Systematic Implications of ndhF Sequence Variation in the ... - BioOne

Systematic Botany (2002), 27(3): pp. 598–609 q Copyright 2002 by the American Society of Plant Taxonomists

Systematic Implications of ndhF Sequence Variation in the Mutisieae (Asteraceae) HYI-GYUNG KIM,1 DENNIS J. LOOCKERMAN,2 and ROBERT K. JANSEN3 Laboratory of Molecular Systematics, Smithsonian Museum Support Center, 4210 Silver Hill Road, Suitland, Maryland 20746; 2 Codis-section of the Crime Laboratory Service, MSC0461, 5805 N Lamar, P. O. Box 4143, Austin, Texas 78765–4143; 3 Section of Integrative Biology and Institute of Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712 1

Communicating Editor: Jerrold I. Davis ABSTRACT. Mutisieae are considered one of the most important tribes for understanding the systematics and evolution of the Asteraceae because of their basal position within the family. The tribe is extremely diverse morphologically and biogeographically, consisting of 84 genera and 900 species. Despite extensive studies using macromorphological, palynological, and molecular approaches, tribal and subtribal delimitation and relationships of Mutisieae remain controversial. Phylogenetic analyses of DNA sequences of the chloroplast gene ndhF were performed using 53 species representing 31 genera of Mutisieae, 11 genera from the remaining five tribes of Cichorioideae, and five genera from the Asteroideae. The ndhF phylogeny of the Mutisieae provides insights into the circumscription and relationships of the tribe, subtribes, and taxonomic placement of several morphologically anomalous genera. The ndhF tree indicates that Mutisieae are polyphyletic. The subtribes Gochnatiinae and Mutisiinae are not monophyletic, whereas the Nassauviinae are monophyletic. The majority of the South American genera and the Chinese genus Nouelia of the Gochnatiinae are positioned in a basal position in the Mutisieae. Furthermore, intergeneric relationships are resolved in several cases.

Mutisieae have a worldwide distribution and consist of 84 genera and approximately 900 species in the three subtribes Gochnatiinae, Mutisiinae, and Nassauviinae (Bremer 1994). Most taxa occur in the New World, mainly from Central to South America, while 11 genera are distributed in Africa and Madagascar, 12 genera in Asia, and one genus in the Hawaiian Islands. The majority of the tribe is found along the southern Andes from Peru to Chile and Patagonia. Many genera are restricted to arid and mountainous habitats. The tribe is morphologically and biogeographically very diverse with many of the genera monotypic or with only a few species. The wide morphological diversity and geographic isolation of many genera has made it difficult to resolve the circumscription and relationships of the tribe. Mutisieae are considered one of the most important tribes for understanding the systematics and evolution of Asteraceae because they are one of the basal lineages in the family. Despite extensive macromorphological (Cabrera 1977; Grau 1980; Hansen 1991a; Karis et al. 1992), palynological (Wodehouse 1929a, 1929b; Para and Marticorena 1968, 1972; Hansen 1991b), and molecular (Jansen and Palmer 1988; Jansen et al. 1991a, 1991b; Kim et al. 1992; Kim and Jansen 1995) studies, tribal and subtribal delimitation and relationships of Mutisieae remain controversial. Much of the controversy is the result of the limited understanding of morphological variation and the difficulty of obtaining complete sampling due to the wide geographic distribution of the tribe. One of the primary systematic issues concerning the

Mutisieae is whether the tribe is monophyletic. Several previous studies suggested that the tribe was polyphyletic based on its diverse floral and pollen morphology and wide geographic distribution (Small 1918; Wodehouse 1929a). Cabrera (1977) considered Mutisieae polyphyletic in his systematic treatment of the tribe. In contrast, monophyly of a recircumscribed Mutisieae has been proposed by several workers (Cronquist 1988; Jansen and Palmer 1988; Jansen et al. 1991b). Hansen (1991a) redefined the Mutisieae by excluding 15 Old World genera. Since the elevation of the Barnadesiinae to subfamily status (Bremer and Jansen 1992), debate on the monophyly of Mutisieae sensu stricto has continued. A second important systematic issue in the Mutisieae concerns their relationship to the other tribes of Asteraceae. The tribe has been suggested to be allied with Cynareae (Bentham 1873; Hoffmann 1890; Cronquist 1955), Heliantheae (Cronquist 1955), and Senecioneae (Small 1918). Recent phylogenetic analyses (Bremer 1987; Jansen et al. 1991b; Kim et al. 1992; Kim and Jansen 1995; Jansen and Kim 1996) did not support their relationship to the Heliantheae or Senecioneae. Several putative synapomorphies support a close relationship to the Cynareae, including pluriseriate phyllaries, caudate anthers, short two-lobed, glabrous or papillate styles, and spiny leaves (Karis et al. 1992). Chloroplast DNA restriction site and rbcL sequence data indicated affinities of the Mutisieae to the Tarchonantheae (Jansen et al. 1991b; Keeley and Jansen 1991) and Vernonieae (Kim et al. 1992). A third controversial issue in the Mutisieae con-

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cerns the circumscription and relationships of the subtribes. Nassauviinae have been considered monophyletic based on the presence of truncate, apically penicillate style branches, whereas the Gochnatiinae and Mutisiinae were artificially delimited primarily by the presence of actinomorphic or bilabiate florets (Bremer 1994). Many genera placed in the Gochnatiinae and Mutisieae have been demonstrated to be closely related (Hansen 1991a; Robinson 1991). Crisci (1974, 1980) transferred several genera, including Lophopappus H. H. Rusby and Proustia Lag., to the Nassauviinae, mainly on the basis of pollen exine structure. However, Proustia and Lophopappus also have rounded stigma lobes and sometimes actinomorphic florets instead of truncate stigma lobes and bilabiate florets, respectively. Since Cabrera’s (1977) classification of the Mutisieae into four subtribes, very few changes have been suggested. The notable exception was the elevation of the Barnadesiinae to subfamilial status (Bremer and Jansen 1992). Monophyly of the Nassauviinae is supported strongly, while many disagreements remain concerning the delimitation of the Mutisiinae and Gochnatiinae. The final systematic issue in the Mutisieae concerns intergeneric relationships. Much effort has been concentrated on clarifying relationships among genera at the several different levels: genus (Cabrera 1936, 1937, 1965, 1970, 1971, 1982; Burkart 1944), generic complex (Jeffrey 1967; Hansen 1985, 1988, 1990) and biogeographic groups (Maguire 1956, 1967; Maguire and Wurdack 1957; Maguire et al. 1957; Jeffrey 1967, 1977; Pruski 1991). These studies have suggested relationships among several genera. Cabrera (1977) developed schemes of interrelationships by identifying a core group of genera within each subtribe. However, no comprehensive study of phylogenetic relationships of genera within the tribe has been completed. In this paper, we conduct phylogenetic analyses of DNA sequences of the 39 end of the chloroplast gene ndhF to address several systematic issues in the Mutisieae. The three objectives are: 1) to test the monophyly of the tribe, 2) to evaluate the circumscription and relationships of the subtribes, and 3) to examine intergeneric relationships. MATERIALS

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DNA Isolation, PCR Amplification, and Sequencing. Sequences of ndhF were examined from 73 species representing 31 genera of Mutisieae, 11 genera from the remaining five tribes of Cichorioideae, and five genera from the Asteroideae (Appendix 1). Total genomic DNAs were isolated from herbarium specimens, fresh leaves or silica-dried leaves. The DNA from fresh leaf material was extracted using a modified 2X CTAB method (Doyle and Doyle 1987), followed by purification in cesium chloride/ethidium bromide gradients (Sambrook et al. 1989). Extractions from the herbarium specimens and silica-dried leaves were performed using the modified 2X CTAB methods of Loockerman and Jansen (1996). DNAs contaminated with high amounts of polysaccharides were

resuspended in 1X TE buffer/1–3M NaCl, followed by precipitation in 100% ethanol and washing in 70% ethanol to remove the residual salt (Jansen et al. 1999). Amplifications were performed in 50 ml reactions, which included 1 ml of unquantified 1/10, 1/50, 1/100 diluted genomic DNA, 20–60 pmole of primer, 10X Tfl polymerase buffer, 0.5 ml of 1 unit/ ml of Tfl polymerase (Epicentre Technologies, Madison, WI), 10 mM MgCl2 and 2.5 mM of each dNTP. The first amplification cycle consisted of 3 min denaturation at 948C, 1 min annealing at 468C, and 1 min 20 sec extension at 728C. This was followed by 34 cycles at 948C for 1 min, 508C for 1 min, and 728C for 1 min and 20 sec with a 3-sec extension/cycle. Amplifications were terminated by a final extension cycle of 728C for 7 min and soaked at 158C. The PCR products were purified by electrophoresis through a 1% agarose gel using 1X TAE buffer. The PCR product was excised from the gel and further purified using the GeneClean II system following the manufacturer’s protocol (Bio101, Inc). The Purified PCR product was sequenced by the snap-chill method (Winship 1989) using Sequenase version 2.0 (USB product No. 70770) and 35S dATP labeling. Amplification of ndhF was performed using two primers (1201 and 1607), and six forward primers were used for sequencing (Jansen 1992). Only the 39 end of ndhF was sequenced because it has been shown to be the most variable region of the gene (Kim and Jansen 1995). All sequences were submitted to GenBank (See Appendix 1 for accession numbers). Phylogenetic Analyses. Parsimony analyses were conducted on a data matrix that included 73 taxa (Table 1) and 984 characters using PAUP* 4.0 (Swofford 1998). DNA sequences were easy to align manually in MacClade (version 3.0, Maddison and Maddison 1992) because of the paucity of insertions/deletions (indels) in ndhF (see Results). All characters were unordered and equally weighted with respect to codon position. The ACCTRAN option was used for character optimization. Gaps were coded as hyphens (-) and treated as missing data in the phylogenetic analyses. We employed a four-step search analysis (Olmstead et al. 1993; Conti et al. 1996) because of the large size of the data matrix. To ensure that the all islands of shortest trees were found, heuristic searches were first performed with Tree Bisection Reconnection (TBR), MULPARS OFF, STEEPEST DESCENT, and MaxTrees set to 5000. All trees generated from each search were saved. Second, all trees in memory were used as starting seeds for heuristic searches with TBR, MULPARS OFF, STEEPEST DESCENT and MaxTrees set to 5000. Third, all trees in memory were utilized as starting seeds for a search using NNI, MULPARS ON and STEEPEST DESCENT. Finally, heuristic searches were completed using all trees in memory with TBR, MULPARS AND STEEPEST DESCENT and MaxTrees set to 5000. Successive approximation weighting (Farris 1969) was performed to facilitate comparison among tree scores according to their rescaled consistency index (RC) based on the best fit characters on any of the trees. Heuristic searches were conducted repeatedly using simple addition, TBR swapping method, STEEPEST DESCENT with MULPARS off, and MaxTrees set to 5000 until the same topologies were obtained consecutively with a base weight of 1000. The amount of support for individual clades was assessed with bootstrap (Felsenstein 1985) and jacknife (implemented in PAUP* 4.0) methods using NNI for branch swapping, simple addition and 100 replicates. Three genera of the subfamily Barnadesioideae were used as outgroups based on previous molecular (Jansen and Palmer 1987; Kim and Jansen 1995) and morphological (Bremer 1987) studies.

RESULTS Variation of ndhF Sequence. Four deletions were detected: 6 base pairs (bp) in Chaetanthera R. & P., 6 bp in Gochnatia Kunth, 12 bp in Pachylaena D. Don, and 9 bp in Hesperomannia Gray. The aligned ndhF sequences contained 984 bp, including 546 invariant sites (55.6%),

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249 parsimony-informative sites (25.3%) and 189 variable but parsimony-uninformative positions (19.2%). Most changes were in the third (50.6%) codon position, with 27.9% and 21.6% in the first and second codon positions, respectively. The ratio of transitions to transversions was 1:1.25. Phylogenetic Analyses. Parsimony analyses of 73 ndhF sequences using the four-step search method identified 5052 equally parsimonious trees (Fig. 1) with 1086 steps, a consistency index (CI) of 0.585 (CI 5 0.480 excluding uninformative characters), and a retention index (RI) of 0.636. Successive differential weighting (SDW) was performed with characters consecutively re-weighted until identical topologies in consecutive analyses were found. The topology of the SDW tree was almost identical to one of the most parsimonious trees with equal weighting, except for the placements of Adenocaulon Hook. and Nouelia Franch. near South American Stifftia J. C. Mikan and at the base of Gerbera L. complex, respectively. Thus, we only present the results of the unweighted analyses. All ndhF trees indicated that Mutisieae were polyphyletic (Fig. 1A-B). Constraining the monophyly of the Mutisieae required 28 additional steps. Despite the weak support for many of the clades, the ndhF tree identified two major groups in the Mutisieae. One (clade A) comprised all taxa of the Nassauviinae, most of the Mutisiinae, and part of the Gochnatiinae, all of which were distributed predominantly in South America. However, this clade was weakly supported with 11% bootstrap and 17% jacknife values (Fig. 1A), and it collapsed in the strict consensus tree (Fig. 2). The second major clade (B) consisted of several genera of Gochnatiinae and Mutisiinae from the Old World, the Tarchonantheae, the Cardueae, the remaining tribes of the Cichorioideae, and the Asteroideae (Fig. 1B). This clade had weak support with 13% bootstrap and 20% jacknife values. The dichotomy between the New World and Old World Mutisieae was the most conspicuous feature of the ndhF tree. The only exceptions were the African genus Piloselloides Less. and the Asian genus Nouelia near the South American group that included Aphyllocladus Wedd., Onoseris Willd., and Plazia R. & P. The Hawaiian endemic genus Hesperomannia, a member of Gochnatiinae, was nested within Vernonieae with strong support (97% bootstrap and 98% jacknife values). The Gochnatiinae and Mutisiinae were polyphyletic in all of the ndhF trees, whereas the Nassauviinae was monophyletic in some. Support for Nassauviinae was weak (Fig. 1A) with 24% bootstrap and 35% jacknife values, and this clade collapsed in the strict consensus tree (Fig. 2). Constraining the monophyly of the Gochnatiinae and Mutisiinae required 37 and 12 additional steps, respectively. The monophyly of a large portion of the Mutisiinae

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(Gerbera complex/Mutisia L. f./Chaetanthera clade/Pachylaena) was weakly supported in the ndhF tree (17% bootstrap and 24% jacknife values, Fig. 1A) and this clade collapsed in the strict consensus tree (Fig. 2). This clade was more closely allied to Nassauviinae than to Gochnatiinae, although support for this relationship was very weak (Fig. 1A). The ndhF tree placed Adenocaulon near the Perezia Lag./Nassauvia Commers. clade within the Nassauviinae (Fig. 1A). However, the position was weakly supported (24% bootstrap and 24% jacknife values). Brachylaena R. Brown and Tarchonanthus L. formed a distinct clade in the ndhF trees with strong support (94% bootstrap and 95% jacknife values). The clade was sister to two African genera of the Mutisieae, Dicoma Cass. and Pasaccardoa O. Kuntze. This relationship was not well supported (34% bootstrap and 34% jacknife values) and collapsed in the strict consensus tree. The clade comprising Brachylaena, Tarchonanthus, and the African Mutisieae was sister to the Cardueae. Relationships among the three groups were not supported strongly with 34% bootstrap and 49% jacknife value (Fig. 1B) and these three clades formed a trichotomy in the strict consensus tree (Fig. 2). Although the ndhF tree was not well resolved and most clades were not strongly supported (Fig. 1A-B), several generic assemblages were evident. The South American genus Stifftia of the Gochnatiinae was positioned as the basal lineage of the Mutisieae (Fig. 1A). The clade including South American genera (Onoseris/ Aphyllocladus/Plazia) and the Asian monotypic genus Nouelia was sister to a large group of South American Mutisieae, including members of the Mutisiinae and Nassauviinae. Gochnatia, one of the largest genera of the Mutisieae, was sister to Cnicothamnus Griseb. (64% bootstrap and 78% jacknife values) and this clade was positioned at the base of a large group that included Old World Mutisieae, Tarchonantheae, the remaining tribes of the Cichorioideae and Asteroideae. However, this clade (clade B) was supported with very low bootstrap (13%) and jacknife (20%) values and it collapsed in the strict consensus tree (Fig. 2). The Asian genus Ainsliaea DC. of the Gochnatiinae was monophyletic and sister to the two Asian genera Pertya Sch.-Bip. of the Gochnatiinae and Myripnois Bunge of the Mutisiinae. The Asian clade formed a strongly supported group with six synapomorphies and high bootstrap (92%) and jacknife (98%) values. The ndhF trees identified three additional, strongly supported generic groupings in the Mutisieae, Mutisia, the Chaetanthera group, and the Gerbera complex. Mutisia was strongly monophyletic with 95% bootstrap and 98% jacknife values. This clade was sister to the Chaetanthera group, which included Duidaea Blake, and Chaetanthera. Chaetanthera was monophyletic and sister to Duidaea. Pachylaena was placed at the base of this

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FIG. 1A. One of the 5056 equally parsimonious trees obtained from analyses of ndhF sequence data matrix (length 5 1086; CI 5 0.585; RI 5 0.636; RC 5 0.372; HI 5 0.415). The tree shows taxa in clade A. Numbers above branches indicate number of character changes. Numbers below branches to left and right of slash indicate bootstrap and jacknife values, respectively. The abbreviated tribal and subtribal names are: Arct. Arctoteae, Card. Cardueae, Goch. Gochnatiinae, Lact. Lactuceae, Liab. Liabeae, Mut. Mutisieae, Tarch. Tarchonantheae, and Vern. Vernonieae. The capital M in parenthesis indicates the tribe Mutisieae. The numbers I and II indicate subfamilies, Barnadesioideae and Cichorioideae.

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FIG. 1B. Taxa of clade B in Fig. 1A. The information on the tree is the same as that of Fig. 1A. The capital M in parenthesis indicates the tribe Mutisieae. The numbers I, II and III indicate the subfamilies, Barnadesioideae, Cichorioideae and Asteroideae, respectively.

clade. However, relationships among the three clades Mutisia, Chaetanthera/Duidaea, and Pachylaena, were not resolved in the strict consensus tree (Fig. 2). The Gerbera-complex, including Chaptalia Vent., Leibnitzia Cass., Piloselloides, and Gerbera, was strongly supported with

high bootstrap (83%) and jacknife (94%) values. The phylogenetic relationships among these four genera remained unresolved in the strict consensus tree (Fig. 2). Three clades were identified in the Nassauviinae in some of the most parsimonious trees (Fig. 1A): (1) Nas-

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FIG. 2. Strict consensus tree of 5056 equally parsimonious trees obtained from PAUP analyses. G, M, and N indicate the subtribes, Gochnatiinae, Mutisiinae and Nassauviinae, respectively. The numbers I, II and III indicate the subfamilies Barnadesioideae, Cichorioideae and Asteroideae, respectively.

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sauvia, Triptilion R. & P., Perezia, and Adenocaulon; (2) Trixis P. Browne, Proustia, and Acourtia D. Don; and (3) Jungia L.f. and Leucheria Lag. The clade with Nassauvia and Triptilion was strongly supported (100% bootstrap and 100% jacknife values), indicating that Nassauvia is paraphyletic (Figs. 1, 2). Perezia appeared at the base of the Nassauvia clade with 67% bootstrap and 78% jacknife values. Adenocaulon was positioned at the base of the clade including Perezia, and Nassauvia/Triptilion (Fig. 1A). However, the basal position of Adenocaulon was not supported in the strict consensus tree (Fig. 2). In a second clade, Proustia was linked with Trixis with 50% bootstrap and 69% jacknife values, and this clade was sister to Acourtia. The third clade in the Nassauviinae included Jungia and Leucheria with support of 34% bootstrap and 50% jacknife values and was sister to clades 1 and 2. Support for this third clade was weak and collapsed in the strict consensus tree (Fig. 2). DISCUSSION This study includes many more taxa of Mutisieae (53 versus 13) than previous molecular systematic comparisons of the tribe (Jansen and Palmer 1988; Kim and Jansen 1995). Thus, the results have the potential to resolve the circumscription and relationships of the major clades of the tribe. Although the low number of synapomorphic characters and low support values for many nodes limit the utility of the ndhF data, several conclusions can be made about the taxonomic circumscription and phylogenetic relationships of the tribe. In some cases, these conclusions are strongly supported, whereas in other instances additional data are needed to confirm or refute the phylogenetic relationships suggested by the ndhF tree. Polyphyly of Mutisieae. The monophyly of the Mutisieae has been questioned for nearly 100 years based primarily on its very diverse morphology (Small 1918; Wodehouse 1929a; Cabrera 1977). Since the elevation of the Barnadesiinae (Cabrera 1977) to the subfamilial level (Bremer and Jansen 1992), the Barnadesioideae have been considered to be the basal member of the Asteraceae. However, the circumscription and relationships of the Mutisieae sensu stricto (excluding Barnadesioideae) have remained uncertain. The ndhF phylogenies (Figs. 1, 2) are consistent with other recent phylogenetic studies (Karis et al. 1992; Bremer 1994; Kim and Jansen 1995) in indicating that the tribe is polyphyletic even after excluding the Barnadesioideae. Furthermore, the trees indicate a dichotomy between the genera from the Old and New World except for the Chinese genus Nouelia and the African genus Piloselloides (Fig. 1A). This biogeographic split suggests a distant relationship between several genera previously placed in the Gochnatiinae and Mutisiinae. The monophyly of the Asian lineage consisting of Myripnois of

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the Mutisiinae and Ainsliaea and Pertya of the Gochnatiinae is strongly supported and this group is sister to an African clade comprising the Tarchonantheae, Dicoma and Pasaccardoa of the Gochnatiinae, and the Cardueae. Circumscription and Relationships of the Subtribes. The ndhF tree is congruent with recent morphological cladograms (Bremer 1987; Karis et al. 1992) regarding the circumscription of the subtribes of the Mutisieae (Figs. 1, 2). The predominantly South American Nassauviinae are monophyletic. Despite the weak support in ndhF trees, this subtribe has been considered monophyletic (Cabrera 1977; Crisci 1974, 1980) based on its truncate (rarely rounded) and penicillate style branches and bilabiate, hermaphrodite flowers. Mutisiinae are characterized by bilabiate disk flowers and styles with rounded apices and without collective hairs. Recent morphological cladistic analyses (Karis et al. 1992; Hansen 1991a) have questioned the monophyly of the subtribe. The ndhF phylogeny supports the polyphyly of the Mutisiinae. All genera of this subtribe except the Old World genus Myripnois form a monophyletic group in the ndhF tree and this clade is sister to the Nassauviinae (Fig. 1A). However, monophyly of the group is not strongly supported and it collapses in the strict consensus tree (Fig. 2). Myripnois is sister to Pertya of the Gochnatiinae and nested within an Asian clade with Ainsliaea of the Gochnatiinae (Fig. 1B). Their close relationship was noted by Mattfeld (1934) based on similar habit and floral characters. Nevertheless, previous taxonomic treatments (Cabrera 1977; Bremer 1994) placed Myripnois in the Mutisiinae because of its bilabiate disk flowers. Our examination of the flowers of Myripnois indicates that it has unevenly split actinomorphic disk flowers instead of the bilabiate condition reported by Yung-chien (1996). A close phylogenetic relationship among these three genera is also supported by two putative morphological synapomorphies, homogamous capitula and heads arranged laterally on the branches. These results suggest two possible systematic changes: (1) removal of Myripnois from the Mutisiinae, and (2) recognition of the Asian clade as a distinct tribe. Neither of these changes are advisable until taxon sampling of the Mutisieae is expanded and more molecular and morphological characters become available. Gochnatiinae have traditionally included 36 genera characterized by actinomorphic disk florets and the absence of axillary spines (Cabrera 1977). This subtribe is clearly polyphyletic in the ndhF tree (Figs. 1A-B, 2). Previous morphological (Hansen 1991a; Karis et al. 1992) and molecular (Jansen and Palmer 1988; Kim and Jansen 1995) phylogenetic studies indicated that Gochnatiinae are not monophyletic. Most South American genera of Gochnatiinae occur in a basal position in the Mutisieae, whereas the African and Asian gen-

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era are in a more derived position with the tribes Cardueae and Tarchonantheae. The one exception to this biogeographic split between the New and Old World Gochnatiinae is the Chinese genus Nouelia, which groups with the South American Gochnatiinae, Onoseris, Plazia, and Aphyllocladus. However, the placement of Nouelia with the South American clade is weakly supported (Fig. 1A) and it collapses in the strict consensus tree (Fig. 2). The New World group has both actinomorphic disk flowers and bilabiate marginal flowers while the Old World group has only actinomorphic disk flowers. The presence of bilabiate marginal florets and actinomorphic disk florets in both Nouelia and the genera of the Gochnatiinae in the South American clade provide additional support for the relationship indicated by the ndhF phylogeny. New Tribal Placement of Hesperomannia. The ndhF phylogeny places the Hawaiian endemic Hesperomannia in a derived position in the Cichorioideae, nested within the Vernonieae (Figs. 1, 2). All previous classifications considered this genus to be closely allied to the South American genera Stifftia, Stenopadus Blake, and Wunderlichia Riedel (Hansen 1991a; Karis et al. 1992; Bremer 1994; Funk and Wagner 1995). Furthermore, Karis et al. (1992) suggested that Hesperomannia is one of the basal lineages in the Cichorioideae, and this placement was used to develop a biogeographic scenario for the Asteraceae (Bremer 1993, 1994). Morphological and chromosomal data provide independent support for the position of Hesperomannia close to the African members of Vernonieae (Kim et al. 1998). Both groups have pollen with a continuous micropunctate tectum, long slender styles with acute or obtuse tips, and a chromosome number of n 5 10. Our results provide evidence for a biogeographic link between Africa and Hawaii and suggest that the biogeographic hypotheses of Bremer (1993, 1994) must be reconsidered. Tribal or Subtribal Placement of Problematic Genera. Several genera whose tribal placements are uncertain have been classified in Mutisieae (Cabrera 1977). The controversy is due primarily to the anomalous morphology and the lack of comprehensive phylogenetic analyses of the tribe. Parsimony analyses of ndhF sequence data provide some resolution of the tribal and subtribal placement of several genera. There has been a considerable controversy regarding the tribal placement of Adenocaulon. The genus has been placed into four different tribes of the Cichorioideae and Asteroideae: Heliantheae (Bentham 1873), Inuleae (Hoffman 1890; Cabrera 1961), Senecioneae (Cronquist 1955), and Mutisieae (Stebbins in Ornduff et al. 1967; Grau 1980; Bittmann 1990; Kim and Jansen 1995). Stebbins (in Ornduff et al. 1967) indicated that this genus has affinities with the Mutisieae based on pollen morphology, the bilabiate tendencies of margin-

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al corollas, the shape and pubescence of the leaves, and the chromosome number of n 5 23. Bittmann (1990) reviewed the systematic position of Adenocaulon and concluded that it belonged in the Mutisieae. Later, Hansen (1991a) excluded the genus from the Mutisieae based on several apomorphic characters, such as petal epidermis pattern, an involucre with few bracts, and 4- or 5-lobed florets with rigid and very broadly widened styles. The ndhF tree of Kim and Jansen (1995) positioned Adenocaulon within the Mutisieae. However, the systematic position in this tribe was not resolved due to limited taxon sampling. In this study, the tribal position of Adenocaulon is confirmed within Mutisieae with expanded sampling (Figs. 1, 2). However, its subtribal placement is still unresolved because of the lack of resolution in the ndhF tree (Fig. 2). Another problematic group includes the two African genera Tarchonanthus and Brachylaena. These genera have been placed in several different tribes: Anthemideae (Skvarla et al. 1977), Astereae (Jeffrey 1978), Inuleae (Bentham 1873), Mutisieae (Leins 1971; Merxmuller et al. 1977; Grau 1980; Bremer 1987; Zdero and Bohlmann 1987; Karis et al. 1992), Vernonieae (Cassini 1828), and Tarchonantheae (Keeley and Jansen 1991). Confusion concerning the tribal position of these genera results from the presence of vernonioid styles, filiform corollas, sagittate-tailed anthers, testa structures, and anthemoid pollen. Despite morphological intermediates suggestive of different systematic positions, Brachylaena and Tarchonanthus are closely related to each other based on dioecy and restricted geographic distribution. A recent molecular study (Keeley and Jansen 1991) indicated that the two genera form a distinct clade at or near the base of the Cichorioideae. This result led to the recognition of this lineage as an independent tribe, the Tarchonantheae. Additional data from ndhF sequences (Kim and Jansen 1995) confirmed that Tarchonanthus is allied more closely with some Cardueae than the genera of Mutisieae examined. Our expanded taxon sampling of ndhF sequence data indicates that Brachylaena and Tarchonanthus are sister to a clade of African Mutisieae that includes Dicoma and Pasaccardoa, although support for the relationship is weak (Figs. 1B, 2). The African clade with Tarchonanthus, Brachylaena, Dicoma and Pasaccardoa is sister to the Cardueae, in agreement with the earlier study by Kim and Jansen (1995). Relationships among these genera collapse in the strict consensus tree (Fig. 2). Intergeneric Relationships. Despite poor resolution of the basal nodes in the ndhF tree, intergeneric relationships in the Mutisieae are resolved in some instances. Gochnatia is a large genus consisting of 68 species that occur predominantly in the New World, with only two species in Asia. The genus is traditionally characterized by apiculate anther appendages and it is considered to be critical for understanding the subtribe

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Gochnatiinae because the generic circumscription is extremely artificial (Bremer 1994). Several genera (Actinoseris (Endl.) Cabrera, Chucoa Cabrera, Cyclolepis Gillies, Hyalis D. Don, and Nouelia) have been suggested to be closely allied with Gochnatia based on apiculate anther appendages (Cabrera 1951, 1970; Bremer 1994). In the ndhF tree, Cnicothamnus is in the same clade as Gochnatia and this position has moderate support (a bootstrap value of 64% and jacknife value of 78%). The Plazia-complex from South America, including Aphyllocladus, was recognized as a natural group on basis of truncate, reddish anther appendages (Hoffmann 1890; Cabrera 1951). Hansen’s (1991a) morphological cladogram supported the monophyly of the Plazia-complex, including Plazia, Aphyllocladus, and Gypothamnium Phil (not sampled). The ndhF tree is congruent with previous work in supporting a close relationship between Plazia and Aphyllocladus. In addition, Onoseris is sister to the Plazia-complex. This genus has been traditionally considered to be a unique taxon within Gochnatiinae because of its zygomorphic disc florets, and it was suggested to be related to the South American monotypic genus Urmenetea Phil. (not sampled). Morphological cladograms of Karis et al. (1992) were inconclusive regarding the relationship between Plazia and Onoseris. The most critical group for understanding relationships in the Mutisieae includes the ten genera from the Guayana Highlands. These genera are divided into two subgroups, one with actinomorphic florets (Chimantaea Maguire, Quelchia N. E. Brown, Stenopadus, and Stomatochaeta (Blake) Maguire and Wurdack), occurring in the Eastern Guayana Highlands, and the other with bilabiate florets (Achnopogon Maguire, Steyermark, & Wurdack, Duidaea, Eurydochus Maguire and Wurdack, Glossarion Maguire and Wurdack, Gongylolepis R. Schomburgk, and Neblinaea Maguire & Wurdack), occurring in the Western Guayana Highlands. The two groups are placed in the Gochnatiinae and Mutisiinae, respectively. Despite their biogeographic isolation and floral dimorphism, the Guayana Highland genera are considered to be monophyletic (Maguire 1956) based on their thick, coriaceous leaves, tree-like habit, large flowers, leafy involucres, and shortly bilobed styles with a continuous stigmatic surface (Pruski 1991; Bremer 1994). Besides the question of the monophyly of this regional assemblage, there is another question regarding its sister relationship. Pruski (1991) indicated that the Guayana Highland genera are more closely related to Stifftia from eastern Amazonia and the Brazilian planalto and Wunderlichia of planaltine Brazil. Unfortunately, only a single Guayana Highland genus, Duidaea, was available for this study, due to the difficulty of obtaining material. The ndhF tree positions Duidaea of Mutisiinae as sister to the

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South American Chaetanthera group of the same subtribe (Figs. 1A, 2). The close relationship of the Asian genera Pertya and Myripnois in the ndhF tree agrees with Mattfeld’s (1934) suggestion that they share a similar habit and sessile, few-flowered capitula. However, the latter genus differs by its unevenly cleft, bilabiate-like or even ligulate flowers and fewer herbaceous involucral bracts. Uncertainty about the systematic position of Myripnois has remained because of the artificial circumscription of the subtribes. The ndhF tree provides strong support for the monophyly of these genera (Figs. 1B, 2). The African group comprises both tropical herbs (Achyrothalamus O. Hoffm., Erythrocephalum Benth., Pasaccardoa, and Pleiotaxis Steetz) and a Madagascan shrub (Gladiopappus Humbert) of the Gochnatiinae. Jeffrey (1967) referred to these as one of the most distinct groupings within the tribe. They have been traditionally grouped together by a peculiar style with subapical hairs (Hansen 1991a). In addition, the sister group relationship of Pasaccardoa within Dicoma was suggested by Bremer (1994). Dicoma and Pasaccardoa are distinguished morphologically by having radiate and discoid heads, respectively. However, in South Africa there are also radiate Dicoma species. Grau (1980) recognized an Old World group with similar testa structure but he excluded Pasaccardoa because it has a testa similar to most other Mutisieae. The ndhF tree strongly supports the sister group relationship between Dicoma and Pasaccardoa (Figs. 1B–2). Despite the worldwide distribution of the Gerberacomplex of the Mutisiinae, it has been considered to be monophyletic on the basis of its monocephalous scape with rosulate leaves and pistillate ray florets (Hansen 1988). It comprises seven genera and nearly 125 species: one large Old World genus, Gerbera, one large New World genus, Chaptalia, and five smaller genera, including Leibnitzia and Uechtritzia Freyn in Asia, Piloselloides and Perdicium L. in Africa, and Trichocline Cass. in South America. Jeffrey (1967) recognized Chaptalia, Gerbera, and the small South African derivative, Piloselloides, and he suggested a sister group relationship of Chaptalia with Piloselloides. Hansen (1985) questioned this relationship and reduced the latter genus to a section of Gerbera. Later, Hansen (1990) performed morphological cladistic analyses of the Gerbera-complex and proposed that its six genera should be merged into a single, large genus. However, the works of Wodehouse (1929a) and Grau (1980) were inconsistent with Hansen (1991a). Our study includes three genera from the New World: Gerbera, Chaptalia, and Leibnitzia, and one genus from Old World, Piloselloides. The monophyly of the Gerbera-complex is strongly supported (Figs. 1A, 2). The most parsimonious ndhF tree (Fig. 1A) is congruent with Hansen’s merger of Gerbera and Piloselloides. However, the strict consen-

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sus tree does not resolve their monophyly because these two genera form a polytomy with Leibnitzia and Chaptalia (Fig. 2). Furthermore, the ndhF tree (Figs. 1A, 2) indicates that the large New World genus Chaptalia is paraphyletic. Additional taxon sampling and molecular data are needed to resolve the circumscription and relationships of the Gerbera complex. Nassauviinae are most diverse in Central and South America, with a few species in the southwestern United States, Mexico, and the West Indies. Crisci (1974, 1980) examined intergeneric relationships in the subtribe using phenetic and cladistic methods. Hansen (1991a) also generated morphological cladograms for this group. However, the clades identified in these studies were not concordant except for the Nassauvia group, which included Macrachaenium Hook. f., Moscharia R & P., Nassauvia, Polyachyrus, and Triptilion. The ndhF tree (Figs. 1A, 2) identifies a clade consisting of Nassauvia and Triptilion. These genera have been considered highly modified because of their secondary inflorescence (a pseudocephalum) and a similar pollen exine (Crisci 1974). Bremer (1994) considered these two genera to be derived members of the Leucheria group, which included three large genera (Nassauvia, Leucheria, and Perezia) and seven small genera (Holocheilus Cass., Pamphalea Lag., Macrachaenium, Moscharia, Polyachyrus, Oxyphyllum Phil., and Triptilion). Several previous workers suggest a close relationship between these two genera (Crisci 1974; Cabrera 1982; Bremer 1994). The ndhF trees (Figs. 1A, 2) suggest that Triptilion should be merged with Nassauvia. Another monophyletic group within the Nassauviinae includes Pleocarphus, Jungia, and Proustia. Proustia has been traditionally regarded as closely related to the North and Central American genus Acourtia, and was treated as a section of Perezia by Vuilleumier (1969). The position of Proustia in the Nassauviinae may be problematic due to the presence of rounded style branches and sometimes actinomorphic florets instead of truncate style branches and bilabiate florets. The ndhF trees (Figs. 1A, 2) place Proustia as sister of Trixis, although support is relatively weak. ACKNOWLEDGEMENTS. We thank the following people for providing plant material: Vicki Funk, Karla Gengler, Doug Goldman, Frank Hellwig, David Keil, Ki-Joong Kim, Youngdong Kim, David Lorence, Jose Panero, Tod Stuessy, and Jun Wen. We thank several herbaria and botanical gardens for granting us permission to use their material, including K, MO, OS, TEX, UC, and US. We also thank Tim Lowrey, Michael Dillon, and Jerrold Davis for helpful suggestions for improving the paper. This paper represents a portion of the Ph.D. dissertation of Hyi-Gyung Kim. I also thank my committee members Stephen Hall, Tom Mabry, Beryl Simpson, and Billie Turner. This work was supported by an NSF grant (DEB-9318279) to RKJ.

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deutung. Mitteilungen der Botanischen Staatssammlung, Munchen 16: 269–332. HANSEN, H. V. 1985. A taxonomic revision of the genus Gerbera (Compositae, Mutisieae) sections Gerbera, Parva, Piloselloides (in Africa), and Lasiopus. Opera Botanica 78: 1–36. ———. 1988. A taxonomic revision of the genera Gerbera sect. Isanthus, Leibnitzia (in Asia), and Uechtritzia (Compositae, Mutisieae). Nordic Journal of Botany 8: 61–76. ———. 1990. Phylogenetic studies in the Gerbera complex (Compositae, tribe Mutisieae, subtribe Mutisiinae). Nordic Journal of Botany 9: 469–485. ———. 1991a. Phylogenetic studies in Compositae tribe Mutisieae. Opera Botanica 109: 1–50. ———. 1991b. SEM-studies and general comments on pollen in tribe Mutisieae (Compositae) sensu Cabrera. Nordic Journal of Botany 10: 607–623. HOFFMANN, O. 1890. Compositae. Pp. 87–391 in Die Naturlichen Pflanzenfamilien, eds. A. Engler and K. Prantl, Berlin. JANSEN, R. K. 1992. Current Research. Plant Molecular Evolution Newsletter 2: 13. ———, D. J. LOOCKERMAN, and H.-G. KIM. 1999. DNA sampling from herbarium material current perspective, Pp. 277–286 in Managing the modern herbarium, ed. D. Metsger. Vancouver: Peanut Butter Press. ——— and J. D. PALMER. 1987. A chloroplast DNA inversion marks an ancient evolutionary split in the sunflower family (Asteraceae). Proceedings of National Academy of Sciences USA. 84: 5818–5822. ——— and ——— 1988. Phylogenetic implications of chloroplast DNA restriction site variation in the Mutisieae (Asteraceae). American Journal of Botany 75: 753–766. ———, H. J. MICHAELS, and J. D. PALMER. 1991a. Phylogeny and character evolution in Asteraceae based on chloroplast DNA restriction site mapping. Systematic Botany 16: 98–115. ———, H. MICHAELS, R. WALLACE, K.-J. KIM, S. KEELEY, L. WATSON, and J. PALMER. 1991b. Chloroplast DNA variation in the Asteraceae: phylogenetic and evolutionary implications. Pp. 252–279 in Molecular systematics of plants, eds. D. Soltis, P. Soltis and J. Doyle. New York: Chapman Hall. ——— and K.-J. KIM. 1996. Implications of chloroplast DNA for the classification and phylogeny of the Asteraceae. Pp 317– 339 in Proceedings of the International Compositae Conference, vol. 1, eds. D.J.N. Hind and H. Beentje. Kew: Royal Botanic Gardens. JEFFREY, C. 1967. Notes on Compositae. II. The Mutisieae in East Tropical Africa. Kew Bulletin 21: 177–224. ———. 1977. Corolla forms in Compositae-some evolutionary and taxonomic speculations. Pp 111–118 in The biology and chemistry of the Compositae, eds. V. Heywood, J.B. Harborne, and B. L. Turner. London: Academic Press. ———. 1978. Compositae. Pp 263–268 in Flowering plants of the world, ed. V. Heywood. London: Mayflower Press. KARIS, P. O., M. KALLERSJO, and K. BREMER. 1992. Phylogenetic analyses of the Cichorioideae (Asteraceae), with emphasis on the Mutisieae. Annals of the Missouri Botanical Garden 79: 416–427. KEELEY, S. C. and R. K. JANSEN. 1991. Evidence from chloroplast DNA for the recognition of a new tribe, the Tarchonantheae, and the tribal placement of Pluchea (Asteraceae). Systematic Botany 16: 173–181. KIM, H.-G., S. C. KEELEY, P. S. VROOM, and R. K. JANSEN. 1998. Molecular Evidence for an African origin of the Hawaiian endemic Hesperomannia (Asteraceae). Proceedings of the National Academy of Sciences USA. 95: 15440–15445. KIM, K.-J. and R. K. JANSEN. 1995. ndhF sequence evolution and the major clades in the sunflower family. Proceedings of the National Academy of Sciences USA. 92: 10379–10383. ———, ———, R. S. WALLACE, H. J. MICHAELS, and J. D. PALMER.

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1992. Phylogenetic implications of rbcL sequence variation in the Asteraceae. Annals of the Missouri Botanical Garden 79: 428–445. LEINS, P. 1971. Pollensystematische studien an Inuleen, I: Tarchonanthinae, Plucheinae, Inulinae, Buphthalminae. Botanische Jahrbucher fur Systematik, Pflanzengeschichte und Pflanzengeographie 91: 91–146. LOOCKERMAN, D. J. and R. K. JANSEN. 1996. The use of herbarium material for molecular systematic studies. Pp 205–220 in Sampling the green world, ed. T. Stuessy and S. H. Sohmer. New York: Columbia University Press. MADDISON, W. P. and D. R. MADDISON. 1992. MacClade: analysis of phylogeny and character evolution, ver. 3.0. Sunderland: Sinauer Associates. MAGUIRE, B. J. 1956. Distribution, endemicity, and evolution patterns among Compositae of the Guayana Highland of Venezuela. Proceedings of American Philosophical Society 100: 467–475. ———. 1967. The botany of the Guayana Highland, part VII: Compositae. Memoirs of the New York Botanical Garden 17: 437– 439. ——— and J. J. WURDACK. 1957. The botany of the Guayana Highland, part II. Memoirs of the New York Botanical Garden 9: 235–392. ———, A. STEYERMARK and J. J. WURDACK. 1957. Botany of the Chimanta Massif, I: Gran Sabana, Venezuela. Memoirs of the New York Botanical Garden 9: 393–439. MATTFELD, J. 1934. Compositae novae sinenses. Notizblatt des Botanischen Gartens und Museum zu Berlin-Dahlem 11: 103– 110. MERXMULLER, H., P. LEINS, and H. ROESSLER. 1977. The Inuleaesystematic review. Pp 577–602 in The biology and bhemistry of the Compositae, eds. V. Heywood, J. B. Harborne, and B. L. Turner, London: Academic Press. OLMSTEAD, R. G., B. BREMER, K. SCOTT, and J. D. PALMER. 1993. A parsimony analysis of the Asteridae sensu lato based on rbcL sequences. Annals of Missouri Botanical Garden 80: 700–722. ORNDUFF, R., T. MOSQUIN, D. KYHOS, and P. RAVEN. 1967. Chromosome numbers in Compositae. IV. Senecioneae II. American Journal of Botany 54: 205–213. PARRA, O. and C. MARTICORENA. 1968. Estudio de los granos de polen de plantas chilenas. Gayana, Botanica 17: 1–54. ——— and ———. 1972. Granos de polen de plantas Chilenas, II: Compositae-Mutisieae. Gayana, Botanica 21: 1–107. PRUSKI, J. F. 1991. Compositae of the Guayana Highland-V. The Mutisieae of the lost world of Brazil, Colombia, and Guyana. Boletim do Museu Paraense Emilio Goeldi serie Botanica 7: 335–392. ROBINSON, H. 1991. Two new species of Stifftia with notes on relationships of the genus (Asteraceae: Mutisieae). Systematic Botany 16: 685–692. SAMBROOK, J., E. F. FRITSCH, and T. MANIATIS. 1989. Molecular clonning, a laboratory manual. New York: Cold Spring Harbor Laboratory Press. SKVARLA, J., B. TURNER, V. PATEL, and A. TOMB. 1977. Pollen morphology in the Compositae and related families. Pp. 141–265 in The biology and chemistry of the Compositae, eds. V. Heywood, J. B. Harborne, and B. L. Turner. London: Academic Press. SMALL, J. 1918. The origin and development of the Compositae. Chapter IV. The corolla. New Phytology 17: 13–40. SWOFFORD, D. L. 1998. PAUP*: Phylogenetic analysis using parsimony, test version 4.0 d64. Sunderland: Sinauer Associates. VUILLEUMIER, B. S. 1969. The systematics and evolution of Perezia Sect. Perezia (Compositae). Contribution of Gray Herbarium 199: 1–163. WINSHIP, P. R. 1989. An improved method for direct sequencing of PCR amplified material using dimethyl sulphoxide. Nucleic Acid Research 17: 1266.

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APPENDIX 1 List of taxa examined for ndhF. Voucher information and GenBank accession numbers follow the species name. Abbreviations for collections from Botanical Gardens are: FTG, Fairchild Tropical Gardens; GOET, Systematisch-Geobotanisches Institut der Universitat Gottingen; K, Kew Botanical Garden; MBG, Matthaei Botanical Garden; MO, Missouri Botanical Garden; UC, University of California Botanical Garden; USDA, United States Department of Agriculture. The subfamilial and tribal classification follows Jansen and Kim (1996). Asteraceae-Barnadesioideae: Barnadesia caryophylla Blake (K001–76–0038, L39396), Chuquiraga jussieui Gmel. (Stuessy et al. 12410 (OS), L39393), Dasyphyllum argenteum H. B. K. (Stuessy & Viteri 12464 (OS), L39392), Doniophyton anomalum (D. Don) Kurtze (Stuessy & Ruiz 12780 (OS), L39396), Schlechtendalia luzulifolia Less. (Stuessy & Katinas 12810 (OS), L39395). Subfamily classification uncertain: MutisieaeGochnatiinae: Ainsliaea acerifolia Sch.-Bip. (Y.-D. Kim s.n. (TEX), L39410), Ainsliaea dissecta Franchet & Savign (K 110–80–00757), AF233813), Ainsliaea reflexa Merr. var. nimborum Hand.-Mazzsp. (Panero & Hsiao 6508 (TEX), AF233845), Aphyllocladus sanmartinianus Molf (Simpson 1-21-86-4 (TEX), AF233804), Cnicothamnus lorentzii Griseb (Nee 36180 (TEX), AF233823), Dicoma carbonaria Humbert (Phillipson (MO), AF233809), Gochnatia hypoleuca Gray (Turner et al. 93–143 (TEX), AF233808), Gochnatia illicifolia Less. (Goldman 426 (TEX), AF233805), Gochnatia paucifolia Jervis (FTG 64– 276, L39397), Gochnatia polymorpha Cabrera (Samuel & Jones 72745 (TEX), AF233806), Hesperomannia lydgatei Forbes (Lorence 7703 (NTBG), AF092584), Hesperomannia arborescens Gray (Wood & Perlman 397 (NTBG), AF092600), Nouelia insignis Franchet (Wu Sugong (no voucher), AF233839), Onoseris hyssopifolia H. B. K.(Jansen 921 (MICH), L39398), Pasaccardoa procumbens (Lisowski) Pope (Philcox et al. 10315 (K), AF233838), Pertya glabrescens Sch.-Bip. (Jun Wen (CSU), AF233832), Pertya phyllicoides Jeffrey (J.F. Jeffen (no voucher), AF233842), Plazia daphnoides Weddell (Hellwig 516 (GOET), AF233840), Stifftia chrysantha Mikan (K 386– 39–38601, L39399). Mutisieae-Mutisiinae: Chaetanthera acerosa Benth. & Hook. (Gengler 49 (OS), AF233831), Chaetanthera flabellifolia Cabrera (Gengler 53 (OS), AF233822), Chae-

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tanthera sp. (Gengler 26 (OS), AF233830), Chaetanthera pusilla (Don) Hook.et Arn. (Zollner 11974 (MO), AF233821), Chaptalia escarpa (Pers.) Baker (Mollin et al. 8655 (US), AF233811), Chaptalia lyratifolia Burkart (Nesom et al. 5981 (TEX), AF233810), Chaptalia nutans (L.) Polak (K 161–83.02018), AF233812), Chaptalia tomentosa Vent (Goldman 497 (TEX), AF233837), Duidaea marahuences Steyerm. (Funk 8199 (US), AF233843), Duidaea pinifolia Blake (Funk 8010 (US), AF233844), Gerbera jamesonii Bolus (Jansen 915 (MICH), L39403), Leibnitzia seemanni Nesom (Nesom 4946 (TEX), AF233815), Mutisia alata Hieron. (Panero 2950 (MSC), AF233833), Mutisia hieronymii Sodiro (Panero 3001 (TEX), AF233834), Mutisia kurtzii Fries (Lavin & Lavin 5855 (TEX), AF233835), Mutisia ledifolia Decne ex Wedd. (Johns 82– 21 (MO), AF233836), Mutisia spinosa Ruiz et Pav. (UC 80–835), AF233818), Mutisia subulata Ruiz et Pav. (Gengler 17 (OS), AF233819), Myripnois dioica Bunge (Lin 340 (UC), AF233846), Pachylaena atriplicifolia Don. (Kiesling et al. 7452 (MO), AF233827), Piloselloides cordata Jeffrey (K 1970–4692), AF233820). Mutisieae-Nassauviinae: Acourtia microcephala DC (Keil 18945 (OBI), L39408), Acourtia runcinata (Lag.) Turner (Nee & Diggs 24539 (TEX), AF233807), Adenocaulon himalaicum Edgew. (K.-J. Kim 13556 (SNU), L39401), Jungia paniculata Gray (Panero 2903 (QCA), AF233816), Leucheria sp. (Gengler 34 (OS), AF233829), Nassauvia digitata Weddel (Gengler 11 (OS), AF233824), Nassauvia gaudichaudii Cass. (K 134–79–01279, L39405), Nassauvia lagascae Meigen (Gengler 16 (OS), AF233826), Perezia multiflora Less. (K 280–64–28004), AF233814), Proustia cuneifolia Don. (Gengler 29 (OS), AF233817), Triptilion spinosum Raiz & Pav. (Gengler 15 (OS), AF233825), Trixis antimenorrhea Mart. (Zardini & Vel. 27635 (TEX), AF233841), Trixis californicum Kellogg (Sundberg & Lavin 2812 (TEX), AF233803). Cardueae: Carlina vulgaris L. (Hellwig s.n. (GOET), L39412), Carthamus tinctorius L. (USDA PI 198990, L39417), Cirsium texanum Buckl. (K.-J. Kim 10693 (TEX), L39418), Echinops exaltatus Koch (Jansen 1001 (MICH), L39411). Tarchonantheae: Brachylaena discolor DC. (D. Haines 235(J), AF233828), Tarchonanthus camphoratus L.(UC 48–0777, L39409). Asteraceae-Cichorioideae: Arctoteae: Arctotis stoechidifolia Bergius (Jansen 920 (MICH), L39425). Lactuceae: Lactuca sativa L. (no voucher, L39389). Liabeae: Liabum glabrum Hemsley (Panero 2554 (TEX), L39421). Vernonieae: Gutenbergia polytrichotoma Wechuysen (King 9982 (US), L39429), Stokesia laevis Greene (K.-J. Kim 13591 (SNU), L39430), Vernonia mesipifolia Less. (Jansen 995 (MICH), L39427). Asteraceae-Asteroideae: Astereae: Aster cordifolius Michaux (Jansen 906 (MICH), L39449). Calenduleae: Calendula officinalis L. (Jansen 903 (MICH), L39439). Coreopsideae: Coreopsis tinctoria Nutt. (K.-J. Kim 12007 (TEX), L39461). Inuleae: Inula helenium L. (K.-J. Kim (SNU), L39453). Senecioneae: Senecio mikanoides Otto (MBG 72326, L39435).