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Molecular Phylogenetics and Evolution 44 (2007) 89–103
www.elsevier.com/locate/ympev
co
p
y
A phylogeny of the “evil tribe” (Vernonieae: Compositae) reveals
Old/New World long distance dispersal: Support from separate
and combined congruent datasets (trnL-F, ndhF, ITS)
Sterling C. Keeley a,¤, Zac H. Forsman b, Raymund Chan a
a
b
Department of Botany, University of Hawaii at Manoa, 3190 Maile Way, Honolulu, HI 96822, USA
Department of Biology, University of Hawaii at Manoa, 2450 Campus Road, Honolulu, HI 96822, USA
al
Received 30 June 2006; revised 21 December 2006; accepted 28 December 2006
Available online 8 January 2007
on
Abstract
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The Vernonieae is one of the major tribes of the largest family of Xowering plants, the sunXower family (Compositae or Asteraceae),
with ca. 25,000 species. While the family’s basal members (the Barnadesioideae) are found in South America, the tribe Vernonieae originated in the area of southern Africa/Madagascar. Its sister tribe, the Liabeae, is New World, however. This is the only such New/Old
World sister tribe pairing anywhere in the family. The Vernonieae is now found on islands and continents worldwide and includes more
than 1500 taxa. The Vernonieae has been called the “evil tribe” because overlapping character states make taxonomic delimitations diYcult at all levels from the species to the subtribe for the majority of taxa. Juxtaposed with these diYcult-to-separate entities are monotypic
genera with highly distinctive morphologies and no obvious aYnities to any other members of the tribe. The taxonomic frustration generated by these contrary circumstances has resulted in a lack of any phylogeny for the tribe until now. A combined approach using DNA
sequence data from two chloroplast regions, the ndhF gene and the noncoding spacer trnL-F, and from the nuclear rDNA ITS region for
90 taxa from throughout the world was used to reconstruct the evolutionary history of the tribe. The data were analyzed separately and in
combination using maximum parsimony (MP), minimum evolution neighbor-joining (NJ), and Bayesian analysis, the latter producing
the best resolved and most strongly supported tree. In general, the phylogeny shows Old World taxa to be basal and New World taxa to
be derived, but this is not always the case. Old and New World species are found together in two separate and only distantly related
clades. This is best explained by long-distance dispersal with a minimum of two trans-oceanic exchanges. Meso/Central America has had
an important role in ancient dispersals between the Old and New World and more recent movements from South to North America in the
New World.
2007 Elsevier Inc. All rights reserved.
Au
1. Introduction
th
Keywords: Vernonieae; Vernonia; Compositae; Old and New World; Phylogeny; ITS; trnL-F; ndhF; Congruence; Mesoamerica: Central America
The Vernonieae is one of the most poorly understood of
the >20 recognized tribes of the Compositae (Funk et al.,
2005). A perplexing array of intergrading morphologies
and overlapping character states juxtaposed with highly
autapomorphic character state combinations (Keeley and
*
Corresponding author. Fax: +1 808 956 3923.
E-mail address: sterling@hawaii.edu (S.C. Keeley).
1055-7903/$ - see front matter 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.ympev.2006.12.024
Turner, 1990; Robinson, 1999a,b) make the Vernonieae
among the most refractory of tribes to elucidating systematic relationships of any members of the family. The frustration felt by those who have attempted to understand
relationships within the Vernonieae has given rise to its
nickname as the “evil tribe” (Funk et al., 2005). No phylogeny has ever been proposed for the tribe and only a few
relationships have been suggested even among the best
known species groups before the present study (Jones,
1977; JeVrey, 1988; Robinson, 1999a,b, 2007). Despite diYculties understanding relationships within the tribe, the
90
S.C. Keeley et al. / Molecular Phylogenetics and Evolution 44 (2007) 89–103
on
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suggesting two separate evolutionary lines within the tribe.
Following this tradition, Robinson (1999a,b, 2007) erected
separate subtribes for Old and New World Vernonieae in
the most complete taxonomic treatments of the tribe to
date.
Despite emphasis on the diVerences between the New
and Old World subtribal lineages, cross-hemisphere relationships have been proposed. Turner (1981) suggested a
possible connection to the Old World for the Central
American Leiboldia (Leboldiinae) group. Their morphologies, chromosome numbers and chemistry did not entirely
Wt with other New World species, but were similar to some
taxa in the Old World. Keeley and Turner (1990), in a cladistic analysis of morphological characters, and Keeley and
Jansen (1994), using cpDNA restriction site data, found
clades containing both New and Old World species suggesting a connection between the hemispheres. In his recent
treatments Robinson (1999a,b, 2007) also pointed out cases
where the New/Old World dichotomy did not seem to hold.
For example, the Old World genus Manyonia was postulated to be close to the New World genus Heterocypsela,
and conversely the New World genera Telmatophila, Acilepidopsis and Mesanthophora were placed in the Old World
subtribe Erlangeinae.
The overall goal of this study was to provide a phylogeny for the Vernonieae for the Wrst time and with it to
clarify New and Old World subtribal relationships.
Within this framework, additional goals were to gain a
better understanding of the potentially important role of
Meso/Central America in connecting the two hemispheres
(Keeley and Jansen, 1994), to ascertain the derived position of the North American taxa, and to further explore
the role of Brazil in the origin of New World taxa, as suggested by Keeley and Jones (1979). Three phylogenetic
markers (the chloroplast non-coding trnL-F and coding
ndhF) and the nuclear rDNA ITS region were chosen to
resolve relationships within the Vernonieae. We also
examined congruence and resolution of these three markers for the tribe worldwide.
Au
th
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monophyly of the Vernonieae has never been in doubt. The
overall circumscription of the tribe has changed little since
its initial description by Cassini (1819, 1828) and Bentham
(1873) and only minor changes have been made as a result
of recent molecular work (Keeley and Jansen, 1994; Kim
et al., 1998). The tribe has been traditionally placed in the
subfamily Cichorioideae, a position reconWrmed in a recent
study of the family (Funk et al., 2005).
The “Vernonia problem” (Bremer, 1994) is one of the
major reasons for the historical diYculties in establishing
relationships within the tribe. Until recently (Robinson,
1999a,b), the vast majority of species (>1000) was found
within a single worldwide core genus, Vernonia. Around
this enormous core genus swirled a cloud of largely monotypic genera with unusual and distinctive morphologies
that made it diYcult to relate these taxa to those with the
more common morphological ground plan of the tribe. For
example, Stokesia, a monotypic endemic of the southeastern US, is the only member of the tribe with zygomorphic
Xorets. Similarly, the monotypic Pacourina edulis from
northern South America has an unusual head morphology
and is the only truly aquatic member of the family, while
Hesperomannia from the Hawaiian Islands has become so
modiWed by adaptation to bird pollination that it was until
recently (Kim et al., 1998) thought to belong to the tribe
Mutisieae. Out of the 121 recognized genera in the Vernonieae 48 are monotypic and another 30 have only two species, leaving most species even now in only a few genera
(Robinson, 1999a,b, 2007). Studies by Robinson and Kahn
(1986) and Robinson and Funk (1987) pointed out the
paraphyly of Vernonia s.l., a Wnding supported by Keeley
and Jansen (1994) in a chloroplast DNA (cpDNA) restriction site study. Robinson (1999a,b, 2007) made sweeping
changes in the circumscription of Vernonia, limiting the
genus to a small group of eastern North American species
that includes the type species for the tribe (Vernonia noveboracensis (L.) Willd.). The remaining taxa were placed in
newly created genera which were in turn placed into one of
approximately 20 subtribes (Robinson, 2007). Few relationships were suggested among subtribes and genera, however,
leaving relationships among tribal members unresolved as
has been the case since the tribe’s original description.
One of the few distinctions generally noted within the
tribe has been that of two geographically separate lineages,
one in the Old World and the other in the New World. This
subtribal dichotomy, initially proposed by Gleason (1906),
was extended by Jones (1977) in an overview of the tribe,
followed by synoptic treatments of Vernonia in the New
World (Jones, 1979) and the Old World (Jones, 1981). The
Old World species of Vernonia were placed into the subgenus Orbisvestus and the New World species into subgenus
Vernonia. In a treatment of African species, JeVrey (1988)
noted features of morphology, chemistry and pollen that
also suggested separate lineages for New and Old World
species. Despite the inclusion of most species in the genus
Vernonia s.l., JeVrey (1988) proposed that the closest relationships were among taxa within each hemisphere, again
2. Materials and methods
2.1. Choice of taxa and regions to be sequenced
Vernonieae taxa from 90 species and 35 genera were
sampled across as wide a geographical range as possible in
the Old and New Worlds. These taxa are listed in Table 1
with GenBank Accession Numbers. Nomenclature is
according to Robinson (1999a,b, 2007). Revision of the Old
World Vernonieae has not yet been completed (Robinson,
pers. comm.), however. Consequently, a number of Old
World species have yet to be formally transferred from
Vernonia s.l. and thus must retain that genus name here
despite the fact that Vernonia s.s is entirely New World
(Robinson, 1999a).
The chloroplast gene ndhF has been used in a variety of
phylogenetic studies at several taxonomic levels (Olmstead
91
S.C. Keeley et al. / Molecular Phylogenetics and Evolution 44 (2007) 89–103
Table 1
Taxa sampled with species codes, geographic locations, and GenBank Accession Numbers
Genus (code)
Species
Author
Geographic
source
Collector, Number,
Herbarium
Genbank Accession Number
Albertinia
brasiliensis
Spreng.
Brazil
Baccharoides (A)
Baccharoides (L)
Bothriocline(L)
Bothriocline(U)
Brachythrix
Cabobanthus
Centauropsis
Centrapalus
Centratherum1
Centratherum2
Chresta
Chrysolaena(F)
adoensis
lasiopus
laxa
ugandensis
brevipapposa
polysphaera
decaryi
pauciXorus
punctatum
punctatum
sphaerocephala
Xexuosa
(Sch.Bip ex Walp.) H. Rob.
(O.HoVm.)H.Rob.
H.Wild & G. Pope
(S. Moore)M.G. Gilbert
H.Wild & G. Pope
(S. Moore) H. Rob.
Humbert
(Willd.)H. Rob.
Cass.
Cass.
DC.
(Sims)H.Rob.
Africa (cult.)
Africa
Rhodesia
Burundi
Tanzania
Zambia
Madagascar
Zimbabwe
Brazil (cult.)
Brazil
Brazil
Paraguay
Chrysolaena(P)
platensis
(Spreng.)H.Rob.
Paraguay
Critoniopsis(H)
Critoniopsis(So)
Cyanthillium
Distephanus(M)
Distephanus(P)
Eirmocephala
huairacajana
sodiroi
patulum
madagascariensis
polygalaefolia
brachiata
(Hieron.) H. Rob.
(Hieron. Ex Sodiro) H. Rob.
(Ait.) H.Rob.
Less.
(Less.) H.Rob.& B. Kahn
(Benth.)H.Rob.
Ecuador
Ecuador
China
Madagascar
Madagascar
Costa Rica
Elephantopus(C)
Elephantopus(E)
Elephantopus(M)
Elephantopus(M)
Elephantopus(N)
Elephantopus(T)
Eremanthus
Eremosis
carolinianus
elatus
mollis
mollis
nudatus
tomentosus
erythropappus
shannoni
Willd.
Gleason
Domin.
Domin
A.Gray
L.
(DC.) MacLeish
(Coult.) H.Rob.
Georgia
Georgia
Hawaii
Singapore
Georgia
Georgia
Brasil
Guatemala
Ethulia
Gorceixia
Gymnanthemum
(Am)
Gymnanthemum
(M)
Hesperomannia
(Arb)
Hesperomannia
(Arr)
Hesperomannia
(L)
Hesperomannia
(SN)
Heterocypsela
Hilliardiella(A)
Hilliardiella(L)
Hilliardiella(O)
Leiboldia
conyzoides
decumbens
amygdalinum
L.
Baker
Sch.Bip. Ex Walp.
Zaire
Brazil
Africa
Stannard, Ganev & Queiroz
51633-US
Kew 453-68-45801-K
Keeley cp-1 (930)-CONN
S. B. Jones 76-114 (Cult.)-G
Reekmans 8820-MO
Carter et al. 2527-K
Philcox et al. 10264-MO
J. L. Zarucchi et al. 7361-MO
Purdue USDA-US
Funk & Keeley 12,443-US
J. Panero-TEX
Azeviedo et al. 533-K
E. Zardini & R. Velasquez
25832-MO
E. Zardini & R. Velasquez
24552-MO
Keeley 4129-US
Keeley & Keeley s.n.-WHIT
Keeley cp-6 (1557)-CONN
P. Phillip 1905-MO
Kew 282-85-03275-K
W. Haber & W. Zuchowski
10536-MO
Urbatsch 6017-LSU
Urbatsch 6051-LSU
Funk& Keeley-US
Lum s.n.-Singapore
Urbatsch 6049-LSU
Coile s.n.-GA
de Moray 661-MO
S.C. Keeley & J. E. Keeley
3161-MO
Pauly 234-K
MGC 935-US
Kew 318-86-02802-K
(Less.) H.Rob.
GH-Africa
Jansen 995-MICH
EF155775 EF155687 EF155863
Hillebr.
Oahu,HI
Ching 11a-DNA library UH
EF155776 EF155688 EF155864
H. Gray
Oahu,HI
Ching A20-DNA library UH AY504696 AY504738 AY504778
Forbes
Kauai,HI
Ching K60-DNA library UH EF155777 EF155689 EF155865
HI
Ching H28-DNA library UH EF155778 EF155690 EF155866
andersonii
aristata
leopoldii
oligocephala
guerreroana
H.Rob.
(DC.) H.Rob.
(Vatke) H.Rob.
(FV.) H.Rob.
(S.B. Jones) H.Rob.
Brasil
South Africa
Ethiopia
South Africa
S. Mexico
Lepidaploa(A)
Lepidaploa(Ba)
Lepidaploa(Bo)
Lepidaploa(C)
Lepidaploa(E)
Lepidaploa(T)
Lepidonia
arborescens
balansae
borinquensis
canescens
ehretifolia
tortuosa
jonesii
(L.) H.Rob.
(Hieron.) H.Rob.
(Urb.) H.Rob.
(Kunth) H.Rob.
(Aristeg.) H. Rob.
(L.) H.Rob.
(B.L.Turner) H.Rob.& V.A.
Funk
Costa Rica
Paraguay
Costa Rica
Costa Rica
Venezuela
Costa Rica
Mexico
A. Salino 3043-US
Funk 12,410-US
Tadesse 7551-MO
Funk 12,429-US
Spooner & Burgos 2625TEX
Keeley 4085-LAM
R. Degen 1606-MO
Keeley 1550-LAM
Keeley s.n.-CONN
Keeley & Keeley 4443F-US
S. Keeley 3252-LAM
Todzia 2835-TEX
ITS
arbuscula
arborescens
sp. nov.
Au
y
EF155745
EF155796
EF155746
EF155747
EF155748
EF155749
EF155750
EF155751
EF155753
EF155754
EF155755
EF155756
co
p
al
on
rs
pe
th
lydgatei
or
's
mesipifolium
NDHF
TRNL-F
EF155744 EF155656 EF155832
EF155657
EF155708
EF155658
EF155659
EF155660
EF155661
EF155662
EF155663
EF155665
EF155666
EF155667
EF155668
EF155833
EF155884
EF155834
EF155835
EF155836
EF155837
EF155838
EF155839
EF155841
EF155842
EF155843
EF155844
EF155757 EF155669 EF155845
EF155821
EF155760
EF155812
EF155761
EF155762
EF155763
EF155733
EF155672
EF155724
EF155673
EF155674
EF155675
EF155909
EF155848
EF155900
EF155849
EF155850
EF155851
EF155764
EF155765
EF155766
EF155767
EF155768
EF155769
EF155770
EF155758
EF155676
EF155677
EF155678
EF155679
EF155680
EF155681
EF155682
EF155670
EF155852
EF155853
EF155854
EF155855
EF155856
EF155857
EF155858
EF155846
EF155772 EF155684 EF155860
EF155773 EF155685 EF155861
AY504695 AY504737 AY504777
EF155779
EF155780
EF155781
EF155782
EF155820
EF155691
EF155692
EF155693
EF155694
EF155732
EF155867
EF155868
EF155869
EF155870
EF155908
EF155783
EF155784
EF155785
EF155786
EF155829
EF155787
EF155788
EF155695
EF155696
EF155697
EF155698
EF155741
EF155699
EF155700
EF155871
EF155872
EF155873
EF155874
EF155917
EF155875
EF155876
(continued on next page)
92
S.C. Keeley et al. / Molecular Phylogenetics and Evolution 44 (2007) 89–103
Table 1 (continued)
Species
Author
Geographic
source
Lessingianthus
Linzia(Ac)
Linzia(G)
Linzia(M)
Linzia(G)
Linzia(M)
buddleiifolius
accommodata
gerberiformis
melleri
gerberiformis
melleri
Mart. Ex DC.
(Wild) H. Rob.
(Oliv. & Hiern.) H. Rob
(Oliv. & Hiern.) H. Rob
(Oliv. & Hiern.) H. Rob
(Oliv. & Hiern.) H.Rob.
Brasil
Zimbabwe
Rwanda
Burundi
Zimbabwe
Malawi
Munnozia
Muschleria
giganteum
sp.
Rusby
Orbivestus
Orbivestus
Parapolydora
Polydora
Sipolesia
Stokesia
Stokesia
Stramentopappus
cinarescens
cinerascens
fastigiata
poskeana
lanuginosa
laevis
laevis
pooleae
Strobocalyx
Tephrothamnus
Vernonanthura (A)
Vernonanthura(B)
Vernonanthura(P)
Vernonia(Ab)
arborea
paradoxa
alamanii
brasiliana
patens
abyssinica
(Sch.Bip.) H. Rob
(Sch.Bip.) H. Rob
(Oliv. & Hiern.) H. Rob
(Vatke & Hillebr.) H. Rob.
Glaz. Ex Oliv.
Greene
Greene
(B.L.Turner) H.Rob.& V.A.
Funk
(Buch.-Ham.) H.Rob.
(Sch.Bip.) H. Rob.
DC.
(L.) H.Rob.
(H.B.K. )H.Rob.
Sch.Bip. Ex Walp.
Vernonia(Al)
Vernonia(An)
Vernonia(Ba)
altissima
angustifolia
baldwinii
Gleason
Michx.
Torr.
Vernonia(Br)
Vernonia(Bu)
brachycalyx
bullata
O.HoVm.
Benth.ex Oerst.
Vernonia(C)
capensis
(Houtt.) Druce
Vernonia(E)
Vernonia(Fc)
elliptica
fasciculata
DC.
Michx.
Vernonia(Fc)
fasciculata
Vernonia(Gi)
gigantea
Vernonia(Hu)
Vernonia(L)
Vernonia(L)
Vernonia(M)
humbloti
lindheimerii
lindheimerii
missurica
Vernonia(N)
noveboracensis
Vernonia(Sp)
Vernonia(SpS)
Vernonia(Su)
Vernonia(T)
Vernoniastrum
th
Drake
A.Gray & Englem.
A.Gray & Englem.
RaWn.
profuga
(L.) Michx.
L.
sp
sp
subplumosa
texana
O.HoVm.
(A.Gray) Small
nestor
(S.Moore) H.Rob.
Genbank Accession Number
ITS
EF155789
EF177478
EF155791
EF155792
EF155752
EF155790
rs
on
al
co
p
R. Anderson 9706-MO
Bayliss 10387-MO
Auquier 3263-MO
Reekmans 10202-MO
Keeley cp-4 (31)-CONN
Christenson & Liperde 1491US
Peru
Dillon s.n.-F
Tanganika
Milne-Redhead & Taylor
9039-K
South Africa
Koekemoer 232-MO
South Africa
M. Koekemoer 232-PRE
South Africa
Koekemoer 225-MO
South Africa
Koekemoer 233-MO
Brasil
Harley et al. 24885-K
SE .US
Kew 068-62-06802 –K (cult.)
SE.USA
Kew 068-62-06802 –K (cult.)
S. Mexico
WoodruV & Todzia
199-TEX
Singapore
Lum s.n.-Singapore
Venezuela
S. Keeley 4500-6-CONN
Texas
Todzia 2869-TEX
Brazil
unknown-MO
Panama
S. Keeley 4685-LAM
Ethiopia
Gilbert, Sebsebe, Vollensen
7408-MO
E.N. America
Keeley cp-2-CONN
South Carolina Coile s.n.-GA
E. North
Kew 643-52-64320-K (cult.)
America
Uganda
Lock 88/16-K
Zambia
Philcox, Pope, Chisumpa,
Ngoma 10265-MO
South Africa
L. E. Watson & J. Panero
94-120-TEX
SE Asia (cult.) Funk & Keeley 12442-US
E North
U. Posnaniensis, Poland,
America (cult.) 573 (cult.)
E North
Kew 590-53-59006-K
America (cult.)
E. North
Kew 611-66-61103-K
America (cult.)
Madagascar
H.J. Beentje 4823-K
Texas
Lievens 4100-TEX
Texas
J. Kim 10573-TEX
E. North
Urbatsch 5870-LSU
America (cult.)
E. North
Kew 000-69-18590-K
America (cult.)
E. North
Keeley cp-5 (1561)-CONN
America (cult.)
Brazil
unknown-US
S. Africa
M. Koekemoer 1973-US
Zambia
Philcox et al. 10296-MO
E. North
Urbatsch 5889-LSU
America
Malawi
Banda, Mwyanyambo
3869-MO
pe
or
's
(Walt.) Trel.
Au
Vernonia(P)
Michx.
Collector, Number,
Herbarium
and Sweere, 1994; Olmstead et al., 2000; Pfeil et al., 2002)
including several in the Asteraceae (Kim and Jansen, 1995;
Kim et al., 1998, 2002) and the Vernonieae (Kim et al.,
NDHF
EF155701
EF177479
EF155703
EF155704
EF155664
EF155702
y
Genus (code)
TRNL-F
EF155877
EF177480
EF155879
EF155880
EF155840
EF155878
AY504697 AY504739 AY504779
EF155793 EF155705 EF155881
EF155795
EF155794
EF155817
EF155797
EF155798
EF155799
EF155800
EF155801
EF155707
EF155706
EF155729
EF155709
EF155710
EF155711
EF155712
EF155713
EF155883
EF155882
EF155905
EF155885
EF155886
EF155887
EF155888
EF155889
EF155774
EF155759
EF155802
EF155827
EF155803
EF155805
EF155686
EF155671
EF155714
EF155739
EF155715
EF155717
EF155862
EF155847
EF155890
EF155915
EF155891
EF155893
EF155806 EF155718 EF155894
EF155807 EF155719 EF155895
EF155808 EF155720 EF155896
EF155809 EF155721 EF155897
EF155810 EF155722 EF155898
EF155811 EF155723 EF155899
EF155813 EF155725 EF155901
EF155815 EF155727 EF155903
EF155816 EF155728 EF155904
EF155818 EF155730 EF155906
EF155819
EF155822
EF155823
EF155824
EF155731
EF155734
EF155735
EF155736
EF155907
EF155910
EF155911
EF155912
EF155825 EF155737 EF155913
EF155826 EF155738 EF155914
EF155814
EF155828
EF155830
EF155831
EF155726
EF155740
EF155742
EF155743
EF155902
EF155916
EF155918
EF155919
EF155804 EF155716 EF155892
1998). Following the work of Kim et al. (1998, 2002) only
the 3⬘ half of the gene was used due to the lack of variation
in the 5⬘end for the Vernonieae. The non-coding trnL-F
93
S.C. Keeley et al. / Molecular Phylogenetics and Evolution 44 (2007) 89–103
Sequence (5⬘–3⬘)
ITS5A
ITS4
trnL C
trnL F
ndhF 1603
ndhF +607
GGA AGG AGA AGT CGT AAC AAG G
TCC TCC GCT TAT TGA TAT GC
CGA AAT CGG TAG ACG CTA CG
ATT TGA ACT GGT GAC ACG AG
CCT YAT GAA TCG GAC AAT ACT ATG C
ACC AAG TTC AAT GYT AGC GAG ATT AGT C
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ence (ILD) test (Farris et al., 1994), implemented as partition homogeneity in PAUP*4.01b.10 (SwoVord, 2002), was
performed. The settings used the nearest neighbor interchange (NNI) method of branch swapping with 100 replications, random addition for 3 replicates, nchuck D 2,
chuckscore D 1; scores are indicated in Table 2. Initial tests
were also performed to determine the eVects of gaps; gaps
excluded, treated as missing data or as a Wfth character.
Treating gaps as a Wfth character resulted in signiWcant heterogeneity, but excluding gaps or treating them as a missing
character resulted in no signiWcant heterogeneity (Table 3).
Including gaps as a missing character resulted in a betterresolved topology without signiWcant topological diVerences, therefore gaps were treated as missing data. Missing
data (no sequence) occurred very rarely for the tailing end
of some sequences and were coded as ambiguous data (N).
Maximum parsimony (MP), neighbor joining (NJ) and
Bayesian methods were used to estimate phylogenies for
each separate data partition, and for all partitions
combined. Maximum parsimony trees were generated in
PAUP*4.01b.10 (SwoVord, 2002) with the following conditions; gaps were treated as missing data in a full heuristic
search with the tree-bisection-and-reconnection (TBR)
branch swapping algorithm, with 10 random additions, and
5 trees held each step, with 1000 non-parametric bootstrap
pseudo-replicates. NJ trees were also estimated in PAUP*
with 1000 non-parametric bootstrap pseudo-replicates,
using the Minimum Evolution criteria. Evolutionary models were selected using the AIC criterion (Akaike Information Criterion, Akaike, 1974) in ModelTest (Posada and
Crandall, 1998). Models for separate and combined data
rs
2.2. Sequence alignment and phylogenetic analysis
Table 3
Primer sequences used for PCR and cycle sequencing
on
including the spacer region also has been used in numerous
phylogenetic studies and for a wide range of plants (for a
summary see Shaw et al., 2005). This region provides varying degrees of resolution, depending on the group. Shaw
et al. (2005) found that even when trnL-F alone was not
suYcient to provide a resolved phylogeny it was eVective
when used in combination with other gene regions. Bayer
and Starr (1998) and Bayer et al. (2000) also found trnL-F
useful at the tribal level in the Asteraceae.
The ITS has been successfully used in studies of the
Asteraceae at the species, tribal and family level (e.g. Baldwin, 1992; Marcos and Baldwin, 2001; Chan et al., 2001;
Baldwin et al., 2002; Roberts and Urbatsch, 2003, 2004).
Work by Kim and Jansen (1995) and Kim et al. (1998,
2002) have shown ITS to resolve relationships at the tribal
and generic levels within the family and within the Vernonieae. These data were also combinable with data
obtained from the ndhF chloroplast region (Kim et al.,
1998). In analyses of two tribes within Asteraceae subfamily Cichorideae, the same subfamily as the Vernonieae
(Panero and Funk, 2002; Funk et al., 2005) Samuel et al.
(2003) and Funk et al. (2004) also found that ITS provided
good resolution.
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To avoid possible problems with ITS alignments
(Álvarez and Wendel, 2003; Goertzen et al., 2003) NJ trees
(implemented in PAUP* with an HKY85 substitution
model for computational speed) were evaluated for three
alternative alignments; one including all data, and two that
excluded columns containing lower than 50% or 20% posterior probability scores generated in ProAlign 0.5a
(Loytynoja and Milinkovitch, 2003). Including all positions
resulted in higher resolution but no signiWcant change in
topology, therefore all positions were included in the Wnal
analyses. The results of these preliminary studies indicated
that the ITS, trnL-F and ndhF would provide good phylogenetic signal for resolving clades within the Vernonieae.
To determine combinability the incongruence length diVer-
ITS-1
5.8S
ITS-2
trnL
trnL-sp
ndhF
ndhF-sp
NUC
ORG
310
234
138
»
0.03
0.25
0.74
0.39
0.33
0.01
»
»
159
17
17
0.01
»
0.53
0.01
0.19
0.01
0.19
»
»
236
193
112
0.34
0.06
»
0.91
0.83
0.81
0.01
»
»
549
23
13
0.55
0.23
0.98
»
0.44
0.39
0.26
»
»
463
47
31
0.28
0.48
0.94
0.81
»
0.19
0.06
»
»
601
91
85
0.15
0.01
0.68
0.63
0.14
»
0.01
»
»
181
58
0
0.99
0.96
1
0.92
1
1
»
»
»
705
444
267
»
»
»
»
»
»
1794
219
129
»
»
»
»
»
»
»
»
»
Au
Size (bp)
PIC gaps D missing
PIC gaps D excluded
ITS-1
5.8S
ITS-2
trnL
trnL-sp
ndhF
ndhF-sp
NUC
ORG
th
Table 2
The size of each data partition and the number of parsimony informative characters (PIC) with gaps treated as missing and gaps excluded for each data
partition are given above the double line
»
0.16
ILD scores are given below the double line with alignment gaps excluded (upper matrix) and alignment gaps treated as missing data (lower matrix). Abbreviations: sp D spacer; NUC D nuclear data partition; ORG, organellar data partition.
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S.C. Keeley et al. / Molecular Phylogenetics and Evolution 44 (2007) 89–103
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The ampliWcation reactions were conducted in a GeneAmp PCR System 9700 (Perkin-Elmer). The PCR proWle
consisted of an initial preheating at 94 °C for 2 min followed by 40 cycles of: 1 min at 94 °C, followed by 1 min at
48 °C (54 °C for cpDNA) and 45 s at 72 °C. Primer extension time was increased by 4 s (7 s for cpDNA) for each subsequent reaction cycle. An additional 7 min extension at
72 °C was added for completion of unWnished DNA
strands. All PCR products were quantiWed by agarose gel
electrophoresis with comparison of an aliquot of products
with a known quantity of a 100-bp DNA ladder (GeneChoice). The remainder was stored at 4 °C until utilized.
PCR products were puriWed for sequencing using an
enzymatic PCR product pre-sequencing kit (USB) following recommendations from the manufacturer. This method
of puriWcation without loss of PCR products (no Wltration,
precipitation, or washes are necessary) is especially important for DNA extracted from herbarium vouchers, which is
sometimes only weakly ampliWed and yields barely suYcient PCR product for sequencing. The cycle sequencing
reactions were done using 96-well microplates in a PTC-100
thermal cycler (MJ Research). Each one-eighth cycle
sequencing reaction cocktail contains 50–150 ng of the
puriWed PCR product, 2 l of a 1-mM concentration of the
sequencing primer, 0.6 l of a 5£ reaction buVer (400 mM
Tris HCl, 10 mM magnesium chloride at pH 9.0), and 1 l
of the reagent pre-mix from the BigDye (Version 2/3) dye
terminator cycle sequencing pre-mix kit (Applied Biosystems). The cycle sequencing program consisted of an initial
preheating at 96 °C for 30 s followed by 25 cycles of: 10 s at
92 °C, followed by 15 s at 55 °C and 4 min at 60 °C. Unincorporated dye terminators were removed by Sephadex
(Sigma) gel Wltration using MultiScreen plates (Millipore).
The puriWed cycle sequencing products were then resolved
by electrophoresis on a 5% polyacrylamide (MJ Research
KiloBasePack) gel using a BaseStation 51 automated DNA
sequencer (MJ Research). Sequences from both strands of
each PCR product were examined, compared, and corrected using Sequence Navigator software (Applied Biosystems). Sequence alignments were generated by ProAlign
0.5a0, and adjusted manually (Loytynoja and Milinkovitch,
2003).
2.3. Extraction and ampliWcation
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sets were: ITS, GTR + I + G; trnL, TVM + G; ndhF,
TVM + I + G; combined data set, TIM + I + G ( D 0.5809,
pinv D 0.5689). Bayesian analysis was performed in MrBayes 3.0b4 (Hulsenbeck and Ronquist, 2001) using the
Markov Chain Monte Carlo analyses (Geyer, 1991), using
four chains sampled every 100 generations. The Wrst 2000
trees were discarded as the burn-in period, this value was
determined empirically from plotting the likelihood values
to determine convergence, and each analysis was run for
one million generations. All trees saved from the independent runs (excluding burn-in) were used to construct 50%
majority-rule consensus trees. Trees were drawn with
PAUP*4.01b.10 (SwoVord, 2002) or with the program
MEGA 3.0 (Kumar et al., 2004).
Outgroup taxa in initial analyses included Munnozia of
the sister tribe, Liabeae, (sequences included in Table 1 for
reference) as well as the yellow-Xowered Madagascan species of the genus Distephanus. The latter taxon was previously shown to belong to the basal group within the
Vernonieae (Keeley and Jansen, 1994). As no diVerence in
topologies or clade composition resulted from the use of
Distephanus alone it was used as the outgroup in the Wnal
analyses for economy of computation. Nucleotide divergence rates were assumed for ndhF and ITS based on estimates provided by Kim et al. (1998). Mean nucleotide
divergence between groups was calculated using the program MEGA 3.0 (Kumar et al., 2004) with 1000 bootstrap
replicates to estimate the standard error and 95% conWdence intervals.
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DNA extractions were performed on herbarium or silica
dried material using Qiagen DNeasy. Plant Mini Kits following the instructions supplied but with an extended incubation period (up to 40 min) for herbarium material. When
necessary, an additional clean up and concentration step
was done using an UltraClean 15 DNA PuriWcation Kit
(Mo Bio Laboratories). Primer ITS5A (Downie and KatzDownie, 1996), based on White et al.’s (1990) fungal primer
ITS5 and corrected at two positions for angiosperms, was
substituted for ITS5 in this study. Primers used to amplify
and sequence the trnL region of cpDNA were designed by
Taberlet et al. (1991) and those used for the 3⬘ end of the
ndhF region were designed by Jansen (1992). All primer
sequences are given in Table 3.
For the PCR ampliWcation reactions, each 25 l PCR
reaction cocktail contained 12.9 l of sterile water, 2.5 l of
10£ PCR reaction buVer A or B (Promega), 2 l of 20 mM
dNTPs (Pharmacia) in an equimolar ratio, 2.5 l of 25-mM
magnesium chloride, 0.5 l of 10 mg/ml Bovine Serum
Albumin (Sigma), 1 l of a 10 M concentration of the forward primer, 1 l of a 10 M concentration of the reverse
primer, 0.1 l of Taq DNA polymerase enzyme (5 U/l from
Promega), and 2.5 l of template DNA. The amount of template DNA was adjusted when necessary to generate suYcient PCR products for DNA sequencing.
3. Results
3.1. Relationship of New and Old World Vernonieae
New and Old World Vernonieae are not entirely distinct.
Taxa from both hemispheres are found together in two
widely separated clades (clade B⬘ and clade 3, Figs. 2 and 3).
Outside of these clades, however, New World and Old
World taxa are in separate monophyletic lineages.
The most signiWcant bi-hemispheric clade includes taxa
from Mesoamerica and southeast Asia (clade B⬘, Figs. 2
and 3). This is not the Wrst time that Mesoamerican taxa
have been found together with Old World species. Keeley
and Jansen (1994) found members of Vernonia subtribe
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S.C. Keeley et al. / Molecular Phylogenetics and Evolution 44 (2007) 89–103
cies apparently arose from a New World Andean-based
lineage, (using the species available here). Once the elephantopoid ancestral line formed in the New World there was
dispersal to the Old World along with continued radiation
within the New World.
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3.2. Individual gene regions
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No single gene region alone was suYcient to provide a
fully resolved phylogeny. Six clades, labeled 1–6, were
strongly supported in all individual gene trees (Fig. 1). Of
the four other clades, labeled A-D, all four were found in
only in the ITS tree, with three and one clades found in the
ndhF and trnL-F trees, respectively. All 10 clades were
present in trees from the combined data (Figs. 2 and 3). The
consistency of these clades suggests that the phylogenetic
signal is similar in both nuclear and organellar DNA (e.g.,
Chen et al., 2003). Taxa in recurring clades are fully
enumerated in Figs. 2 and 3, and are summarized in Fig. 1
to save space. Bayesian trees consistently had higher resolution and support values for each data partition (Fig. 1) and
for the combined analyses (Fig. 2) than MP (Fig. 3) and NJ
trees. Only Bayesian results are shown for the individual
regions.
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Leiboldiinae (Lepidonia jonesii and Stramenopappus pooleae), plus the North American monotypic genus Stokesia,
at the intersection of the Old and New World lineages, as
they are here (Fig. 2). In Keeley and Jansen (1994) these
three New World taxa were derived above a group of Old
World species (clades 5 and 6 here) and together formed the
base from which all other New World species arose. Similarly, in Kim et al. (1998) Lepidonia jonesii and Stokesia
were placed between Old and New World species in trees
constructed using ndhF and ITS sequence data.
The aYnities of the Leiboldiinae to both New and Old
World taxa is also shown by the the somewhat labile aYnities of the individual taxa of this group (clade B⬘). The New
World Leiboldiinae and Stokesia, and the Old World taxa
Strobocalyx arborea and Vernonia elliptica, are the only
taxa to switch positions between New and Old World polytomies in the individual gene trees (Fig. 1). Clade B⬘ is also
the only one to change position in the combined analyses
(Fig. 2 versus Fig. 3). Regardless of the position of this
clade, the taxa are more closely related to each other than
they are to other species in their respective hemispheres.
A diVerent connection between New and Old World
taxa is seen in the monophyletic genus Elephantopus (clade
3, Figs. 2 and 3). In this case both Old and New World spe-
Fig. 1. Phylograms generated by Bayesian analyses for ITS, ndhF and trnL-F regions. Posterior probability scores higher than 70 are indicated by an asterisk. Analyses were run for 1 million generations, log-likelihood scores: ITS ¡14495.97, ndhF ¡2623.95, trnL-F ¡3057.80 (see Section 2 for individual data
set models). Numbered and lettered brackets indicate recurring clades. Darkened triangles are collapsed clades; a full listing of taxa in these clades is given
in Fig. 2. Asterisks next to the collapsed clades indicate the number of groupings within the clade with posterior probability scores higher than 70.
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S.C. Keeley et al. / Molecular Phylogenetics and Evolution 44 (2007) 89–103
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Fig. 2. Bayesian analysis of the combined data set (ITS, ndhF, trnL-F) run for 1 million generations, with model TIM + I + G, log-likelihood score
¡20656.5. Posterior probability values are given above the line, bootstrap values (1000 replicates) from a majority rule consensus NJ tree (minimum
evolution) constructed using the same model are given below the line.
3.2.1. ITS
The ITS tree was the most resolved and best supported
of the individual trees (Fig. 1). Overall the tree was divided
into two general sections, one New World (clades 1, 2, 3, A,
B) and the other Old World (clades C, D, 4, 5, 6). While no
New World taxa were found in the Old World portion,
some Old World taxa were found intermixed in the primarily New World portion of the tree. Two geographically
anomalous species, Vernonia elliptica and Strobocalyx
arborea from southeast Asia were found among the New
World species, and clade 3 consisting of the mostly North
American species in the genus Elephantopus, also included
one widespread Old World taxon. E. mollis.
Several distinct clades (clades 1, A, B) contained taxa
that occupy a similar geographic region within the New
World. Clade 1, for example, was primarily composed of
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S.C. Keeley et al. / Molecular Phylogenetics and Evolution 44 (2007) 89–103
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Fig. 3. Majority rule consensus tree of 1000 bootstrap pseudoreplicates. Bold lines indicate the topology of the strict consensus of 672 most parsimonious
trees for the combined data set (ITS, ndhF, trnL-F). Bootstrap values are provided above the nodes, values below 50% are not shown. Tree length 3127,
CI D 0.395, RI D 0.783.
North American Vernonia species (Robinson, 1999a), with
a small number of South and Central American taxa (see
Fig. 2 for the expanded clade). Clade A was composed of
Brazilian taxa and clade B of Central American species.
The majority of Old World taxa were found in a large polytomy below the New World groups. This polytomy
included clades C and D from south and east Africa and a
group formed from clade 4 (South Africa) paired with
Centauropsis (Madagascar). Clades 5 and 6 formed a sister
group to the other Old World clades. Clade 5 included taxa
from Africa, Madagascar and Hawaii, supporting the
relationship of Hawaiian and African species reported in
Kim et al. (1998). Clade 6 included east African species in
the genus Linzia, recently described by Robinson (1999b).
3.2.2. ndhF
The ndhF region included nine of the 10 clades present
in the ITS tree (Fig. 1). New World clades 1, 2, 3 and A were
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S.C. Keeley et al. / Molecular Phylogenetics and Evolution 44 (2007) 89–103
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3.2.3. trnL-F
The trnL-F region had the fewest informative characters
of the three regions (Table 2) and correspondingly produced the least resolved tree (Fig. 1). There was no statistical support for a distinction between Old and New World
taxa, other than for the basalmost clades 5 and 6. Smaller
clades identiWed in the other trees were present, i.e., clades 1,
2, 3, 4 and B, but their relationship to other taxa was unresolved.
tive estimates of clade support compared to the Bayesian
posterior probability values. Posterior probability and
bootstrap values are both provided, as they may provide
estimates of upper and lower limits of statistical support.
As in the individual analyses the overall pattern was that
of basal groups consisting of primarily Old World taxa and a
derived group containing most New World taxa. All 10
clades were present and an additional one was recognized
(clade B⬘, Figs. 2 and 3). The formation of this clade provided
resolution for the somewhat ambiguous positions of the New
World Stokesia and clade B taxa (subtribe Leiboldiinae taxa,
Leiboldia, Lepidonia,Stramentopappus) and the Old World
pair Strobocalyx/Vernonia elliptica. The taxa in clade B⬘ are
most closely related to each other rather than to other genera
in their respective geographical areas. Clade 3, with both Old
and New World Elephantopus species, remained with New
World taxa as in individual analysis. The major topological
diVerence between MP and Bayesian analyses was in the
position of clade B⬘ versus the grade including clades 2 and 3
at the intersection of the large Old World group and the predominately New World group (Figs. 2 and 3). There was no
diVerence in relationships of the Old World clades to each
other in any of the analyses. The Old World basal portion of
the tree consisted of sister clades 5 and 6 and a large group
including clades C, D, and clade 4 (plus Centauropsis) (Figs. 2
and 3). Bootstrap support and posterior probability values
were similar for these groups as well, underscoring the stability of the clades in this portion of the tree.
on
found in the New World polytomy, as they were in the ITS
tree, but their relationships were slightly diVerent. Clade A
(Brazilian) and clade 1 (primarily North American) were
derived from the same branch with Tephrothamnus
(Venezuelan) and Eirmocephala (Central American) at the
base of these clades. This changed the relationships
between these clades from independent lines, as was the
case for ITS, to ancestral and derived positions. Chresta and
Heterocypsela (both Brazilian) were on separate branches
in the polytomy, again suggesting a more distant relationship to taxa in clade A, similar to the Wndings for ITS.
Clade 3 was composed of New and Old World Vernonieae
and remained with the New World taxa.
The major diVerence between the ndhF and ITS trees
was the position of Central American taxa of clade B in the
subtribe Leiboldiinae (Leiboldia, Lepidonia, Stramentopappus) and the North American Stokesia in the otherwise Old
World polytomy that included clades 4, C and D. Additionally, Strobocalyx arborea and Vernonia elliptica, found with
New World species in the ITS tree, were this time placed
with other Old World taxa. Old World clades 5 and 6,
remained in the same basal position as in the ITS tree.
3.3. Combined analyses
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Results of ILD tests (partition homogeneity in PAUP*)
showed that the partitions were congruent except for the
ndhF spacer region, and the 5.8S ribosomal gene (Table 3).
The ndhF spacer could not be reliably aligned and was
excluded from further analyses. The 5.8S ribosomal gene
contributed a small number of informative characters
(Table 2), and excluding it did not alter the tree topology
(data not shown). It was left in the Wnal analysis in an eVort
to obtain as much phylogenetic information as possible.
Combined analyses using Bayesian (Fig. 2), NJ (not shown)
and MP (Fig. 3) methods produced similar topologies. The
Bayesian and NJ methods produced trees with higher resolution and statistical support than MP, which only uses a
subset of the data (parsimony informative characters).
Bayesian posterior probability values were considerably
higher than NJ (values given in Fig. 2) or MP bootstrap
values (Fig. 3). There was no conXict between the Bayesian
analysis and clades supported by the other methods. The
interpretation of Bayesian posterior probability values is
controversial, and may overestimate statistical support (e.g.
Simmons et al., 2004; Suzuki et al., 2002). We consider the
NJ and MP bootstrap values as potentially more conserva-
4. Discussion
Turner (1981) was the Wrst to suggest a connection
between Old and New World Vernonieae involving Leiboldiinae taxa. Based on morphology Turner (1981, p. 403)
said that section Lepidonia (now a genus within the Leiboldiinae Robinson (1999a)) “ƒ is as closely related to some
of the Old World sections (e.g. Cyanopsis) as to those of the
New World sections.” He also noted that the chromosome
number, n D 19 for Stramentopappus pooleae supported an
African relationship. Old World Vernonieae typically have
a chromosome number of n D 9 or 10 while New World
Vernonieae are typically n D 17 (Jones, 1977; Turner, 1981).
Chemical data were somewhat equivocal, however. Gershenzon et al. (1984) found the Old World type of nonglaucolide germacranolides in Lepidonia jonesii while the
New World type of glaucolide was found in Stramentopappus pooleae. Robinson and Funk (1987) disagreed with
Turner (1981) based on a morphological cladistic analysis
saying the Leiboldiinae were autochthonous New World
elements not related to Old World taxa. If Turner’s reasoning is followed then Stokesia, found with the Leiboldiinae
taxa here as well as in these other studies, may similarly
have ties to the Old World. With a chromosome number of
n D 7 Stokesia is anomalous among New World taxa. A
count of n D 7 is otherwise known only from the Madagascan species, Vernonia appendiculata (Rabakonandrianina
and Carr, 1987). It is also tempting to consider
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S.C. Keeley et al. / Molecular Phylogenetics and Evolution 44 (2007) 89–103
n D 7 + n D 10 as a possible source of the n D 17 common in
New World Vernonieae today.
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Zavada and de Villiers (2000) reported the earliest
unequivocal record of the Compositae (Asteraceae) at ca.
60 MYA from Paleocene-Eocene sediments of South
Africa. This new date nearly doubles the most widely
accepted earlier estimates of 30–40 MYA (Raven and Axelrod, 1974) and 42–47 MYA recently proposed by Kim
(2005). The family is undoubtedly Gondwanan (Bremer
and Gustafsson, 1997; Zavada and de Villiers, 2000) and
South American in origin (Jansen and Palmer, 1987;
Bremer, 1994). However, several near-basal tribes, for
example the Vernonieae, Arctoteae and Cichorieae in subfamily Cichorioideae, are Old World in origin. Given the
relatively young age of the family with respect to continental separations (90–120 MYA (Scotese, 2002)) and its
worldwide distribution, long distance dispersal must have
played an important role in creating current distribution
patterns.
For the Vernonieae long distance dispersal is likely to
have been especially important. The Vernonieae originated
in the region of southern Africa/Madagascar. The sister
tribe to the Vernonieae, the Liabeae (Keeley and Jansen,
1994; Robinson, 1999a), is entirely New World. This is the
only such New/Old World sister relationship found among
tribes anywhere within the Compositae (Funk et al., 2005).
Vernonieae taxa are now found from Argentina to Canada
in the New World and throughout Africa, south and southeast Asia, and Australia in the Old World, and on island
chains in both hemispheres. Using the rates of 0.0007 nucleotide changes/MY for ndhF and 0.0078 nucleotide changes/
MY for ITS established by Kim et al. (1998) for the Vernonieae, we estimate a date of ca. 14–20 MY for the major
radiation of Old to New World for the Vernonieae (ITS
18.9 § 1.4 MY, ndhF 17.1 § 4.2 MY), values that are in
agreement with those of Tremetsberger et al. (2005) for the
age of the tribe. South America and Africa were already in
their current positions by this time (Scotese, 2002).
Long distance dispersal has been shown for several
tribes in the Compositae. Undoubtedly the best-documented case is that of the Hawaiian silversword alliance
(tribe Heliantheae) which originated 5–7 MYA from a Californian ancestor (Baldwin, 1992; Baldwin and Sanderson,
1998; Baldwin, 2003). In addition to the original colonization from the mainland, there has been spectacular adaptive radiation (3 genera, 27 species) involving multiple
inter-island dispersals (Carlquist et al., 2003). The Hawaiian islands are volcanic, originating from an undersea hot
spot and have never been connected to land (Clague and
Dalrymple, 1987). They are also among the most distant
land masses in the world today (ca. 4000 km from the edge
of western North America the nearest land). Other recently
reported examples of long distance dispersal in the Compositae include that of Hypochaeris (tribe Cichorieae) from
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4.1. Long distance dispersal and the role of Mesoamerica
Africa to South America (Tremetsberger et al., 2005) and
Senecio (tribe Senecionieae) from Mediterranean Europe to
western North America (Coleman et al., 2003).
The Hawaiian endemic Hesperomannia (Vernonieae),
like the silverswords, came from a distant mainland ancestor, in this case from east Africa/Madagascar (Kim et al.,
1998) and this paper (Figs. 2 and 3), a distance of almost
12000 km. Since initial colonization there has also been dispersal between the islands. Additionally, there are >30
endemic Vernonieae in the West Indies (Keeley, 1978). The
Compositae is not the only group where long distance dispersal is important. There are numerous other cases of long
distance dispersal for both plant and animal groups whose
young age precludes vicariance as an explanation for present day distributions. (e.g., Melastomataceae, Renner et al.,
2001; Renner, 2004a; and other trans-Atlantic disjunct
plant genera, Renner, 2004b; monkeys, Xightless insects,
frogs, baobabs etc. summarized in de Queiroz, 2005; land
snails, Cowie and Holland, 2006).
Meso/Central America is an important region connecting Old and New World taxa in the Vernonieae (Figs. 2 and
3). Over the past 15–16 MY a chain of volcanic islands
formed proto-Central America. When the islands joined
together to form a solid landmass and the Panamanian
isthmus closed 3–3.5 MYA (Keigwin, 1982; Coates et al.,
2003; Morley, 2003; Sanmartin and Ronquist, 2004) a landbased corridor for dispersal became established between
North and South America. Dispersal overland once North
and South America were connected is well known (e.g., the
Great American Exchange (Marshall et al., 1979; Morley,
2003)). Not all species would have moved at the same rate
or necessarily in the same direction, however, and some
may not have moved at all. Changes in climate and Xuctuations in sea level likely created disjunct populations especially in some of the higher elevation areas of Mesoamerica
(particularly in southern Mexico and northern Guatemala).
In turn, these isolated areas may have become refugia.
Rzedowski (1993) pointed out that many of the lineages
now found in the Neotropics might have arrived in Mexico
and the Neotropics from other parts of the world, with
some subsequently becoming extinct in important parts of
their original area of distribution. Taxa whose histories
show this pattern include, for example, northern
hemisphere Liriodendron, Nyssa, and Tilia and southern
hemisphere Ayenia, Coccoloba and Enterolobium (Graham,
1993; Rzedowski, 1993).
Not all relicts leave clear fossils, but their past presence
in an area can be reasonably inferred on the basis of their
current distributions and phylogenetic relationships. The
Mesoamerican Vernonieae of subtribe Leiboldiinae (clade
B⬘, Figs. 2 and 3) are rare and restricted to small, widely
separated populations in cloud forests and on wet mountain slopes of volcanoes from 800 to 2400 m (in southern
Mexico above the Isthmus of Tehuantepec (Oaxaca, Guererro) and northern Guatemala (Alta Vera Paz)) and in the
mountains of central Costa Rica (Cartago) (Turner, 1981).
These areas were likely high enough to have remained
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S.C. Keeley et al. / Molecular Phylogenetics and Evolution 44 (2007) 89–103
on
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y
Hesperomannia, it is clear Lepidaploa species can disperse
over signiWcant distances.
Given the multiple possible pathways for movement
between North and South America one route, not yet mentioned, does not seem to apply to the Vernonieae. This is
the Boreotropic pathway (TiVney, 1985). In this scenario an
east Asian (Laurasian) ancestral lineage is postulated to
have crossed into North America via one of the land
bridges formed during the Tertiary with subsequent southward movement to tropical South America. Climatic events
after these initial radiations may have resulted in the elimination of North American taxa thus leaving behind the
extant members in South America. For taxa with African/
South American connections formed in this way (i.e. dahlbergioid legumes (Lavin et al., 2000)) the most derived species would then be South American. The distribution and
phylogenetic histories of the Vernonieae precludes the tribe
from having this kind of history. The tribe is not found in
east Asia and is also lacking in Europe making a Laurasian
ancestry unlikely. Vernoniae are also found in south and
southeast Asia, i.e., India, Thailand, Viet Nam, and parts of
Malayasia and adjacent southern China, but not north of
the Himalayas. In addition, the most derived New World
taxa are in eastern North America, contrary to the predictions of the Boretropic hypothesis.
rs
above water as parts of the volcanic island chain that made
up proto-Central America were variously submerged and
uplifted. It is these relict taxa that show the greatest aYnity
to Old World species (clade B⬘) and if the topology of Fig. 2
is correct, established the basal lineage of Vernonieae in the
New World from Old World ancestors.
Movement of Vernonieae taxa in the New World
appears to be from South to North America, with Meso/
Central America once again playing an important role. For
example, the Costa Rican Vernonieae species, Eirmocephala
brachiata, groups with the Venezuelan genus Tephrothamnus and in a position between Brazilian taxa (clade A) and
the Mesoamerican/Brazilian/North American taxa of clade
1. The genus Eirmocephala includes two other species (not
sampled) that range from Panama, to Colombia and Venezuela and into northern Ecuador. Given the derived position of the North American clade it is not hard to imagine
that South American species migrated northward through
Central America. A somewhat more complex history is suggested by the Mesoamerican taxon, Vernonanthura alamanii
in clade 1. This species is found at the base of the clade and
above which are derived Brazilian (and northern South
American) species, on the one hand, and North American
species on the other. The Brazilian derivatives may reXect a
back dispersal or perhaps V. alamanii is a relict taxon now
isolated from its southern relatives. In any case, the history
of Mesoamerican taxa is important in understanding the
current distribution of the Vernonieae. Rzedowski (1993)
noted the importance of southern elements in the Xora of
Mexico (and mega-Mexico including Mesoamerica) concurring with Raven and Axelrod (1974) that an important
part of the Mexican Xora must have originated in Central
and South America.
Understanding the history of the tribe will likely require
looking at the interplay of land-based radiations and long
distance dispersals in Meso/Central America. This is suggested, for example, by the contrasting geographical distributions of the Mesoamerican taxa in the relictual subtribe
Leiboldiinae, previously discussed, and the species of the
widespread genus Lepidaploa. In the latter case, Lepidaploa
tortuosa and L. borinquesis are both restricted to montane
Costa Rica, but are not each other’s closest relatives. Lepidaploa tortuosa is basal to its congeners (at least those used
here), including Lepidaploa ehretifolia from Venezuela. The
specimen of the latter species was collected by the Wrst
author from the top of Auyantepui, one of several isolated
remnants of the Guyana shield. Given its position in the
tree, L. ehretifolia is more recently derived than a number
of other New World taxa and is apparently not a relict
taxon which might otherwise be suggested by its geographical location. Other Lepidaploa species are derived from a
Brazilian/Paraguayan group with roots in the Andes. As
mentioned earlier, there are also endemic species of Lepidaploa in the West Indies, particularly in the older islands of
Cuba and Jamaica, and one species, Lepidaploa arborescens, is present on several islands in the West Indies and in
both South and Central America (Keeley, 1978, 1982). Like
4.2. New World relationships
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Within the New World lineages (clades A, 1, 2, 3) several
diVerent patterns can be seen that create a complex network of relationships involving South, Central and North
American taxa. The relative positions of the clades remain
the same with the exception of clade B⬘ discussed above
(Figs. 2 and 3). Brazilian taxa of clade A form a sister group
to other Brazilian species and the grade of American taxa
that culminates in clade 1. Within clade 1 itself, Brazilian
taxa are also in the sister group to the monophyletic North
America Vernonia s.s (Robinson, 1999a). The basal most
taxon of clade 1 is the morphologically distinctive V. alamanii from southern Mexico (Mesoamerica) whose characters are such that it may deserve separate generic
recognition (Robinson,pers. comm.).
The sister group to the combination of clade A and the
grade including clade 1 (discussed above) is composed of
Andean species (Critoniopsis) plus clades 2 and 3 (Fig. 2).
This relationship suggests two separate major New World
lineages despite the fact that both contain Brazilian taxa. In
the Wrst case with dispersal from Brazil through Mesoamerica to North America, with back radiation to Brazil, and in
the second case radiation of taxa from western South
America with dispersal east to Brazil, and up to Central
and North America. Secondary radiations and exchanges
may also have occurred. For example, Lepidaploa species
are found widely distributed in South America (Argentiana,
Bolivia, Brazil, Colombia, Ecuador, Peru, Venezuela), Central America and southern Mexico, and across the West
Indies (Keeley, 1978, 1982; Robinson, 1999a). If these
101
S.C. Keeley et al. / Molecular Phylogenetics and Evolution 44 (2007) 89–103
originated in the Andes then there could have been long
distance dispersal directly to the West Indies and overland
spread to Brazil and nearby regions or overland spread
may have occurred Wrst followed by dispersal to the islands.
The events surrounding the uplift of the Andes 5–15 MYA
(Funk et al., 2005) are also likely to have aVected the Vernonieae by separating and perhaps recombining species populations and providing new pathways for dispersal. In
addition, the geological history of the Antilles and protoCentral America is complex (Keigwin, 1982; Coates et al.,
2003) providing opportunities for dispersal away from
South America at one time, and in the reverse direction at
others.
It is apparent from the other long distance dispersal
events discussed above, and the relationship among Elephantopus species that an exchange of taxa occurred
between the New and Old Worlds on at least two occasions
and in two directions. One dispersal was from southeast
Asia to the Americas (clade B⬘) and the other from the
Americas to the Old World (clade 3).
al
co
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y
(1) New and Old World lineages are not separate. Here
they occur together in two diVerent and distantly
related clades.
(2) Long distance dispersal has been important in New/
Old World exchange and has occurred at least twice
and at least once in each direction.
(3) Mesoamerica has played an important role in both
ancient and modern dispersals, and is a refuge for
ancient taxa.
(4) The direction of movement in the New World has been
predominately from South America to North America.
(5) North American species originated from at least two
widely separated lines of New World taxa, and both
are recently derived within the tribe.
(6) The Vernonieae are Gondwanan and dispersal did
not follow a Boretropic route.
The Old World tribe Vernonieae is the only member of
the Compositae to have a New World sister tribe (the Liabeae (Funk et al., 2005)). Therefore, it is reasonable to suspect a more visible connection between New and Old
World members of the Vernonieae than may exist for most
other tribes in the family. The phylogeny of the tribe points
to the family wide need to focus on speciWc biogeographically important regions where taxa from Old and New
Worlds meet. Long distance dispersal is important in the
family and there are many possible source areas, times and
pathways to be further investigated. The Vernonieae oVer
an unusually good system to understand the history of the
family; no mean feat for an evil tribe.
Au
th
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rs
The Vernonieae originated in the Old World in the
region of Madagascar/southern Africa (Keeley and
Turner, 1990; Keeley and Jansen, 1994; Kim et al., 1998).
The several African lineages sampled here provide better
resolution of the relationships among them. For example,
Keeley and Jansen (1994) found Distephanus (as Vernonia
populifolia) in a basal clade that also contained clade 5 species, Baccharoides adoensis, Gymnanthemum amygdalinum,
G. mesipifolium (all considered species of Vernonia at that
time) and one species of clade 6, Linzia melleri (as Vernonia glabra). Distephanus is now a separate genus (Robinson, 1999b) and has been shown to be basal in the tribe, as
Wrst suggested in a morphologically-based study by Keeley
and Turner (1990). The position of taxa in clades C, D and
4 (Figs. 2 and 3) is also better resolved as a sister group to
the combination of newly circumscribed genera Linzia,
Gymnanthemum,and Hesperomannia (Robinson, 1999a,
2007). The taxa in clades C and D are found broadly in
eastern Africa (Burundi, Botswana, Ethiopia, Malawi,
Rwanda, Zimbabwe) while those in clade 4 are found only
in South Africa. Additionally, taxa shared by the present
study and that of Keeley and Jansen (1994) remain in
much the same relationship to each other. For example,
Centrapalus galamensis and Parapolydora fastigiata (clade
D) remain sister taxa and the three species, Orbivestus cinarescens, Baccharoides lasiopus and V. brachycalyx (previously treated as species of Vernonia) are members of the
same clade as in Keeley and Jansen (1994). None of the
Hilliardiella (clade 4) species were included in Keeley and
Jansen (1994) so their position was not previously
described. Robinson (1999b) placed taxa from these clades
(C, D, 4) within a broadly constructed subtribe, the Erlangeinae. Relationships within the Old World taxa sampled
to date are stable.
The data in this study reveal several important features
of Vernonieae phylogeny and biogeography.
on
4.3. Old World relationships
5. Conclusions
Acknowledgments
We thank Vicki Funk, Harold Robinson, Elizabeth Zimmer, Kimberley Peyton and the reviewers for helpful comments and technical support, and the individual collectors
and the curators of K, MO and US for materials. Support
was provided by National Science Foundation Grant DEB0075095 to S.C.K., the Laboratory of Molecular Systematics and the Department of Botany, National Museum of
Natural History, and by the College of Natural Sciences,
University Research Council and the Department of Botany, University of Hawaii.
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