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For exceptions, permission may be sought for such use through Elsevier’s permissions site at: http://www.elsevier.com/locate/permissionusematerial 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 or 's pe rs 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 al co p y 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 or 's pe rs 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 y Name al co p 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. or 's pe 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. 94 S.C. Keeley et al. / Molecular Phylogenetics and Evolution 44 (2007) 89–103 on al co p y 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 pe rs 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. Au th or 's 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 95 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. y 3.2. Individual gene regions on al co p 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. Au th or 's pe rs 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. 96 th or 's pe rs on al co p y S.C. Keeley et al. / Molecular Phylogenetics and Evolution 44 (2007) 89–103 Au 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 97 th or 's pe rs on al co p y S.C. Keeley et al. / Molecular Phylogenetics and Evolution 44 (2007) 89–103 Au 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 98 S.C. Keeley et al. / Molecular Phylogenetics and Evolution 44 (2007) 89–103 y co p al pe rs 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 Au th or 's 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 99 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. Au th or 's co p al on pe rs 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 y 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 100 S.C. Keeley et al. / Molecular Phylogenetics and Evolution 44 (2007) 89–103 on al co p 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 Au th or 's pe 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 p 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 or 's pe 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. 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