwide, wherecompetition is reduced. They can be encountered along

roadsides, on stream banks, in fields and pastures or in

open forests in most parts of the northern hemisphere.

Two main regions of diversification are recognized: one

in the Old World and another in the New World. The

Old World species are centered in the Mediterranean

basin and radiate in a northeasterly manner. About 25

diploid and polyploid species occur in this region(Zeltner, 1970). All New World species (�30 spp.) are

polyploids and most occur in western North America

from Mexico to Canada (British Columbia), with cen-

ters of high diversity in California and Texas. Some

species extend southward to Central and South

America.

Centaurium appears to be the largest genus of the

Chironieae, even though the exact number of species isnot yet clearly established. The species circumscription

within Centaurium is difficult because traditional mor-

phological characters discriminate species poorly.

Moreover, a large amount of morphological plasticity

occurs due to varying environmental conditions and

natural hybridization that may obscure species bound-

aries (Melderis, 1931; Zeltner, 1970). The abundance of

polymorphisms found within different taxa has led tothe naming of many species in Centaurium and different

nomenclatural opinions among authors (e.g., Gilg, 1895;

Grisebach, 1839; Hegi, 1966; Melderis, 1931; Robyns,

1954; Zeltner, 1970). Based on field experience and ex-

amination of different herbaria collections, a good esti-

mate of the actual species number appears to be 55–60

(Mansion, unpublished).

The phylogenetic position of Centaurium withinChironieae is not clear. Molecular trees inferred from

combined sequences of the matK and trnL intron re-

gions support its inclusion in the subtribe Chironiinae,

close to the genus Sabatia (Struwe et al., 2002). ITS

results placed Centaurium in the same subtribe, but as a

sister clade to Chironia plus Orphium (Thiv et al.,

1999a). Only a few species of Centaurium have been

investigated so far with molecular tools, and somegenera such as Exaculum were not sampled. The previ-

ous molecular-based hypotheses of Chironieae phylog-

eny do not reflect the whole range of diversity within the

subtribe. Consequently, the intergeneric relationship of

Centaurium has remained obscure. No cladistic studies

have previously been performed on the entire genus, and

the monophyly of Centaurium has not been established.

In addition, only a few morphological studies have beenperformed on the Gentianaceae, and little is known

concerning the evolution of vegetative and floral char-

acters within the subtribe Chironiinae and particularly

Centaurium.

The main goals of this study were (1) to resolve the

phylogenetic position of the subtribe Chironiinae

within the tribe Chironieae, (2) to investigate the in-

tergeneric relationships within the subtribe Chironiinae,(3) to test the monophyly of Centaurium, and (4) to use

the phylogenetic reconstruction of the Chironiinae as a

framework to infer biogeographic scenarios and char-

acter evolution in this group. For these purposes se-

quences of the internal transcribed spacer (ITS) of

nrDNA and a combination of the trnL UAA intron

with the trnL–F spacer (trnLF region) of cpDNA were

used for cladistic analyses. These regions have beenwidely used to infer phylogenetic relationships within

the Gentianaceae at both tribal, generic and species

levels (Chassot et al., 2001; Gielly and Taberlet, 1996;

Struwe et al., 2002; Thiv et al., 1999a,b; von Hagen and

Kadereit, 2001; Yuan and K€upfer, 1995, 1997; Yuan

et al., 1996, 2003).

2. Materials and methods

2.1. Sampling

In total, 80 taxa were included in the ITS data set and

76 in the trnL intron+ trnL–F spacer matrix (hereafter

called trnLF). To investigate the phylogenetic position

of the Chironiinae within the Chironieae, several ac-cessions representing neighboring tribes and subtribes

recognized by Struwe et al. (2002) were included in the

analyses (Table 1). One of the more basal tribes in the

family, Exaceae, represented by the genera Exacum and

Sebaea, was chosen as a functional outgroup (Struwe

et al., 2002; Yuan et al., 2003). Within the Chironieae,

subtribe Canscorinae was represented by Canscora,

Hoppea, Microrphium, Schinziella, and subtribe Cou-toubeinae by Coutoubea, Deianira, Schultesia, and

Symphyllophyton. Most of the genera comprising sub-

tribe Chironiinae, i.e., Blackstonia, Centaurium, Cicen-

dia, Chironia, Eustoma, Exaculum, Geniostemon,

Ixanthus, Orphium, and Sabatia, were sequenced for

several species for the ITS (58 accessions) and trnLF

regions (48 accessions). Only the rare Brazilian genus

Zygostigma is missing due to lack of suitable materialfor molecular studies. The Caribbean endemic Bis-

goeppertia was excluded since recent results indicate that

Bisgoeppertia, tentatively included by Struwe et al.

(2002) in the subtribe Chironiinae, is more closely re-

lated to Lisianthius and thus fits better in the Potalieae

(M. Thiv, pers. comm.). Most of the Centaurium species

included in this study were collected and determined in

Table 1

Origin of plant material, voucher information, and GenBank accessions for DNA sequences used in this paper

Taxa Voucher information Location GenBank Accession Nos. References

ITS1 ITS2 trnL (UAA)

intron

trn L-F spacer

Anthocleista amplexicaulis Baker S. Wohlhauser SWPBZT (NEU) Madagascar n/a n/a AJ490189* n/a Yuan et al.

(2003)

Anthocleista grandiflora Gilg M. Callmander s.n. (NEU) Madagascar AJ489864* AJ489864* AJ490190* AY251777 Yuan et al.

(2003)/This study

Anthocleista vogelii Planch. S. A. Thompson & J.E. Rawlins 1399

(NY)

n/a AY251688 AY251718 AF102377 n/a Struwe et al.

(1998)

Blackstonia acuminata (W.D.J. Koch)

Domin

Licht (MJG) Italy AJ011468* AJ011477* n/a n/a Thiv et al.

(1999a,b)

Blackstonia grandiflora Maire G. Mansion 010832 (NEU) Spain AY251684 AY251714 AY251742 AY251768 This study

Blackstonia imperfoliata (L.F.) Samp. G. Mansion 010833 (NEU) Spain AY251685 AY251715 AY251743 AY251769 This study

Blackstonia imperfoliata (L.F.) Samp. M. Thiv (HBM) Italy AJ011470* AJ011480* n/a n/a Thiv et al.

(1999a,b)

Blackstonia perfoliata (L.) Huds. G. Mansion 98712 (NEU) France AY047793 AY047878 AF402198 AF402254 This study

Blackstonia perfoliata (L.) Huds. L. & N. Zeltner 2050 (NEU) Spain AY251686 AY251716 n/a n/a This study

Canscora alata (Roth) Wallich J. C. Piso, S. Wohlhauser & L. Zeltner

MO24 (NEU)

Madagascar AJ489865* AJ489865* AJ490191* n/a Yuan et al.

(2003)

Canscora andrographioides Griff. P. Chassot 99-234 (NEU) Thailand AJ489866* AJ489866* AJ490192* n/a Yuan et al.

(2003)

Canscora diffusa (Vahl.) Roem.

& Schult.

Kokou s.n. (TOGO) n/a AY256386 AY256391 AF102389* AY251779 Struwe et al.

(1998)/This study

Canscora diffusa (Vahl.) Roem.

& Schult.

P. Chassot 99-231 (NEU) Thailand AJ489867* AJ489867* AJ490193* AY251780 Yuan et al.

(2003)/This study

Centaurium cachanlahuen (Molina)

B. L. Robinson

L. Zeltner 020501 (NEU) Chile AY251694 AY251724 AY251749 AY251786 This study

Centaurium cachanlahuen (Molina)

B. L. Robinson

L. Zeltner 020502 (NEU) Chile AY251695 AY251725 AY251750 AY251787 This study

Centaurium erythraea Rafn Anonymous 717367 (MEL) Australia AY251669 AY251699 AY251729 AY251753 This study

Centaurium favargeri Zeltner L. & N. Zeltner 2044 (NEU) Spain AY251670 AY251700 AY251730 AY251754 This study

Centaurium gypsicola (Boiss. et Reut.)

Ronniger

L. & N. Zeltner 2081 (NEU) Spain AY251671 AY251701 AY251731 AY251755 This study

Centaurium littorale (D. Turner) Gilmour L. & N. Zeltner 1816 (NEU) England AY251672 AY251702 AY251732 AY251756 This study

Centaurium maritimum (L.) Fritch G. Mansion 98904 (NEU) France AY251673 AY251703 AY251733 AY251757 This study

Centaurium pulchellum (Sw.) Druce G. Mansion 98505 (NEU) France AY047787 AY047872 AY251734 AY251758 This study

Centaurium scilloides (L. fil.) Samp. L. & N. Zeltner 981111 (NEU) France AY251675 AY251705 AY251737 AY251761 This study

Centaurium tenuiflorum (Hoffmgg.

& Link) Fritsch

L. & N. Zeltner 1767 (NEU) Morocco AY047773 AY047858 AY251735 AY251759 This study

Centaurium tenuiflorum (Hoffmgg.

& Link) Fritsch

L. & N. Zeltner 1766 (NEU) Morocco AY251674 AY251704 AY251736 AY251760 This study

Chelonanthus alatus (Aubl.) Pulle F. Bretagnolle & J. Piguet C3 (NEU) Ecuador AJ489868* AJ489868* AJ490194* AY251775 Yuan et al.

(2003)/This study

Chelonanthus angustifolius Gilg F. Bretagnolle & J. Piguet T11 (NEU) Ecuador AJ489869* AJ489869* AJ490195* AY251776 Yuan et al.

(2003)/This study

Chelonanthus purpurascens (Aubl.)

Struwe, S. Nilsson, & V. Albert

F. Bretagnolle & J. Piguet C92 (NEU) Colombia AJ489870* AJ489870* AJ490196* n/a Yuan et al.

(2003)

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Table 1 (continued)

Taxa Voucher information Location GenBank Accession Nos. References

ITS1 ITS2 trnL (UAA)

intron

trn L-F spacer

Chironia baccifera L. M. Callmander & S. Wohlhauser A005

(NEU)

South Africa AY251690 AY251720 AY251746 AY251783 This study

Chironia laxa Gilg M. Callmander & S. Wohlhauser A003

(NEU)

South Africa AY251691 AY251721 n/a n/a This study

Chironia linoides L. M. Callmander & S. Wohlhauser A004

(NEU)


Cicendia filiformis (L.) Delarbre M. Thiv 2156 (MJG) France AJ011463* AJ011473* AF102403* n/a Thiv et al.

(1999a,b)

Cicendia quadrangularis (Lam.) Griseb. P. Maas 8154 (U) n/a AY251682 AY251712 AF102404* AY251765 Struwe et al.

(1998)/This study

Coutoubea minor H.B.K. G. A. Romero 1684 (NY) n/a n/a n/a AF102407* n/a Struwe et al.

(1998)

Coutoubea ramosa Aubl. B. Hoffman & C. Capellaro 984 (NY) n/a n/a n/a AF102408* n/a Struwe et al.

(1998)

Coutoubea spicata Aubl. S. Mori 24349 n/a SM24075 SM24349 AY251745 AY251778 This study

Coutoubea spicata Aubl. S. Mori 24075 (NY) French Guiana n/a n/a AF102409* n/a Struwe et al.

(1998)

Deianira pallescens Cham. & Schlecht. W. A. Anderson 9385 (NY) n/a n/a n/a AF102410* AY251782 Struwe et al.

(1998)/This study

Eustoma exaltatum (L.) Salisb. L. & N. Zeltner 980610-1 (NEU) Mexico AY251697 AY251727 n/a n/a This study

Eustoma exaltatum (L.) Salisb. L. & N. Zeltner 980528-1 (NEU) Mexico AY251698 AY251728 AY251752 AY251789 This study

Eustoma russelianum G. Don G. Mansion s. n. (NEU) Switzerland

(cultivated)

AY251696 AY251726 AY251751 AY251788 This study

Exaculum pusillum Caruel P. K€upfer s. n. (NEU) Italy AY251681 AY251711 AY251740 AY251764 This study

Exacum affine Balf. Miller et al. 8238 Yemen, Socotra AJ489877* AJ489877* AJ490202* AY251770 Yuan et al.

(2003)/This study

Exacum caeruleum Balf. Miller et al. 11356 Yemen, Socotra AJ489882* AJ489882* AJ490207* AY251771 Yuan et al.

(2003)/This study

Exacum tetragonum Roxb. Ludin & Klackenberg 332 India AJ489907* AJ489907* n/a n/a Yuan et al.

(2003)

Fagraea fragrans Roxb. C.-H. Tsou 207 (NY) n/a AY251689* AY251719* AF102421* n/a Struwe et al.

(1998)

Geniostemon gypsophilum B.L. Turner G. Nesom et al. 7621 (LL) Mexico n/a n/a AF102429* AY251766 Struwe et al.

(1998)/This study

Gyrandra brachycalyx (Standley & L.O.

Willams) Mansion

G. Mansion, L. & N. Zeltner 990239

(NEU)

Mexico AY047770 AY047855 n/a n/a This study

Gyrandra brachycalyx (Standley & L.O.

Willams) Mansion


(NEU)

Mexico AY047771 AY047856 AF402184 AF402240 This study

Gyrandra tenuifolia (Martens & Galeotti)

Mansion


(NEU)


Hoppea dichotoma Willd. C.D.K. Cook RHT307 (MJG) n/a n/a n/a AF102440* n/a Struwe et al.

(1998)

Ixanthus viscosus (Aiton) Griseb. M. Thiv s.n. Spain, Tenerife AJ011471* AJ011481* n/a n/a Thiv et al.

(1999a,b)

Ixanthus viscosus (Aiton) Griseb. P. K€upfer s.n. (NEU) Spain, Tenerife AY251683 AY251713 AY251741 AY251767 This study

954

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Macrocarpaea macrophylla (Kunth) Gilg R. Callejas & H. Balslev 1030 (NY) Colombia AY256385 AY256390 AF102455 * n/a Struwe et al.

(1998)/This study

Macrocarpaea angelliae J.R. Grant

& Struwe

J.R. Grant 02-4289 (NEU) Ecuador AY397760 AY397761 n/a n/a Yuan et al.

(2003)

Megacodon stylophorus (C.B. Clarke)

H. Smith

Y.-M. Yuan 93-142 (NEU) China Z48109* Z48137* AJ315200* AY251773 Yuan et al.

(2003)/This study

Microphium pubescens C.B. Clarke P. Chassot 99-243 (NEU) Thailand AJ489916* AJ489916* AJ490241* AY251781 Yuan et al.

(2003)/This study

Orphium frutescens E. Meyer M. Callmander & S. Wohlhauser A001

(NEU)


Sabatia angularis (L.) Pursh. T. Ghammers 4860 (NY) USA AJ011467* AJ011476* n/a n/a Thiv et al., 2000

Sabatia campestris Nutt. G. Mansion, L. & N. Zeltner 97706

(NEU)

USA AY256382 AY256387 AY255692 AY255696 This study

Sabatia dodecandra (L.) Britton,

Sterns & Poggenb.

J. Grant 97-2858 (NEU) USA AY256383 AY256388 AY255693 AY255697 This study

Sabatia stellaris Pursh. J. Grant 97-2871 (NEU) USA AY256384 AY256389 AY255694 AY255698 This study

Schenkia australis (R. Br.) Mansion L. & N. Zeltner 001203 (NEU) Australia AY251676 AY251706 n/a n/a This study




Schenkia clementii (Domin.) Mansion L. & N. Zeltner 001201 (NEU) Australia AY251680 AY251710 AY251739 AY251763 This study

Schenkia spicata (L.) Mansion G. Mansion 981005 (NEU) France AY047791 AY047876 AF402196 AF402252 This study

Schenkia spicata (L.) Mansion L. & N. Zeltner 1756 (NEU) Morocco AY047792 AY047877 AF402197 AF402253 This study

Schinziella tetragona (Schinz) Gilg Malaisse 13852 (BR) n/a n/a n/a AF102479* n/a Struwe et al.

(1998

Schultesia guianensis (Aubl.) Malme C.C. Berg & A.J. Henderson BG 661

(NY)

n/a n/a n/a AF102480* n/a Struwe et al.

(1998)

Sebaea brachyphylla Griseb. J. Raynal 19414 Madagascar AJ489920* AJ489920* AY251744 AY251772 Yuan et al.

(2003)/This study

Sebaea madagascariensis Klack. J. C. Piso, S. Wohlhauser & L. Zeltner

MO18 (NEU)

Madagascar AJ489921* AJ489921* n/a n/a Yuan et al.

(2003)

Swertia perennis L. P. K€upfer s.n. (NEU) Switzerland AY251687 AY251717 AY255695 AY251774 This study

Symphyllophyton caprifolioides Gilg Ratter 6742 (E) Brasil AJ011462* AJ011472* AF102490* n/a Thiv et al.

(1999a,b)

Zeltnera abramsii (Munz) Mansion F. Bretagnolle & G. Mansion 990903

(NEU)

California AY047712 AY047797 AF402145 AF402201 This study

Zeltnera arizonica (A. Gray) Mansion G. Mansion, L. & N. Zeltner 97726

(NEU)

Texas AY047725 AY047810 AF402155 AF402211 This study

Zeltnera beyrichii (Torr. & A. Gray)

Mansion


(NEU)


Zeltnera calycosa (Buckl.) Mansion G. Mansion, L. & N. Zeltner 96519

(NEU)


Zeltnera glandulifera (Correll) Mansion G. Mansion, L. & N. Zeltner 96518

(NEU)


Zeltnera madrensis (Hemsl.) Mansion G. Mansion, L. & N. Zeltner 990230

(NEU)


Zeltnera martinii (Broome) Mansion G. Mansion, L. & N. Zeltner 990215

(NEU)


Zeltnera multicaulis (B.L. Robinson)

Mansion


(NEU)

Arizona AY047726 AY047811 AF402156 AF402212 This study

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Table

1(continued)

Taxa

Voucher

inform

ation

Location

GenBankAccessionNos.

References

ITS1

ITS2

trnL(U

AA)

intron

trnL-F

spacer

Zeltneraquitensis(K

unth)Mansion

G.Mansion,L.&

N.Zeltner

990201

(NEU)

Mexico

AY047766

AY047851

AF402176

AF402232

Thisstudy

Zeltnerasetacea(Benth.)Mansion

G.Mansion,L.&

N.Zeltner

990232

(NEU)

Mexico

AY047746

AY047831

AF402182

AF402238

Thisstudy

Zeltnerastricta(Schiede)

Mansion

G.Mansion,L.&

N.Zeltner

990217

(NEU)

Mexico

AY047758

AY047843

AF402180

AF402236

Thisstudy

Zeltneratexensis(G

riseb.)Mansion

G.Mansion,L.&

N.Zeltner

97714

(NEU)

Texas

AF047734

AF047819

AY402161

AY402217

Thisstudy

Zeltneratrichantha(G

riseb.)Mansion

G.Mansion,L.&

N.Zeltner

96504

(NEU)

California

AY047710

AY047795

AF402143

AF402199

Thisstudy

Zeltneravenusta(A

.Gray)Mansion

G.Mansion,L.&

N.Zeltner

96514

(NEU)

California

AY047713

AY047798

AF402146

AF402202

Thisstudy

Zeltnerawigginsii(Broome)

Mansion

G.Mansion,L.&

N.Zeltner

990234

(NEU)

Mexico

AY047753

AY047838

AF402171

AF402227

Thisstudy

Sequencesdirectlyretrieved

from

Genbankare

marked

withanasterisk.n/a,notavailable.


the field by G. Mansion or L. Zeltner (the latter a long-time Centaurium specialist). Voucher specimens are de-

posited in the herbarium of the University of Neuchatel

(NEU). The taxonomic names in Table 1 and through-

out this paper reflects the new generic classification

proposed by Mansion (in press), which was based on

these phylogenetic results.

2.2. DNA extraction, amplification, and sequencing

Total DNA was extracted from leaf tissue using ei-

ther the CTAB method (Doyle and Doyle, 1987) or the

DNeasy kit (Qiagen). Double-stranded nrDNA and

cpDNA were amplified by symmetric PCR using a

Whatman Biometra T Gradient or T3 thermocycler. The

PCR parameters were as follows: 1 cycle of 3min at

94 �C, linked to 30 cycles of 10 s at 94 �C, 20 s at 50 �C(or 55 �C), 1min 30 s at 72 �C, followed by 4min at 72 �Cto complete primer extension. Primer ITS1 (GGA AGT

AGA AGT CGT AAC AAG G) and ITS2 (TCC TCC

GCT TAT TGA TAT GC), respectively, binding to the

30 end of the 18S rRNA gene and 50 end of the 26S

rRNA gene (White et al., 1990), were optimally used at a

final concentration of 0.2 pmol/ll. Using the same PCR

conditions, primers �c� (CGA AAT CGG TAG ACGCTA CG) and �d� (GGG GAT AGA GGG ACT TGA

AC) were employed to amplify the chloroplast trnL

(UAA) intron; for the trnL–F spacer, primers �f� (ATT

TGA ACT GGT GAC ACG AG) and �e� (GGT TCA

AGT CCC TCT ATC CC) were used (Taberlet et al.,

1991). PCR products were run on a 1% agarose gel

stained with ethidium bromide, in order to evaluate the

quality and quantity of the amplified templates, andthen purified using the QIAquick PCR purification kit

(Qiagen). For difficult specimens (e.g. herbarium mate-

rial), a lower annealing temperature (50 �C) was used,

providing multiple ITS fragments of different lengths.

The latter were separated directly on the gel, by cutting

and subsequent purification of the desired region with

the QIAEX II Gel Extraction Kit (Qiagen). Purified

PCR products were automatically sequenced on anApplied Biosystems 310 DNA sequencer, using the

dideoxy chain termination technique and a BigDye

Terminator Cycle Sequencing Ready Reaction Kit

(Applied Biosystems). A 5 ll sequencing labeling reac-

tion was performed with 2 ll of premix provided by the

manufacturer (containing the four labeled terminators,

the deoxynucleotide triphosphates, the AmpliTaq DNA,

MgCl2, and Tris–HCl buffer), 0.2 ll of primer (10 ng/ll)and 2.8 ll of amplified DNA (about 10 ng/ll). The cyclesequencing program was performed on a thermocycler

(Biometra) and consisted in 30 cycles of (10 s

[96 �C]+ 4min [50 �C]). The accession numbers of the

sequences submitted to GenBank are listed in Table 1.

Previously published sequences that were retrieved from

GenBank are marked with an asterisk in Table 1.

G. Mansion, L. Struwe / Molecular Phylogenetics and Evolution 32 (2004) 951–977 957

2.3. Maximum parsimony analyses

DNA sequences were initially aligned using the pro-

gram Clustal W (Higgins et al., 1996). The obtained

multiple alignment was manually adjusted by sequential

pairwise comparisons. Several parameters such as se-

quence length, transition/transversion ratio, GC con-

tent, and number of informative characters and pairwise

distance divergence values were computed using version4.0b10 of PAUP (Swofford, 2002).

Maximum parsimony (MP) analyses were first con-

ducted for each set of ITS and trnLF sequences sepa-

rately. Heuristic searches were performed under the

Fitch parsimony criterion using the following options in

Winclada version 1.00.08 (Nixon, 2002) and version 2.0

of NONA (Goloboff, 1999): 100 random repetitions

with five starting trees (seed¼ 0), saving at most 10,000most parsimonious (MP) trees, and using the mult*

max* option for branch swapping. Branch support was

performed using the jackknifing method (Farris et al.,

1996) as implemented in Winclada/NONA, with the

following settings: 100 repetitions with five replicates

each search, five starting trees (seed¼ 0). Homoplasy

measures were computed using the consistency index

(CI), excluding all uninformative characters, and theretention index (RI) as implemented in Winclada/

NONA.

2.4. Assessment of congruence

Three alternative strategies have been proposed for

handling multiple data sets, and this topic has given rise

to considerable debate regarding the advantages ordisadvantages of the so-called combined, the condi-

tional, or consensus approaches, respectively (De Que-

iroz et al., 1995; Huelsenbeck et al., 1996). The real

issues are how to determine the source of conflict be-

tween data sets, and more important, if there are any

evolutionary explanations for any differences between

data sets. Several statistical approaches have recently

been proposed to assess whether or not data are a merepartition of a global data set, thus sharing the same

natural history and evolutionary processes (Johnson

and Soltis, 1998).

The incongruence length difference test (ILD; Farris

et al., 1995) was used first to examine the phylogenetic

congruence between the respective nrDNA and cpDNA

data sets. This test, implemented in PAUP as the par-

tition homogeneity test (PHT), was performed with 100replicates of heuristic searches (invariant and ambigu-

ous characters excluded, Maxtree¼ 500 and TBR

branch swapping). It has been suggested that PHT p

values greater than p ¼ 0:01 reflect congruent data sets

that, if combined, will either improve or will not neg-

atively affect phylogenetic accuracy (Cunningham et al.,

1998).

Second, the strict consensus trees, obtained from theseparate analyses of each data set, were compared for

topology and branch support, allowing a possible

identification of potential localized sources of conflict.

2.5. Maximum likelihood analyses

Once the placement of the tribe Chironieae within the

Gentianaceae was clarified, a smaller 53-taxon data set,including only the Chironiinae and its neighboring sub-

tribes (Canscorinae and Coutoubeinae), was used to

perform full maximum likelihood (ML) analyses. In the

53-taxon data set chosen, the respective ITS and trnLF

partitions were combined following the total evidence

approach (Kluge, 1989; Section 3). Heuristic searches

were performed under theML criterion, using the general

time reversible (GTR) model of sequence substitutionwith the following settings: assumed nucleotide frequency

A¼ 0.2559, C¼ 0.2672, G¼ 0.2364, T¼ 0.240; substitu-

tion rates between A–C¼ 0.9152, A–G¼ 1.7496,

A–T¼ 0.8489, C–G¼ 0.4627, C–T¼ 2.5705, shape pa-

rameter gamma¼ 4.8303, assumed number of invariable

positions¼ 0. The best model was evaluated through the

Hierarchical Likelihood Ratio Test procedure as imple-

mented in Modeltest version 3.06 (Posada and Crandall,1998). The resulting ML tree was aimed to be used as a

framework for subsequent molecular clock estimations,

phenotypic characters mapping, and biogeographic

reconstructions.

2.6. Molecular clock and divergence time calculation

The estimation of divergence time using a molecularclock is highly controversial and should be treated with

caution (Sanderson, 1998). Nevertheless, it is the only

way of inferring diversification rates and lineage age

when fossil data are lacking, which is usually the case for

annual plants such as Centaurium and relatives.

To test the assumption of a clockwise molecular

evolution of DNA sequences for the subtribe Chiro-

niinae, a likelihood ratio test (LRT) was performed onthe 53-taxon data set, under the general time revers-

ible (GTR) model of sequence substitution. The LRT

was calculated as 2 lnðLclock=Lno�clockÞ, and assumed to

follow a v2 distribution with (n� 2) degree of free-

dom, where n is the number of taxa (Sanderson,

1998).

If the LRT failed to support a clock-like evolution of

the data set, relative branching time was estimated fromnon-parametric rate smoothing analyses (NPRS, clock-

independent method), using the default settings in

TreeEdit version 1.0a8 (Rambaut and Charleston, 2001;

Sanderson, 1997). The NPRS method does not assume

an underlying molecular clock and will score better than

a clock-based method if the sequences are not evolving

in a clock-like fashion (Sanderson, 1997).


To estimate absolute diversification rates, it is neces-sary to fix at least one node on the cladogram to a known

date, which can be done by using either paleobotanical

records or geological events (Sanderson, 1998). Only two

fossil pollen records are known from the Gentianaceae,

and neither of these are from Chironieae (Graham, 1984;

Scott, 1995), so we used geological dating based on the

age estimate of Gran Canaria of the Macaronesian Is-

lands, ca. 15 million years (MY) (Juan et al., 2000). Thisgeological estimation may be seen as the maximum age

of the divergence between the disjunct sister genera

Ixanthus (an endemic of Gran Canaria and more recent

islands of the Macaronesian Archipelago) and Blacks-

tonia (of Mediterranean origin) (Thiv et al., 1999a).

2.7. Phenotypic character optimization

To detect synapomorphic features supporting the

clades obtained with molecular data, and to better un-

derstand the state transformation of various important

characters within the Chironiinae, 27 phenotypic char-

acters (mainly morphological and phytochemical ones,

Table 2) were added to the 53-taxon molecular matrix.

These characters were mapped on the combined ML

cladogram, using the trace Character function andACCTRAN optimization as implemented in MacClade

(Maddison and Maddison, 1997). The character coding

for Centaurium species was largely based on personal

observations on living and herbarium material; for other

Table 2

Morphological, palynological, and phytochemical characters and their chara

1. Life form: 0¼ tree, shrub, or suffrutescent herb; 1¼perennial, non-suffr

2. Stem-x-section: 0¼ terete; 1¼ quadrangular; 2¼winged

3. Inflorescence: 0¼dichasial; 1¼monochasial; 2¼ cluster of sessile flowe

4. Calyx: 0¼ polymerous [8–12-merous]; 1¼ 5-merous; 2¼ 4-merous; 3¼ 2

5. Fusion of sepals: 0¼ scarcely; 1¼half; 2¼ almost completely

6. Abaxial side of calyx lobes: 0¼ smooth; 1¼keeled; 2¼winged

7. Calycine colleters: 0¼ absent; 1¼present

8. Corolla color: 0¼ green, yellow, or white; 1¼ blue, lilac, pink, or red

9. Corolla merosity: 0¼polymerous [8-12–merous]; 1¼ 5-merous; 2¼ 4-m

10. Corolla shape: 0¼ rotate (saucer-shaped); 1¼ funnel-shaped (infundibu

11. Petal fusion: 0¼ scarcely; 1¼half; 2¼ almost completely

12. Floral nectaries: 0¼ none (or rudimentary); 1¼on the corolla; 2¼ gyno

13. Anther shape: 0¼ non-sagittate; 1¼ sagittate

14. Anther shape at anthesis: 0¼not twisted; 1¼ recurved; 2¼helically twi

15. Filament bases: 0¼ not united; 1¼united by a membrane

16. Stamen insertion: 0¼near the base of the corolla tube; 1¼ between the ba

tube

17. Ovary shape: 0¼ globular; 1¼oval; 2¼ elliptic, long

18. Stigma: 0¼ simple, capitate, or subcapitate (to slightly bilobed); 1¼bilo

19. Fruit type: 0¼ capsular; 1¼ baccate

20. Pollen when released: 0¼monad; 1¼ tetrad; 2¼polyad

21. Pollen aperture: 0¼ colpi; 1¼ colpori; 2¼ pori

22. Secoiridoid biosynthesis end-product: 0¼ sweroside; 1¼ swertiamarine;

23. Xanthone O-glycosides: 0¼ absent; 1¼ present

24. Xanthone 3-oxygenated: 0¼ absent; 1¼ present




cases literature resources were used (M�esz�aros et al.,2002, with further additions and modifications).

2.8. Biogeographic reconstructions

During the past decade, several analytical biogeo-

graphic methods have become available to test alterna-

tive biogeographic hypotheses (Morrone and Crisci,

1995). Dispersal biogeography represents an approachthat emphasizes dispersal from source areas (ancestral

area or center of origin), by coding areas as a multistate

character and optimizing states on a taxa cladogram,

using either the Camin–Sokal or Fitch parsimony cri-

terion (Bremer, 1992; Maddison et al., 1992; Morrone

and Crisci, 1995). In contrast to dispersalism, vicariance

biogeography assumes a correspondence between taxo-

nomic phylogeny and area relationships and allows theconstruction of area cladograms, showing hypotheses of

historical relationships between areas (van Veller et al.,

2001). Lastly, dispersal–vicariance analysis (Ronquist,

1997) is a compromise method that permits both dis-

persal and vicariance hypotheses. This approach, im-

plemented in DIVA (Ronquist, 1996), reconstructs

ancestral distribution in a given phylogeny without any

a priori assumptions about area relationships, minimizesdispersal and extinction and allows reticulate relation-

ships among areas (Ronquist, 1997).

The historical biogeography of the Chironieae was

inferred using character optimization methods based

cter states used in this study

utescent herb; 2¼ annual or biennial herb

rs; 3¼ solitary

-merous

erous

lar) to campanulate; 2¼ salver-shaped (hypocrateriform) or tubular

ecial (disk?)

sted

se and the mouth of the corolla tube; 2¼near the mouth of the corolla

bed, the lobes well-separated; 2¼decurrent

2¼ gentiopicroside


either on dispersalism (ancestral areas method; Bremer,1992; Fitch optimization; Maddison et al., 1992) or on

dispersal vicariance approach (DIVA; Ronquist, 1997).

Unlike vicariance-based methods, character optimiza-

tion methods allow the reconstruction of ancestral dis-

tributions without the constraint of an area-cladogram

(Bremer, 1992; Maddison et al., 1992).

Bremer�s ancestral area method was performed with

PAUP, by considering each area as a binary character,and optimizing the number of gains and losses for each

character on the taxon cladogram, using the Camin–

Sokal parsimony criterion.

Fitch optimization was achieved with MACCLADE,

on a data matrix with geographic areas coded as a single

multistate character. In this method, polymorphism is

restricted to terminal taxa (i.e., widespread species)

whereas ancestors are reconstructed as monomorphic(occurring in a single area).

For DIVA, the data matrix was constructed by

scoring the taxa for presence or absence in each area.

DIVA method is similar to Fitch optimization but al-

lows the treatment of widespread ancestors (Ronquist,

1997). Since the DIVA 1.1 computer program requires a

limited number of taxa and a fully resolved cladogram,

the reference 53-taxon matrix was reduced to a 25-taxonmatrix, joining the sister species occurring in the same

area into one terminal component. The polytomy en-

countered in Zeltnera (Section 3) was arbitrary resolved

by grouping the Z. multicaulis clade with the Z. martinii

clade. Alternative solutions gave the same results (not

shown). DIVA optimizations were then conducted with

either an unrestricted number of areas assignated to

each node, or with a number restricted to two (for most

Table 3

Alignment and sequence characteristics of the different nrDNA and cpDNA

Characteristics ITS data set (ITS1/ITS2) trn

Sequence length range (bp) 398–469 682

(222–238/194–247) (37

Sequence length mean (bp) 457 778

(230/227) (40

G+C content (range%) 55.5–70.3 33.

(54–71/56–71) (34

G+C content (mean%) 62.2 34.

(61/63) (36

Aligned sequence length (bp) 507 114

(254–257) (60

Parsimony informative characters 311 224

(158/153) (85

Constant characters 136 787

(68/68) (44

Uninformative characters 64 134

(28/36) (75

Most-parsimonious trees (N) 48 30

Tree length 1428 548

CI 0.46 0.7

RI 0.78 0.8

RC 0.36 0.7

of the taxa investigated do not occur in more than twoareas).

The geographic areas defined for these analyses were:

the Mediterranean (A), southern Africa (including the

Cape region (B), Australia (C), Western North America

(D), Eastern North America (E), Central America (F),

South America (G), and Southeastern Asia (H).

3. Results

3.1. DNA sequence variation

The length of the unaligned ITS1 and ITS2 sequences

ranged between 222–238 and 194–247 base pairs (bp),

respectively. The GC content varied from 54–71%

(ITS1) to 56–71% (ITS2), with respective mean values of61% (ITS1) and 63% (ITS2). The alignment of 80 ITS

sequences produced a matrix of 507 characters with 311

(61.3%) of these being parsimony informative. The

boundaries of ITS regions were identified based on a

comparison with reference sequences of Gentiana frigida

ITS1 and ITS2 (GenBank Accessions Nos. Z48053 and

Z48084). One region covering 12 ambiguous positions

was poorly alignable and was experimentally excludedfrom all analyses. For this exclusion did not affect the

results (not shown), all the characters were kept for

parsimony statistics calculation (Table 3).

In the trnLF region, sequence length ranged from 682

to 873 bp (378–490 bp for trnL intron and 290–451 bp

for trnL–F spacer), with minimum and maximum GC

values of 33.5 and 36.9%, respectively (Table 3). The

limits of the trnL intron and trnL–F spacer was defined

regions investigated in the present study

LF data set (trnL intron/trnL–F spacer) Combined data set

–873 831–1341

8–490/290–451)

1189

4/374)

5–36.9 43.9–51.3

–38/31–37)

3 45.4

.2/33.2)

5 1656

7–538)

507

/139)

962

7/340)

187

/59)

20

1890

9 0.56

8 0.77

1 0.43


by comparison with Gentiana frigida (GenBank Acces-sion Nos. X77883 and AJ315277, respectively). This

matrix included 75 sequences and 1145 aligned charac-

ters, of which 224 (20%) were phylogenetically infor-

mative. Most of the indels necessary for the matrix

alignment do not contain any phylogenetic information.

Nevertheless, some indels appear to be informative for

the tribe Exaceae (position 337–462 in Exacum and 363–

462 in Sebaea) or the subtribe Coutoubeinae (position567–583).

A summary of the main characteristics for the re-

spective regions is presented in Table 3.

3.2. Phylogenetic analyses

3.2.1. MP analysis of the ITS data set

Heuristic searches performed on the ITS matrix re-sulted in 48 most parsimonious (MP) trees of 1428 steps,

with a CI of 0.46, and a RI of 0.78. One arbitrary chosen

cladogram is shown in Fig. 1, where the sign ‘‘Ø’’ in-

dicates the nodes that collapse on the strict consensus

tree. Jackknife values (J) for node supports are indicated

below the branches (when equal to or greater than 50%).

The ITS strict consensus tree is well resolved, with good

branch support for most of the tribes and subtribesdefined by Struwe et al. (2002). Nevertheless, the

monophyly of the tribe Chironieae is not supported

(branch collapsing on the ITS strict consensus tree). Yet,

the respective subtribes are well delimited, with high

jackknife values for the Canscorinae (J ¼ 100) and

Coutoubeinae (J ¼ 100), but weak support for Chiro-

niinae (J < 50). Within the subtribe Chironiinae, all the

species of Centaurium (except C. cachanlahuen) are dis-tributed into four independent, well-separated clades,

each well-supported and here named C1 (J ¼ 87), C2

(J ¼ 100), C3 (J ¼ 99), and C4 (J ¼ 90; see Fig. 1).

Some clades are well supported, such as (Blacksto-

nia+ Ixanthus; J ¼ 100), (Orphium+Chironia+Centau-

rium cachanlahuen; J ¼ 78), (Sabatia+Centaurium clade

C2; J ¼ 71), and (Exaculum+Centaurium clade

C3+Centaurium clade C4; J ¼ 100). Finally, themonophyly of several additional genera in the Chiro-

niinae is established by having maximum node support

of J ¼ 100 (e.g., Blackstonia, Cicendia, Eustoma, and

Sabatia).

3.2.2. MP analysis of the trnLF data set

Phylogenetic analyses performed on the trnLF data

set, under the MP criterion, gave 30 MP trees of 548steps (CI¼ 0.80, RI¼ 0.89). One MP phylogram is

shown in Fig. 2 (with jackknife branch support). In-

ferred trees from the trnLF sequences analyses are

generally well resolved at the intergeneric level, and

usually received good branch support. The groups

identified on the ITS cladogram such as subtribes

Canscorinae (J ¼ 99), Coutoubeinae (J ¼ 91), or the

(Blackstonia+ Ixanthus) clade (J ¼ 99), are also stronglysupported by the cpDNA analysis. Furthermore, tribe

Chironieae and subtribe Chironiinae received significant

branch support (J ¼ 100 and J ¼ 97, respectively). The

polyphyly of Centaurium is partially confirmed, with

high jackknife values for the clades C1 (J ¼ 71, but

excluding C. maritimum), C3 (J ¼ 96), and C4 (J ¼ 96),

respectively, but not for the unresolved clade C2. The

intergeneric relationships within the Chironiinae arerather poorly solved in the trnLF result (Fig. 2). Some

clades depicted by the ITS analysis such as (Chiro-

nia+Orphium+Centaurium cachanlahuen), (Gyran-

dra+Sabatia), or (Exaculum+Schenkia+clade C4) are

not resolved in the trnLF strict consensus tree.

3.2.3. Assessment of congruence

The partition homogeneity test, performed on acombined data set of 65 sequences (with constant and

ambiguous characters excluded), indicate incongruence

between the ITS and trnLF data sets (p ¼ 0:01). Nev-

ertheless, a comparison of the cladograms obtained for

each separate partition suggests a general congruence in

topologies and branch supports. In both the ITS and

trnLF analyses: (1) subtribes Canscorinae, Chironiinae,

and Coutoubeinae are monophyletic; (2) most of thegenera, within the Chironiinae, received quite good

branch support; and (3) Centaurium s.l. is polyphyletic.

Thus, the rejection of the ILD test may be due to

minor conflicts in species placement. Some sister rela-

tionships observed on the ITS cladogram such as (C.

pulchellum+C. maritimum) and (Gyrandra tenuifo-

lia+G. brachycalyx) are not supported by the trnLF

topology (Figs. 1 and 2). The latter argues for two al-ternative clades, (C. pulchellum+C. tenuiflorum) and (G.

tenuifolia+Sabatia dodecandra), respectively. The ex-

clusion of these species in the ILD test increases the

support for congruence between the data set

(P ¼ 0:949).

3.2.4. Combined MP and ML analyses

Once the conflicting species have been detected, wecombined the two data sets in a ‘‘total evidence’’ ap-

proach (Kluge, 1989; Fig. 3). Sequences of the combined

region range from 831 to 1341 bp, with a mean GC

content of 45.4%. The combined matrix of 65 taxa

comprised 1656 aligned characters with 507 (ca. 31%)

being parsimony informative.

Cladistic analyses performed on the combined matrix

resulted in 20 MP trees of 1890 steps, with CI¼ 0.56 andRI¼ 0.77 (Fig. 3). The strict consensus tree is similar to

the ITS one, with generally a better resolution and

branch support, except for some unresolved clades such

as (Centaurium+Chironia) or (Exaculum+Schen-

kia+Zeltnera). This lack of resolution may be the

consequence of weak conflict between the nrDNA and

plastid data sets, in the relationships of these taxa.

Fig. 1. One of the 48 most parsimonious trees (L¼ 1428/CI¼ 0.46/RI¼ 0.78) obtained from the ITS data set. Jackknife values >50% are given below

the branches. Numbers 1–5 correspond to the tribe Exaceae, Potalieae, Gentianeae, Helieae, and Chironieae, respectively. Clades C1–C4 represent

the polyphyletic genus Centaurium: C1¼Centaurium, C2¼Gyrandra, C3¼Schenkia, and C4¼Zeltnera. Branches that collapse in the strict con-

sensus tree are indicated by Ø.


Fig. 2. One of the 30 most parsimonious trees (L¼ 548/CI¼ 0.79/RI¼ 0.88) obtained from the trnLF data set. Jackknife values >50% are given

below the branches. Numbers 1–5 correspond to the tribe Exaceae, Potalieae, Gentianeae, Helieae, and Chironieae, respectively. Clades C1–C4

represent the polyphyletic genus Centaurium: C1¼Centaurium, C3¼Schenkia, and C4¼Zeltnera. Clade C2 is not supported. Branches that collapse

in the strict consensus tree are indicated by Ø.


Fig. 3. One of the 20 most parsimonious trees (L¼ 1890/CI¼ 0.56/RI¼ 0.77) obtained from the combined (ITS+ trnLF) data set. Jackknife values

>50% are given below the branches. Numbers 1–5 correspond to the tribe Exaceae, Potalieae, Gentianeae, Helieae, and Chironieae, respectively.

Clades C1–C4 represent the polyphyletic genus Centaurium: C1¼Centaurium, C2¼Gyrandra, C3¼Schenkia, and C4¼Zeltnera. Branches that

collapse in the strict consensus tree are indicated by Ø.



Finally, hard conflicting species, responsible of the ILDfailure, are resolved as in the ITS topology.

ML analyses (GTR model) performed on the 53-

taxon data set (with constant and ambiguous characters

Fig. 4. Single ML ultrametric tree (� ln ¼ 6133:56) of the subtribe Chironiin

set. Outgroup comprises seven species of subtribes Canscorinae and Coutou

method. The calibration point used (grey ellipse: 15 MYR) is a possible maxim

and the Mediterranean Blackstonia, and is based on geological events (maxim

C1–C4 represent the polyphyletic genus Centaurium: C1¼Centaurium, C2¼

excluded) yield to one single tree (� ln ¼ 6852:32). Thisinferred ML cladogram differs from the MP strict con-

sensus tree in that Cicendia is no longer sister group to

(Blackstonia+ Ixanthus) clade, and is the basal most

ae (53-taxon data set), obtained from the combined (ITS+ trnLF) data

beinae. Branch lengths and node ages were computed using the NPRS

um age of the divergence between the Macaronesian endemic Ixanthus

um age estimate of Gran Canaria of the Macaronesian islands). Clades

Gyrandra, C3¼Schenkia, and C4¼Zeltnera.


genus in the subtribe Chironiinae (Fig. 4). Only onepolytomy have been encountered in Zeltnera (Node C4).

3.3. Molecular dating

By enforcing a molecular clock on the 53-taxon data

set, we obtained a ML tree with a log-likelihood score of

6908.83. Further comparison with the non-clock model

(phylogram with a score of 6852.32) suggested therejection of the MC assumption (LRT¼ 113,

df ¼ 51; P < 0:01). The respective branch lengths were

thus calibrated using the clock-independent NPRS

method, with the Blackstonia+ Ixanthus node fixed to

Fig. 5. Character mapping of morphological characters on the fully resol

polytomy in Zeltnera has been resolved arbitrary: see text). Character states a

text. Illustrated species examples are noted in brackets after each character st

1¼Perennial herb [Centaurium scilloides, scale bar¼ 1 cm]; 2¼Annual herb

(0¼Terete, not winged stem [Blackstonia perfoliata]; 1¼Quadrangular, not

Scale bar¼ 5mm). 3. Corolla color (0¼Yellow corolla [Cicendia filiformis

merosity (0¼Polymerous corolla [Blackstonia perfoliata]; 1¼Pentamerous c

lum]. Scale bar¼ 1 cm). 5. Corolla shape (0¼Corolla rotate [Sabatia camp

shaped [Zeltnera trichantha]. Scale bar¼ 1 cm). Arrows represent the length

twisted (not illustrated); 1¼Recurved anther (a. Eustoma, b. Sabatia); 2¼H

Stamen insertion (0¼Stamens inserted between the base and the mouth

brachycalyx]. Scale bar¼ 5mm). Arrows show the respective stamen insertio

ovary [Gyrandra pauciflora]; 2¼Elliptic and long ovary [Zeltnera maryanna

shape (0¼ Stigma simple, capitate or subcapitate [e.g., Zeltnera]; 1¼ Stigma b

The arbitrarily chosen character corolla color (3) is optimized on the cladog

15 million years ago (MYA). By using this calibration,the maximum age of the tribe Chironieae and subtribe

Chironiinae was estimated to the early Miocene (23.3

and 22.1 MY, respectively, Fig. 4).

3.4. Phenotypic characters variation

Among the 27 phenotypic characters investigated

(Table 2), only a few appeared to contain some phylo-genetic information when optimized on the 53-taxon

cladogram (Fig. 5). The phylogenetic importance and

evolution of these nine selected characters will be

discussed.

ved ML cladogram obtained from combined 53-taxon data set (the

nd illustrations of the main morphological features are discussed in the

ate. 1. Life form (0¼Tree or shrub [e.g., Fagraea sp., scale bar¼ 2m];

[Zeltnera martinii, scale bar¼ 1 cm]). 2. Stem shape in cross-section

winged stem [Zeltnera glandulifera]; 2¼Winged stem [Canscora alata].

]; 1¼Pink corolla [Exaculum pusillum]. Scale bar¼ 1 cm). 4. Corolla

orolla [Eustoma exaltatum]; 2¼Tetramerous corolla [Exaculum pusil-

estris]; 1¼Corolla funnel-shaped [Exacum affine]; 2¼Corolla salver-

of the corolla tube. 6. Anther shape at anthesis (0¼Not recurved or

elically twisted anther (c. Chironia, d. Zeltnera). Scale bar¼ 1mm). 7.

[Zeltnera venusta]; 1¼Stamens inserted near the mouth [Gyrandra

ns. 8. Ovary shape (0¼Globular ovary [Chironia baccifera]; 1¼Oval

and Centaurium rhodense, respectively]. Scale bar¼ 5mm). 9. Stigma

ilobed, the lobes well-separated [e.g., Centaurium]. Scale bar¼ 20mm).

ram (Fitch and ACCTRAN optimization).

Table 4

Results of the ancestral area analysis of the Chironiinae

Area Gain Loss G/L (%) Coefficient

Mediterranean basin 5 6 83.4 1.00

Western North America 4 7 57.1 0.68

Central America 5 10 50 0.60

South America 3 7 42.9 0.51

Southern Africa 1 5 20.0 0.24

Eastern North America 1 7 14.3 0.17

Australia 1 8 12.5 0.15


Most of the vegetative and floral characters (e.g., type

of inflorescence, occurrence of calycine colleters or floral

nectaries, petal fusion) are highly homoplastic. On the

other hand, palynological (pollen release and type of

aperture) and phytochemical (secoiridoid biosynthesis)

features were poorly investigated at the tribal level, andgenerally remained uninformative.

3.5. Biogeographic reconstruction

Both the ancestral areas method (Bremer, 1992) and

Fitch optimization (Maddison et al., 1992), performed

on 53-taxon data set, support a Mediterranean origin

for the subtribe Chironiinae (Table 4, Fig. 6).DIVA analyses resulted in multiple combination of

optimal reconstructions. Constraining the number of

maximal areas to two resulted in a unique Mediterra-

nean ancestral distribution for the subtribe Chironiinae

(instead of a widespread ancestral distribution in the

unconstrained analysis; see Fig. 4). Overall, DIVA

analyses require either 11 (unconstrained analysis) or 13

(constrained analysis) dispersal events, and suggest arather complicated biogeographical history for the

subtribe. One possible scenario involved several dis-

persal from the Mediterranean to South Africa, Aus-

tralia, or the Americas (Fig. 6).

4. Discussion

4.1. Utility, limits, and congruence of the molecular data

sets

Sequences of the ITS and trnLF regions have pro-

vided good results in previous cladistic analyses per-

formed on the Gentianaceae at both generic and specific

levels (Chassot et al., 2001; Gielly and Taberlet, 1996;

Thiv et al., 1999a,b; von Hagen and Kadereit, 2001;Yuan and K€upfer, 1995; Yuan et al., 1996, 2003). The

current study confirms the utility of the ITS sequences to

resolve interspecific relationships within the Gentiana-

ceae, and particularly in the Chironiinae. In general,

strict consensus trees show well-resolved groups, and

branch support is high. On the other hand, trnLF

sequences evolve slower than the ITS region, but theyprovide good resolution at the generic level. Yet, most of

the intergeneric relationships within the Chironiinae,

inferred with ITS sequences are supported by the trnLF

data set.

Nevertheless, interspecific relationships are poorly

resolved on the trnLF cladograms, and may result in

soft incongruence between the respective data sets

(Wendel and Doyle, 1998). Pruning taxa with low res-olution on the trnLF cladogram (e.g., C. maritimum or

C. cachanlahuen, Fig. 2) do not change the results of the

ILD test (results not shown). Alternatively, two con-

flicting nodes (with J > 70) have been identified in the

respective ITS and trnLF cladograms as source of hard

incongruence. Their exclusion of the combined data set

resulted in the acceptation of the ILD test.

It is likely that some of the taxa present in the con-flicting clades have separate histories for the nuclear

(ITS) and the plastid (trnLF) genomes. Thus, evolu-

tionary (reticulate) processes known to perturb cladistic

reconstruction in plants, such as hybridization, intro-

gression, lineage sorting, gene duplication or interlocus

concerted evolution may be considered to explain the

present phylogenetic incongruence (Doyle, 1992; Wen-

del and Doyle, 1998).In Centaurium, topological incongruence between

phylogenetic cladograms inferred from ITS and trnLF

data sets have proven to be good tools for estimation of

reticulate evolution and detection of allopolyploidy

(Mansion et al., unpublished). Several examples of in-

terspecific hybridization involving, e.g., C. maritimum,

C. tenuiflorum, or C. pulchellum have been documented,

based on karyological grounds (Zeltner, 1970), andrecently confirmed with molecular tools (Mansion

et al., unpublished). Regarding the importance of in-

terspecific gene flow within Centaurium, hypothesis of

nuclear introgression could be evoked for the mor-

phologically distinct species C. maritimum and C. pul-

chellum that have a sister relationship with the ITS tree,

but divergent positions in the trnLF cladogram (Figs. 1

and 2).Introgressive hybridization is an important trend in

plant evolution known to contribute in intraspecific

variation (Anderson, 1948). Introgression have been

reported in Sabatia (Bell and Lester, 1978), and artificial

hybridization also revealed a high intersectional cross-

ability between species (Perry, 1971). Since cytoplasmic

introgression is not uncommon in taxa capable of in-

terspecific hybridization (Rieseberg, 1995), the sisterrelationships depicted between Gyrandra tenuifolia and

Sabatia dodecandra (Fig. 2) suggest possible episodes of

chloroplast capture. In this case, one explanation for its

occurrence may be long-distance dispersal since Gyran-

dra tenuifolia occurs in Mexico (Broome, 1973), and

Sabatia dodecandra is encountered from Connecticut to

Louisiana (Wilbur, 1955).

Fig. 6. Biogeographic patterns in the Chironiinae. (A) DIVA reconstruction mapped on the fully resolved ML cladogram obtained from combined

53-taxon data set (the polytomy in Zeltnera has been resolved arbitrary: see text). The outgroup includes seven species of subtribes Canscorinae and

Coutoubeinae. Letters A to H are the defined geographic areas for the analysis: A¼Mediterranean basin and surrounding areas, B¼ Southern

Africa, C¼Australia, D¼Western North America, E¼Eastern North America, F¼Central America, G¼ South America, H¼Southeastern Asia

Letters in parentheses are DIVA optimization with number of areas constrained to two. Branch patterns are Fitch optimization (ACCTRAN) of the

character ‘‘repartition’’ on the cladogram. (B) A possible scenario for the biogeographic history of the Chironiinae (dashed lines¼ dispersal; full

lines¼ vicariance). Dispersalism methods and DIVA (maxareas¼ 2) argue for a Mediterranean origin (A) for the Chironiinae, with a primary

dispersal into South Africa (B: Chironia, Orphium) and South America (G: Centaurium cachanlahuen, Cicendia quadrangularis), followed by several

later episodes of colonization into North America (D,E,F) or Australia (C).



4.2. Generic delimitation of Centaurium s.l. and system-

atic implications

One of the most unexpected results of this study is the

polyphyly of Centaurium. This genus has always been

regarded as a morphologically well-delimited assem-

blage by most authors of systematic treatments (e.g.,

Broome, 1973; Gilg, 1895; Grisebach, 1839; Zeltner,

1970). Our molecular analyses depict four major clades:clade C1 which mainly clusters the Eurasian species of

Centaurium; clades C2 and C3, comprising some Mexi-

can species, Mediterranean and Australian ones, re-

spectively; and clade C4 which includes the remaining

native American centauries.

In the early classifications of Centaurium, established

by Schmidt (1828), Grisebach (1839, 1845), and Gilg

(1895), no segregation between Old World and NewWorld species of centauries was undertaken. Only

Grisebach (1845, 1853) placed one species from Mexico

in his new genus Gyrandra (G. chironioides), and a spe-

cies from Hawaii in another new genus Schenkia (S.

sebaeoides). In Gilg�s (1895) taxonomic treatment, he

presented a classification based mainly on pollen char-

acters, and this classification was used for over a cen-

tury. He recognized the genus Erythraea (withCentaurium and Gyrandra in synonymy) and placed it in

subtribe Erythraeinae, close to Sabatia. The classifica-

tion of Struwe et al. (2002) combined phylogenetic ap-

proaches and traditional data, and close affinities were

proposed between Centaurium, Sabatia, and Chironia.

Even if such relationships had been previously advo-

cated (Broome, 1973; Weaver and R€udenberg, 1975;

Wilbur, 1955), the apparent polyphyly of Centaurium

has never been proposed earlier. Similar cases, where

molecular analyses do not support an apparent mor-

phological homogeneity, have been reported in the

Polemoniaceae within the genus Linanthus (Bell and

Patterson, 2000). In that genus, which has mainly di-

verged in response to the spreading drought that oc-

curred in California during the Pliocene, no clear

morphological characters were available to segregatebetween the different molecular-based clades. Several

other examples may be cited as well, particularly Lotus

(Fabaceae), which is a group showing a strikingly sim-

ilar geographical distribution to Centaurium, and in

which two very distinct North American and Eurasian

clades have been depicted by ITS analyses (Allan and

Porter, 2000).

This polyphyly not only disrupts the infragenericclassification of the group, but intergeneric relationships

as well. A new classification has recently been proposed

to resolve the problem of the polyphyly of Centaurium

(Mansion, in press). In this classification, several taxo-

nomic changes are made as a result of the findings in this

paper. Clade C1 represents the Eurasian species of the

genus Centaurium Hill, and the �true� Centaurium. The

genera Gyrandra and Schenkia, both described byGrisebach (1845, 1853), on the basis of G. chironioides

(¼Centaurium chironioides) and S. sebaeoides (¼Cen-

taurium sebaeoides), are resurrected and extended to

include additional species. Finally, a new genus Zeltnera

was proposed for the remaining American species cor-

responding to clade C4 (Mansion, in press). This no-

menclatural treatment, dividing Centaurium into four

genera, is followed in the present discussion.

4.3. Phylogenetic relationships within the Chironiinae

This study, based on ITS and trnLF data sets, sup-

ports the monophyly of the tribe Chironieae, and con-

firms the subtribes Canscorinae and Coutoubeinae as

sister clades with the Chironiinae, as suggested by

Struwe et al. (2002). The relationships described below,unless otherwise stated, are based on the ITS cladogram

that allows a better resolution at the specific level

(Fig. 1).

4.3.1. The (Blackstonia + Ixanthus) clade

A close relationship between Blackstonia and Ixan-

thus was suggested by Anderson (1948), Grisebach

(1839), and more recently by Thiv et al. (1999a).Blackstonia comprises four annual, diploid or tetraploid

species of mainly Mediterranean distribution (Zeltner,

1970). The monotypic Ixanthus, one of 28 endemic

genera of the Canary Islands (Kunkel, 1993), is a pe-

rennial, basally woody, and sparsely branched herb,

which reaches a height of up to 2m. These genera share

some morphological features such as the presence of

perfoliate bracts, calycine colleters, and a yellow corolla.A more detailed comparison, including anatomy–his-

tology, morphology, karyology, and ecological charac-

ters, was presented by Thiv et al. (1999a). In our study,

the relatively basal position and sister group relationship

between Blackstonia and Ixanthus are reinforced.

4.3.2. Large phylogenetic distance between Cicendia and

Exaculum

The phylogenetic position of Cicendia within the

Chironiinae remains problematic. MP analyses suggest

an intermediate placement of Cicendia between the basal

(Blackstonia+ Ixanthus) clade and the remaining genera

(Figs. 1 and 3) whereas the inferred ML cladogram

supports a basal most position of Cicendia within the

subtribe (Fig. 4). Earlier hypotheses of close affinities

between the Mediterranean genera Cicendia and Exac-

ulum (Grisebach, 1839), and to a lesser extent with the

Mexican genus Geniostemon (Thiv and Kadereit, 2002a),

are not supported by our molecular analyses (Fig. 2). All

the inferred cladograms (Figs. 1–3) reveal a large phy-

logenetic distance between Cicendia and Exaculum.

These genera share a similar morphological appearance

(slender herbs, sparsely branched, with minute flowers


and untwisted anthers after pollen release) and an affinityfor the same type of open habitats (Favarger, 1960). In

contrast toCicendia, the flowers ofExaculum are pink and

possess a short calyx tube with the calyx lobes exceeding

the corolla tube in length, and the corolla tube is longer

than the corolla lobes (vs. equal length in Cicendia). In

Cicendia, the stigma is subcapitate whereas Exaculum

possesses a bilobed stigma (Vald�es et al., 1987). Lastly, thechromosome number differs between these taxa, withn ¼ 13 for Cicendia filiformis and n ¼ 10 for Exaculum

pusillum (Favarger, 1960).

The present cladistic analyses support phylogenetic

affinities of Exaculum with either Zeltnera (ITS, Fig. 1)

or Schenkia (trnLF, Fig. 2), or an unresolved position

(combined analysis, Fig. 3). Schenkia comprises plants

with spicate, cymose inflorescences, centered in the

Mediterranean basin, but is also found in Russia, Japan,Australia, and on the Hawaiian Islands. Molecular,

morphological (spike-like cyme inflorescence, subsessile

flowers, and subcapitate stigma) and karyological

(n ¼ 11) evidence support the generic identity of

Schenkia and argue against the inclusion of Exaculum in

it. The genus Zeltnera also differs from Exaculum in

several aspects such as the chromosome number

(n ¼ 17; n ¼ 20; n ¼ 21; and n ¼ 22; Mansion andZeltner, in press), stamen insertion (in the upper half of

the corolla tube in Zeltnera vs. in the throat of the co-

rolla in Exaculum), coiling of the anthers (absent in

Exaculum), and geographic repartition (North and

Central America for Zeltnera). Thus, molecular, kary-

ological and morphological data support the recognition

of a monospecific genus Exaculum.

4.3.3. The South African (Chironia +Orphium) clade and

Centaurium

Most species of Chironia (ca. 30 species) are found in

grassy places of southern Africa, mainly in the Fynbos

vegetation of the Cape region, where the endemic

monotypic genus Orphium also occurs. Orphium is a

suffrutescent herb, which closely resembles the pubes-

cent species of Chironia, such as C. baccifera. Fewmorphological differences (mainly the keeled calyx of

Orphium) discriminate between the two genera; hence,

all the generic characters described for Orphium are also

encountered in Chironia. Both ITS and trnLF support a

close relationship between Orphium and Chironia bac-

cifera (Figs. 1–3), suggesting either a broader circum-

scription of Chironia (including Orphium) or a

paraphyletic genus Chironia.Phylogenetic analyses suggest a derived position of

Chironia and Orphium compared to the Mediterranean

genus Centaurium (Figs. 1 and 4), or failed to resolve the

position of the respective clades (Fig. 3). Chironia and

Orphium share morphological features with Centaurium

such as pink to purple corollas (rarely white or yellow)

and anthers coiling after dehiscence (Marais and Verd-

oon, 1963). The main morphological difference betweenthese genera is the corolla shape, i.e., rotate to funnel-

shaped with the corolla tube shorter than the corolla

lobes in Chironia vs. salver-shaped, with the corolla tube

equaling or exceeding the corolla lobes in Centaurium.

Furthermore, perennial species with a basally woody

stem are missing in Centaurium, but frequently en-

countered in Chironia. It is noteworthy that the Cape

flora has a surprisingly low proportion of annuals (ca.7% of the species) compared to other regions with

Mediterranean climates (Goldblatt and Manning, 2000).

Indeed, the Gentianaceae, with about 18 annual species

(mainly Chironia and Sebaea), is one of the families with

the highest number of annuals in the Cape flora

(Goldblatt and Manning, 2000, p. 15).

4.3.4. Position of the South American Centaurium cachan-

lahuen

The unique phylogenetic position ofC. cachanlahuen is

of interest because it groups with Chironia and not with

any other Centaurium species (Figs. 1 and 3), which im-

plies a striking South American–South African disjunc-

tion (see below). This important medicinal herb (Molina,

1787; Schneider, 1974), endemic to the Andean areas of

Chile and Argentina, is frequent from the Pacific littoralto the Andean pre-Cordillera and from the Atacama

Province in the North to the Chilo�e Province in the South(Gunckel, 1924). The gross morphology of C. cachan-

lahuen evokes Zeltnera quitensis. However, the style dif-

fers in being very short (i.e., as long as the stigma lobes)

and the divided stigma much resembles that of Chironia

krebsii (Mansion, personal observations), whereas Zelt-

nera species generally share undivided stigma (Mansion,in press).

Further comparative morphological studies are

planned to resolve the taxonomic status of C. cachan-

lahuen and the relationships with the South American

Chironiinae taxa (Zygostigma australe and Centaurium

ameghinoi) that were not included in the present study

(Mansion and Zeltner, in progress).

4.3.5. Relationships among the (Gyrandra+Sabatia)

clade and Eustoma

The (Gyrandra + Sabatia) clade received strong

branch support in the ITS result (J ¼ 71). Sabatia is a

rather species-rich genus within the Chironiinae (ca. 20

taxa), comprising distinctive taxa classified in several

sections on the basis of morphological and karyological

data and incompatibility among hybrids (Perry, 1971;Wilbur, 1955). The present study includes one species

each from four out of seven sections, and gives pre-

liminary support for the monophyly of Sabatia.

The genus Gyrandra was first described by Grisebach

(1839), and contains five species (Mansion, in press).

The ITS and combined cladograms (Figs. 1, 3, and 4)

support the sister position of Gyrandra to Sabatia,


whereas trees based on cpDNA characters (Fig. 2) arguefor the inclusion of Gyrandra speciosa in Sabatia.

Eustoma is a small genus occurring in the southern

United States, Mexico, and Greater Antilles with two

species (NatureServe, 2003), E. russellianum (Hook.) G.

Don (including E. grandiflora (Raf.) Shinners) and E.

exaltatum (L.) Salisb. (cf. Shinners, 1957). This genus

was first placed in the tribe Tachiinae (Gilg, 1895), close

to Lisianthius, due to similar pollen features (reticulatepollen).

It is noteworthy that the taxa belonging to the (Eus-

toma+Gyrandra+Sabatia) clade share the chromosome

number of 2n ¼ 72 (with a high dysploid variation in

Sabatia), a larger-sized corolla (except Sabatia arenicola

and Gyrandra brachycalyx) than other Chironiinae, and

stamen insertion near the sinus of the corolla lobes

(Mansion, in press). Moreover, their respective geo-graphic distribution (Caribbean, Mexico and eastern

North America) may support a common ancestral origin

and vicariant patterns. One morphological difference is

the degree of anther coiling after anthesis, being helically

twisted in Gyrandra, slightly coiled in Eustoma and re-

curved to circinnately coiled or half-twisted laterally in

Sabatia. Another difference is stigma shape, which is

subcapitate to slightly bilobed in Gyrandra, bilobed andwell separated in Eustoma, and deeply bilobed and

twisted around each other at anthesis in Sabatia.

4.4. Character evolution in the Chironiinae

The Chironieae have been divided in three subtribes

based on molecular data (Struwe et al., 2002). None-

theless, no morphological cladistic analysis has beenperformed on the whole tribe, except some more general

works covering the Gentianaceae (M�esz�aros et al., 1996,2002). The latter study failed to detect synapomorphic

characters supporting current tribe or subtribe delimi-

tations. This lack of synapomorphies for above-generic

ranks is also noted in this analysis combining molecular

data and structural characters, with some exceptions

where a few distinct characteristics support supragenericgroupings or genera.

4.4.1. Morphological characters

Vegetative characters appear to contain poor, if any,

phylogenetic information at the supergeneric level. Most

of the Chironiinae investigated are annual herbs, and

the suffrutescent habit found in Chironia, Coutoubea,

Ixanthus, Orphium, and Symphyllophyton has developedindependently several times (Fig. 5, character 1). It

seems that the basal woodiness in Ixanthus is best in-

terpreted as secondary, and has either evolved as a

consequence of the perennial life cycle or as an adap-

tation to the laurel forest habitat (Thiv et al., 1999a).

The suffrutescent habit encountered in Chironia and

Orphium could be a derived character, when compared

to the herbaceous condition found in Centaurium. Thus,the cause for woodiness in insular Ixanthus and conti-

nental genera (Chironia and Orphium) may differ. The

stems are mainly quadrangular in cross-section within

the Chironiinae (Fig. 5, character 2), except for the basal

genera (Blackstonia, Cicendia, Ixanthus) and Eustoma,

which are all terete.

Corolla color varies significantly within Chironieae.

Yellow, white or green corollas appear to be a plesio-morphic state (Fig. 5, character 3). The pink to lilac

corollas seem to be synapomorphic for most of the

genera of the Chironiinae, except for the basal Blacks-

tonia, Cicendia, and Ixanthus, and some species of

Centaurium (C. maritimum: yellow corollas) or Sabatia

(white corollas). Most of the pink-flowered genera often

include certain populations with white-flowered indi-

viduals (Mansion, pers. obs.). Pentamerous corollas aremost common within the tribe Chironieae and have been

regarded as the ancestral merosity state for the family

(M�esz�aros et al., 2002). Yet, tetramerous flowers are

also frequent in several genera of Chironieae, either

consistently (e.g., Canscora, Cicendia, Coutoubea,

Cracosna, Exaculum, Hoppea, or Schinziella) or occa-

sionally (e.g., Centaurium, Ixanthus, Schenkia or Zelt-

nera), as well as polymerous ones (Blackstonia, Sabatiasection Dodecandrae, and outside of the Chironieae:

Anthocleista, Potalia, and Urogentias). Our combined

analysis supports the pentamery of the corolla as a

plesiomorphic state for the Chironieae. On the other

hand, the tetramerous flowers appear to be synapo-

morphic for a part of the subtribe Canscorinae (Fig. 5,

character 4), as recently found by Thiv and Kadereit

(2002b), and based on a cladistic analysis of morpho-logical characters. Most of the genera in the tribe have a

salver-shaped corolla. Among the exceptions are

Blackstonia, Gyrandra, and Sabatia, all three with a

rotate corolla, and (Chironia+Orphium) with a funnel-

shaped corolla (Fig. 5, character 5).

An unusual specialization of the androecium is the

coiling of the anther after pollen release, hitherto con-

sidered as a typical characteristic of Centaurium s.l. Withthe generic break up of Centaurium into four separate

genera, this character is no longer a possible synapo-

morphy. Such spiralization or recurvation of the thecae

is also present to various degrees in other genera of the

Chironiinae, such as Chironia, Eustoma, Orphium, and

Sabatia, but is absent within the basal genera (Blacks-

tonia, Cicendia, Geniostemon or Ixanthus) and in Exac-

ulum (Fig. 5, character 6). Within the subtribe, thestamens are basifixed and generally inserted in the upper

half of the corolla tube, except for the (Sabatia+Gyr-

andra+Eustoma) clade where the insertion occurs

higher up in the corolla throat, near the sinuses of the

rotate or funnelform corolla (Fig. 5, character 7).

The gynoecium of the Chironieae is bicarpellate, with

a superior ovary variable in shape (Fig. 5, character 8),


and surmounted by a style of variable length. The latteris either slightly divided (apically divided in Blackstonia,

twisted in Sabatia, or slightly branched in Centaurium or

Chironia spp.) or oblong-capitate (terminally hooked in

Chironia spp. and Orphium, or subcapitate or slightly

bilobed to funnelform in Gyrandra, Schenkia or Zelt-

nera). The degree of style division is generally a good

discriminating character at the generic level (Fig. 5,

character 9). Another character of importance in thesystematics of the Chironiinae is the stigma morphol-

ogy. This feature is particularly useful to discriminate

between Chironia species (Marais and Verdoon, 1963).

Within the centauries, stigmas with fan forms generally

appear in Zeltnera, whereas Centaurium species mainly

have elliptic or reniform ones (Mansion, in press).

4.4.2. Palynology

Within subtribe Chironiinae, 3-colporate and finely

reticulate pollen grains have previously been reported in

14 species of Centaurium s.l. (K€ohler, 1905). Further-more, similar pollen morphology (defined as ‘‘Centau-

rium pulchellum’’ pollen type) has been encountered in

six species of Centaurium and in Schenkia spicata (Punt

and Nienhuis, 1976). This preliminary pollen study does

not support the distinctive phylogenetic distance be-tween Centaurium and Schenkia as found by us.

Recently, Nilsson (2002) described striate to striato-re-

ticulate pollen grains in both Chironia and Orphium,

thus supporting the possible inclusion of Orphium into

Chironia.

The palynology of Zeltnera has been poorly studied.

Elias and Robyns (1975) reported pollen characters for

Zeltnera quitensis that do not differ much from themorphology found in the ‘‘Centaurium pulchellum’’

pollen type. Nonetheless, variation has been observed

regarding the pollen shape (subprolate in Zeltnera vs.

sphaeroidal in Centaurium) and exine features (lirae

irregularly interlaced vs. lirae parallel to colpi and

sometimes abruptly changing direction; Rao and

Chinnappa, 1983).

Overall, palynological data remain fragmental withinthe subtribe Chironiinae, but some features may con-

stitute possible generic synapomorphies. A palynologi-

cal study that includes all representative taxa is greatly

needed.

4.4.3. Phytochemistry

Phytochemical characters have been used extensively

for taxonomic purposes in the Gentianaceae, and aresometimes helpful to discriminate between tribes, genera

or even sections (Gottlieb, 1982; Hostettmann-Kaldas

et al., 1981; Jensen and Schripsema, 2002). Particular

compounds that are known to occur in aerial parts and

roots of many gentians are xanthones (often yellow) and

secoiridoids (very bitter). The xanthones are biosyn-

thetically of mixed acetate and shikimate origin, and

two main hydroxylation patterns (or ‘‘parent patterns’’)can be found (Carpenter et al., 1969; Hostettmann and

Wagner, 1977; Jensen and Schripsema, 2002). Xant-

hones can be encountered in most of the genera of the

Chironieae, except in some species of Cicendia (C. fili-

formis), Centaurium (C. maritimum) and Sabatia (S.

angularis, S. elliotii; Schaufelberger, 1986). These com-

pounds also occur in some Texan species of Zeltnera

(Mansion and Rodriguez, unpublished data).Diverse oxidation patterns of the parent xanthones

have been recorded in the Gentianaceae, and are not

distributed equally within the family. The variation in

oxidation patterns has been used for cladistic recon-

structions by Gottlieb (1982), van der Sluis (1985), and

M�esz�aros (1994), in which the polarity of this character

has been expounded in different ways. A more satisfac-

tory proposition has been recently suggested by Jensenand Schripsema (2002), who arranged the xanthones of

the Gentianaceae in four biosynthetic groups based on

how �primitive� they are in the biosynthetic chain. In our

analysis, xanthones of group 1, the most primitive state

(1-3-7 and/or 1-3-7-8 substituted xanthones), constitute

a homoplastic state since they are randomly distributed

in five genera within the respective tribes. They appear,

for example, in Blackstonia and Orphium, but not intheir respective sister genera Ixanthus and Chironia, re-

spectively. On the other hand, particular hexa-substi-

tuted xanthones, mainly corresponding to group 4 of

Jensen and Schripsema (2002), are frequent in the Chi-

ronieae and may be a synapomorphy for this group (not

shown).

4.5. Biogeography and evolutionary history of the Chiro-

niinae

Extant species of the Chironieae are distributed in

three main geographic regions. The Canscorinae and

Coutoubeinae are of Paleotropical and Neotropical dis-

tributions, respectively, whereas theChironiinae aremore

frequent in the subtropical to temperate northern hemi-

sphere. The Chironiinae particularly occurs in the WestPalearctic (Blackstonia, Centaurium, Chironia, Cicendia

p.p., Exaculum, Ixanthus, and Schenkia p.p.) or in the

southern part of the Nearctic (Cicendia p.p., Eustoma,

Geniostemon, Gyrandra, Sabatia, and Zeltnera). Some

genera are encountered in the southern hemisphere as

well, mainly in Argentina, Brazil, and Chile (Centaurium

cachanlahuen, Zygostigma australe), South Africa (Chi-

ronia, Orphium), and western Australia (Schenkia p.p.).Our ancestral area analysis strongly supports a

Mediterranean origin for the Chironiinae with sub-

sequent dispersal into the Americas, South Africa, and

Australia (Table 4). Constrained DIVA analyses and

Fitch optimization also argue for a Mediterranean ori-

gin for the subtribe, but with a primary dispersal into

South Africa (Chironia, Orphium) and South America


(C. cachanlahuen), followed by several episodes of col-onization into North America or Australia (Fig. 6).

Furthermore, DIVA results suggest that the most recent

common ancestor (MRCA) of the (Eustoma+Gyran-

dra+Sabatia) clade first colonized Central America

(giving rise to Eustoma and Gyrandra) and then eastern

North America (becoming Sabatia). Later, a possible

dispersal to Australia would explain the distributional

patterns in the extant genus Schenkia. Coinciding withthis was a large species diversification in Zeltnera in

western North America (Fig. 6). Alternatively, a Medi-

terranean taxon may have evolved locally providing the

MRCA of Schenkia and Exaculum, and the MRCA of

Zeltnera, which further dispersed to western North

America. Based on this, several alternative dispersal

hypotheses can be inferred, suggesting dispersal from

the Mediterranean area to either (1) North America,Central America, and South America, (2) South Africa

and Cape region, or (3) Australia and the Pacific Is-

lands. Particularly disjunct patterns such as the amphi-

tropical repartition of Cicendia quadrangularis, or the

unusual transatlantic connection between Chironia and

C. cachanlahuen will be discussed below.

4.5.1. Connections between regions with Mediterranean

climate

Most of the genera in the Chironiinae are restricted to

either the Mediterranean basin and surrounding areas

(Blackstonia, Cicendia, Centaurium, Exaculum, Ixanthus,

and Schenkia) or to diverse regions with mainly Medi-

terranean climates, such as southwestern North America

(Cicendia, Eustoma, Geniostemon, Zeltnera), South Af-

rica (Chironia, Orphium), Australia (Schenkia) and Chile(C. cachanlahuen). In these latter regions, introduced

Chironieae species from the Mediterranean are also

frequent (e.g., Blackstonia, Centaurium, or Cicendia). A

few genera are restricted to smaller climatic regions,

such as tropical climates with summer rain Gyrandra or

Zeltnera in Mexico, or hot and humid climates (Sabatia

in eastern North America).

Biogeographic patterns inferred from the DIVAanalysis indicate that several migrations to the Americas

have occurred in the Chironiinae. Two major routes are

known to have provided connections between the Old

and New World, namely, the Bering Land Bridge (BLB)

and the North Atlantic Land Bridge (NALB). The first

bridge (BLB) between northwestern North American

and northeastern Asia was present throughout the

Tertiary with repeated formation over the past 20 mil-lion years during the Miocene, Pliocene, and Pleistocene

(Graham, 1999; Tiffney, 1985a; Wen, 1999), and has

permitted many possibilities for exchange of plants and

animals between Eurasia and North America. During

the Miocene, the BLB appears to have been the main

connection for many temperate, deciduous plants cur-

rently present in North America and Asia. However, the

temperatures at these high latitudes were too cool formost herbaceous groups (Tiffney, 1985a). Movements

via the BLB still remained possible by successive colo-

nization of disturbed sites (or alternatively by a southern

Aleutian bridge), or spread by random long-distance

dispersal (Tiffney, 1985a). Considering that many of the

Chironiinae taxa are frequently found in disturbed sites

(e.g., Blackstonia, Centaurium, Schenkia or Zeltnera)

where competition is weak, the first solution appears tobe more likely. Nevertheless, the current lack of Chiro-

niinae in eastern Asia (except Centaurium pulchellum in

China and Schenkia japonica in Japan) provides a weak

support for the BLB hypothesis. The second alternative

bridge (NALB) involved at least four geographical links:

two between North America and Greenland, one be-

tween Fennoscandia and Greenland, and one between

Greenland and the British Isles (McKenna, 1983). Thelatter link broke by the early Eocene (ca. 50 MYA),

whereas some other connections between Eurasia and

North America remained available until the end of the

Eocene (ca. 38 MYA) allowing plant migrations (Tiff-

ney, 1985b; Tiffney and Manchester, 2001). These ranges

are not congruent with current age estimations for the

subtribe Chironiinae (22.1 MY; Fig. 4). Even with a

larger estimation of the divergence time to 30 MY, be-tween Ixanthus and Orphium (based on a matK–trnL

data set; Thiv, unpublished), the NALB does not ap-

pear to have played a major role in the Chironieae

diversification.

The relationship found between the Mediterranean

native Centaurium and the South African genera Chi-

ronia and Orphium (Fig. 3), exemplifies a distributional

pattern known in several other groups of plants (e.g.,genera in the Dipsacaceae, Papaveraceae, and Thym-

eleaceae). Only 4% of the genera encountered in South

Africa are thought to have disjunct affinities with Eur-

asian taxa (Goldblatt, 1978). The direction of these ex-

changes is largely unknown, with some South African

genera being either basal or derived compared to their

Mediterranean counterparts (e.g., Crassulaceae [Mort

et al., 2001] or Colchicaceae [Caujape-Castells et al.,2002]). Our study weakly support a derived position of

Chironia relative to Centaurium (Figs. 1 and 4).

One significant feature of the Tertiary history of Af-

rica is the union of the African-Arabian block with

Eurasia, thereby closing the Tethys Sea, by the mid-

Miocene (ca. 15 MYA; Axelrod and Raven, 1978). Di-

rect migration of the Eurasian flora into Africa became

then possible, and two main routes have been proposed(Quezel, 1978); (1) incursion into central Sahara via the

Ahaggar and Tibesti mountains, and (2) incursion of

Mediterranean woodland vegetation via the Red Sea

hills of Egypt and Somalia (Wickens, 1976). The pres-

ence of Centaurium pulchellum, along with the enigmatic

Monodiella flexuosa Maire (¼Centaurium flexuosum

(Maire) Lebrun & Marais), in the Tibesti Mountains


(between southern Libya and northern Chad) supportsthis view. A new uplift in East Africa by the Miocene

caused the formation of cooler and drier areas, and

provided additional routes for migration and exchange

between Mediterranean and South African floras. By

the Pliocene and Pleistocene, exchanges with South

Africa seem to be impossible due to a tropical–equato-

rial barrier (Quezel, 1978). Therefore, a probable Mio-

cene origin (5–25 MY) may be proposed for a (Chironia+ Orphium) divergence from a Mediterranean ancestor,

which is congruent with the node age estimation of this

clade using our geological calibration point (7.1 MY;

Fig. 4). By the Miocene, the development of a cold

circum-Antarctic current (around 11–14 MY) favored

the aridification of the Cape region (Richardson et al.,

2001). This was an important factor in initiating the

transformation of the Miocene subtropical forest to theFynbos vegetation of today (Axelrod and Raven, 1978).

The Canarian fauna and flora is related to that of

other Atlantic islands, such as Madeira and, to some

lesser extent, that of the Azores and the Cape Verde

Islands. This flora has affinities mainly with the Medi-

terranean region, although some elements are related to

more remote regions such as South America or Aus-

tralia (Juan et al., 2000). The genus Ixanthus occurs inthe most recent islands of the Canary archipelago such

as (from east to west) Gran Canaria (14–16 MY), Ten-

erife (11.6 MY), La Gomera (10 MY), La Palma (2

MY), and El Hierro (1 MY), where it grows in the un-

derstory of laurel forests (Bramwell and Bramwell,

1990). The genus is absent from the two eastern islands,

Fuerteventura (20 MY) and Lanzarote (15.5 MY).

Relative ages of the respective islands are based on po-tassium argon dating (Juan et al., 2000). As the Canary

Islands are of oceanic origin (Juan et al., 2000), long-

distance dispersal is required for plant colonization.

Thus, one can expect a single introduction of either the

MRCA of the (Blackstonia+ Ixanthus) clade or Ixanthus

on Gran Canaria, followed by a stepwise colonization

sequence from this relatively older to the younger is-

lands in the chain (i.e., from Gran Canaria, to Tenerife,and the more western islands). This type of single col-

onization pattern has been demonstrated for many en-

demic Macaronesian genera, including Argyranthemum

(Francisco-Ortega et al., 1997), Pericallalis (Panero

et al., 1999), or Sonchus (Kim et al., 1996) for the As-

teraceae, Echium (Boraginaceae, Bohle et al., 1996),

Crambe (Brassicaceae, Francisco-Ortega et al., 1999),

Aenium (Crassulaceae, Mes et al., 1996), Bencomia

(Rosaceae, Helfgott et al., 2000), and Sideritis (Lamia-

ceae, Barber et al., 2002). Alternatively, Ixanthus may

have colonized older arid eastern islands such as Fu-

erteventura (20 MY), or arose directly in continental

laurel forests, and became extinct locally due to the

aridification of the habitat or volcanic activity (Juan

et al., 2000).

The presence of some Blackstonia, Centaurium orCicendia species in Australia can be interpreted as an-

thropozoogenic introduction, but an alternative sce-

nario may be proposed for Schenkia. Schenkia is

widespread in damp and sandy places and occurs in all

the Australian states (Adams, 1996). The current dis-

tribution of Schenkia shows that diploid taxa are com-

mon around the Mediterranean basin and radiate

throughout Asia, and tetraploid species are distributedin western Australia, Hawaii, and the Easter Islands

(Zeltner, pers. comm.). A long-distance dispersal of an

ancestral diploid of Mediterranean origin to Australia

and Pacific islands (Hawaii or Easter Islands) remains

possible. The rare endemic Hawaiian species S. sebaeo-

ides is tetraploid (Carr, 1978; Zeltner, pers. comm.), and

morphologically closely related to S. australis. Relative

maximum age estimation within Schenkia is 5.4 MY(Fig. 4), i.e., by late Miocene. The migration of diploid

or tetraploid populations accompanying the invasion of

Asian plants in northeastern Australia, could have been

possible during the late Miocene, by the New Guinea

bridge permitting the junction of the Australian conti-

nent with the Philippine Sea plate (Hill, 1994; Renner

et al., 2001). The occurrence of S. japonica, a morpho-

logically close relative to S. australis (not included in ouranalyses), in Taiwan and Japan supports this view. Such

recent colonization (the end of Miocene to Pliocene) of

the Australasian region has been documented for the

genus Myosotis (Winkworth et al., 2002).

4.5.2. Genera with disjunct repartition

Among 18 North American–European disjunct an-

giosperm genera (Qian, 1999), four are bispecific genera(Cicendia, Corema, Hottonia, and Mespilus). All of them

are disjunct between the western Old World and the

western New World, especially the Mediterranean and

the Madrean regions (Takhtajan, 1986). Likewise, some

species of Centaurium (C. erythraea, C. pulchellum or C.

tenuiflorum) or Schenkia (S. spicata) from Europe, are

naturalized in North America. Node age estimations of

the Cicendia split (ca. 14.7 MY, Fig. 4) indicate a pos-sible introduction to North America by the middle

Miocene, more likely via the BLB. Recent discoveries

suggest that the Neotropical climates were unstable over

the past 2 million years during the Pleistocene (Whit-

more and Prance, 1987). Cyclic glacial events led to

periods of cooler and/or drier climate in which

(rain)forest species may have withdrawn to small refugia

pockets (Haffer, 1982). Other recent geological phe-nomena that have been suggested for driving Neotrop-

ical speciation are the uplift of the northwestern Andes

(ca. 5 MY) and the bridging of Isthmus of Panama,

achieved ca. 3.5 MY (Gentry, 1982). Thus, one can

hypothesize for Cicendia quadrangularis a migration to

South America via the existing Panama land bridge,

between 3.5 and 2 MY. Alternatively, a dispersal event


from North to South America by long-distance migra-tion, via an intermediate series of appropriate ‘‘way

stations’’ such as Peru or Bolivia (Raven, 1963), remains

probable. Such a pattern has been recently documented

in the amphitropical disjunct genus Larrea (Lia et al.,

2001).

The transatlantic disjunct pattern described between

South African Chironia and New World C. cachanlah-

uen, has been encountered in only four other genera ofthe Gentianaceae, namely Enicostema, Neurotheca

[Potalieae], Schultesia [Chironieae], and Voyria. Several

hypotheses have been proposed, according to the rela-

tive age of the groups investigated, and sometimes

evocating possible Gondwanic vicariance processes or

boreotropic taxon interchange (Struwe et al., 2002). The

last connection between Africa and South America is

estimated to 105 MY, but many of the angiosperm lin-eages have radiated more recently in the Tertiary, and

do not extend back to the Cretaceous. Therefore, vi-

cariant species resulting from the break-up of the wes-

tern Gondwana cannot satisfactory explain these

disjunct angiosperm lineages. Moreover, both conti-

nents continued to be connected via a series of volcanic

islands until the end of the Eocene (ca. 38 MY), when

they were separated by 1400 km and fewer islands re-mained (Raven and Axelrod, 1974). Nevertheless, dis-

persal directly across the Atlantic Ocean (although

perhaps feasible in some cases) seems unlikely to be a

general explanation. An alternative migratory road has

been proposed for some groups, such as the Malpighi-

aceae (Davis et al., 2002). Members of this family have

migrated from South America into North America via

scattered continental or volcanic islands that connectedboth the continent at various times in the Tertiary.

From North America these lineages may have migrated

eastward to Laurasia via a series of connections across

the North Atlantic Land Bridge, subsequently diversi-

fying in Africa (Madagascar), and in some cases in

Eurasia. In addition, the Bering Land Bridge facilitated

the spread of northern hemisphere biota during periods

of climatic warming in the late Paleocene-early Eocene.This pattern is also encountered in Melastomataceae,

and in the opposite direction, in Annonaceae and

Lauraceae. Nonetheless, such explanations may not be

available for more recently disjunct groups such as

(Chironia+C. cachanlahuen).

To summarize, our work based on molecular and

morphological data sets strongly supports the mono-

phyly of the subtribe Chironiinae, with all genera exceptone (Zygostigma) investigated. These results also argue

for a polyphyletic genus Centaurium, split up into four

well-supported clades. From a systematic and biologist�spoint of view, species of Centaurium are best recognized

in four distinct genera, namely Centaurium (ca. 20 spp.),

Gyrandra (5 spp.), Schenkia (c. 5 spp.), and Zeltnera (c.

25 spp.). Intergeneric relationships revealed by molecu-

lar data significantly change the established classifica-tion of the subtribe and may have further important

evolutionary consequences. From a biogeographic point

of view, the Mediterranean origin of the Chironiinae is

clearly established in the light of the current investiga-

tion, but some unresolved patterns remain to be inves-

tigated. For instance, the transatlantic connection

between the southern African (Chironia+Orphium), and

the Chilean C. cachanlahuen, cannot be determinedwithout including the remaining South American species

belonging to the Chironiinae (e.g. Zygostigma australe

or Centaurium ameghinoi). Such incomplete sampling of

the subtribe may also explain the difficulty in finding

good morphological synapomorphies or other distinc-

tive traits such as chromosomal, palynological, or sec-

ondary chemistry features. A better comprehension of

the evolutionary trends within the subtribe Chironiinae,and the Gentianaceae as a whole, definitively requires

the phylogenetic resolutions of the four newly proposed

genera, along with the hitherto poorly investigated ones

(Chironia or Sabatia).

Acknowledgments

The authors gratefully thank Jason R. Grant for help

with grammar in the early stages of the manuscript,

Mike Thiv for providing DNA samples, Louis Zeltner

for sharing unpublished results, Kristin Mylecraine and

Cynthia Frasier for comments on an earlier version of

the manuscript, and Philippe K€upfer for general super-vision of G.M.�s PhD thesis. This study was financially

supported by the Fonds National Suisse de RechercheScientifique (Grant No. 31-52885.97). L.S. thanks Rut-

gers University for funding during the final stages of

manuscript preparation.

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